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Molecular cloning and characterization of a novel zinc finger protein: brain expressed ring finger protein… El-Husseini, Alaa El-Din 1997

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M O L E C U L A R CLONING A N D CHARACTERIZATION OF A N O V E L ZINC FINGER PROTEIN: B R A I N EXPRESSED RING FINGER PROTEIN (BERP) by A L A A EL-DIN EL-HUSSEINI B.Sc , Ain Shams University M S c , The University of Manitoba A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Neuroscience) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A August 1997 © Alaa el-Din el-Husseini, 1997 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Colurffbia Vancouver, Canada Date +33-Abstract A novel mammalian RING finger protein which is highly expressed in the brain (Brain expressed RING protein; BERP) was discovered. RING finger proteins are a new family of zinc finger proteins which are involved in processes that regulate cell cycle, growth and differentiation. BERP belongs to a subgroup of RING finger proteins known as the "RING finger-B-box-Coiled Coil (RBCC) proteins. Several RBCC proteins were identified as oncogenes. The human BERP gene mapped to chromosome 1 lpl5, an area containing several tumor suppressor genes. Northern blotting and in situ hybridization analysis indicated that BERP is expressed in all brain regions examined. Immunohistochemical analysis showed that BERP is expressed in several neuronal populations throughout the brain with a punctate pattern in the soma and dendrites. In addition, transiently expressed BERP in human embryonic kidney (HEK) cells is found in the cytoplasm with a punctate distribution. However, the expression of a truncated form of BERP which contains only the N-terminal RBCC domain was found in the nucleus. These results suggest that the C-terminal region of BERP may interact with cytoplasmic proteins while the N-terminus may mediate signals in the nucleus. The yeast two-hybrid assay was used to identify proteins which might interact with BERP. An unconventional class V myosin, interacted with the C-terminus of BERP in yeast and co-immunoprecipitated with BERP. Type V myosins have been implicated in vesicular transport. These data suggest that BERP associates with class V unconventinal myosins and may be involved in vesicular transport. Pheochromocytoma (PC 12) cells transfected with the N-terminus RBCC domain of BERP did not differentiate following nerve growth factor (NGF) treatment while cells expressing the full-length protein responded to NGF treatment and developed neurites. Hence, expression ii of the truncated BERP may act as a dominant negative mutant of endogenous BERP and block its in vivo functions. The suggested involvement of BERP in neuronal differentiation is consistent with the role of class V myosins in transporting vesicles to sites of filopodial growth. In conclusion, I have discovered a novel RING finger protein, BERP, which may be required for vesicular transport fostering the differentiation and maintenance of neuronal cells. iii Table of Contents Page# Abstract ii Table of Contents iv List of figures viii List of tables xi List of abbreviations xii Acknowledgements xiv Introduction 1 I. RING finger family 3 II. Structure of the RING finger 3 1. The RING finger is a unique zinc binding motif. 3 2. RING finger variants 4 3. Location of the RING finger motif 5 4. Structural comparison of the RING finger motif with other known zinc fingers 6 III. Signals mediated by RING finger proteins 7 IV. Proteins containing the RING finger motif 9 1. Viral RING finger proteins 9 2. Oncogenic RING finger proteins 10 3. RING finger proteins in transcription regulation 11 4. RING finger proteins in vesicular transport 12 5. RING finger proteins in signal transduction pathways 13 V. Putative role (s) of the RING finger domain 15 1. The RING finger is a putative protein-protein interaction motif 15 VI. The RBCC group of RING finger proteins 17 Materials and Methods I. Reverse transcription (RT) reactions 19 II. Polymerase chain reaction (PCR) 19 III. Library screening 20 1. Cloning of the rat BERP 20 2. Cloning of the human BERP 21 a. Cloning of the 3' end of human BERP cDNA 21 b. Cloning of the 5' end of human BERP cDN A 21 IV. RNA extraction and Northern Blotting 22 V. In situ Hybridization 23 VI. Fluorescence in situ hybridization FISH analysis 25 iv VII. Yeast two hybrid analysis 25 1. Construct preparation, library amplification, and yeast strain phenotype verification 26 2. Yeast transformation and library screening 27 a. Competent cell preparation: 27 b. Small scale transformations: 28 c. Large scale transformations 28 d. P-galactosidase filter lift assay 29 e. Yeast two hybrid analysis of true and false positives 29 VIII. Cell transfection 30 1. Transient transfection of human embryonic kidney cells (HEK) 293 30 a. Transfection procedure 30 2. Transient and stable transfection of rat pheochromocytoma (PC 12) cells 31 IX Immunocytochemistry analysis of transfected cells 32 X. Immunohistochemistry 33 XI. Western blotting analysis and Immunoprecipitation 34 1. In vitro translation 34 2. Generation of antibodies against BERP 34 3. Western blotting 35 4. Immunoprecipitation (IP) 35 a. Preparation of Protein A-Sepharose beads (CL4B, Pharmacia) for IP 36 b. Immunoprecipitation procedure 36 X. Constructs generated for transfection mammalian cells 37 1. Full length BERP (FBERP) 37 2. N-terminal region of BERP (NBERP) 37 3. C-terminal region of BERP (CBERP) 38 4. Actinin 39 5. His-myr6 39 Results I. Cloning of a novel RING finger protein 42 1. Molecular cloning of rat BERP 42 2. Molecular cloning of the Human BERP 46 3. Sequence analysis of BERP 50 a. BERP contains the RING finger-B box-Coiled-Coil (RBCC) domain (amino acids 1-285) 50 i. RING finger motif (amino acids 22-65) 50 ii. B box (amino acids 115-146) 54 iii. Coiled coil region (amino acids 147-285) 56 iv. BERP contains a domain that is present as several repeats in ABP-120 and ABP-280 (ABP-like repeat) 59 v v. BERP contains a putative nuclear localization signal 62 vi. BERP contains a unique C-terminus 62 II. Gene expression of BERP 67 1. Northern analysis 67 a. Rat tissues 67 b Human tissues 67 2. In situ hybridization 68 3. Immunohistochemical analysis 72 a. Generation and characterization of BERP antibodies 72 i. Crude serum from an immunized rabbit detects an ~82 KDa band that corresponds to BERP 72 ii. Anti-BERP antibodies recognizes BERP in HEK cells transfected with BERP cDNA 74 iii. Crude as well as affinity purified anti-BERP antibodies immunoprecipitate BERP from HEK cells transfected with BERP 74 iv. Immunocytochemical detection of BERP protein in HEK cells transfected with BERP cDNA, using the affinity purified anti-BERP antibodies 78 b. Distribution of BERP protein in the brain 80 III. Cell transfection experiments 86 1. Transient expression in HEK cells 88 a. Distribution of the full length BERP protein in HEK cells transfected with BERP cDNA 8 8 b. Subcellular distribution of truncated versions of BERP protein 90 i. Localization of a truncated form of BERP that contains the C-terminal region (CBERP) 90 ii. Localization of a truncated form of BERP that contains the N-terminal RBCC domain (NBERP) 91 2. Stable expression of full length and truncated BERP sequences in PC 12 cells 94 a. Generation of stably transfected PC 12 cells 95 b. Expression of CBERP 95 c. Expression of full length BERP 98 d. Expression of NBERP 98 e. NGF treatment of stably transfected PC 12 cells 102 IV. Yeast two-hybrid analysis 105 1. Using the yeast two-hybrid system to identify proteins interacting with the RBCC domain 105 a. Background 105 b. Positives obtained with RBCC domain as a bait 109 2. Using the yeast two-hybrid system to identify proteins vi interacting with BERP C-terminal region 109 a. Background 109 b. Positives obtained with the C-terminal bait (CBERP 1) 110 3. Analysis of various domains of BERP for interaction with myr 6 and actinin 111 a. Interaction with alpha actinin 111 b. Interaction with myr 6 112 4. Using the yeast two hybrid to examine BERP oligomerization 116 a. Background 116 i. A role for RBCC domain in protein oligomerization 116 ii. A role for BERP C-terminal region in protein oligomerization 117 b. BERP oligomerization as determined by the yeast two hybrid 117 5. Using the yeast two hybrid to test for interactions between BERP and the HIV regulatory protein Tat 121 V. Using immunoprecipitation to identify proteins interacting with BERP 123 1. Background 123 2. Construct preparation 124 3. Alpha actinin immunoprecipitation 126 4. Myr 6 immunoprecipitation 129 5. BERP co-immunoprecipitates with rat dilute 129 VI. FISH analysis and chromosomal mapping of the human BERP gene 136 Discussion 139 Bibliography 175 vii List of Figures page Figure 1. Nucleotide sequence of the rat BERP. 44 Figure 2. Amino acid sequence of rat BERP. 45 Figure 3. Alignments of the nucleotide sequence of the human and rat BERP. 47 Figure 4. Sequence alignments of the deduced amino acids for human and rat BERP. 49 Figure 5. Schematic representation of the various structural motis present in BERP. 51 Figure 6. Sequence comparison of the human BERP with HT2 A. 52 Figure 7. Sequence of RING finger motif. 53 Figure 8. Sequence of the B box. 55 Figure 9. The Coiled-coil motif. 58 Figure 10. BERP contains a domain that is present as several repeats in two actin binding proteins, the yeast ABP 120, and the human ABP 280. 61 Figure 11. The C-terminal region of BERP contains repeats with a (3 strand structure. 64 Figure 12. Secondary structure comparison of BERP and CRAF1. 65 Figure 13. Northern blot analysis of rat BERP mRNA. 69 Figure 14. Northern blot analysis of human BERP mRNA. 70 Figure 15. In situ hybridization analysis of BERP mRNA expression in the brain. 71 Figure 16. Western blot analysis of BERP expression in cerebellum. 73 Figure 17. Western blot analysis of BERP expression in various brain regions. 75 Figure 18. Western blot analysis of BERP expression in transiently transfected HEK cells 76 Figure 19. Immunoprecipitation of BERP from HEK cells transfected with a FLAG-tagged BERP. 77 Figure 20. Immunodetection of BERP protein in HEK cells transiently transfected viii with BERP cDNA using the DAB/ABC method. 79 Figure 21. Immunohistochemical detection of BERP in the nucleus of the trapezoid body. 82 Figure 22. BERP is expressed in several neuronal populations in the brain. 83 Figure 23. BERP is expressed in several neuronal populations with a unique ' punctate pattern. 84 Figure 24. High power magnification of some brain stem neurons stained positive for BERP. 85 Figure 25. Expression of the full-length BERP in HEK cells. 87 Figure 26. Expression of the full length BERP in HEK cells. 89 Figure 27. Staining of HEK cells transiently expressing a truncated BERP (CBERP). 92 Figure 28. Staining of HEK cells transiently expressing a truncated BERP (NBERP). 93 Figure 29. Western blot analysis of expression of truncated forms of BERP. 96 Figure 30. Stable expression of a truncated form of BERP (CBERP) in PC 12 cells. 97 Figure 31. Stable expression of full length BERP in PC 12 cells. 99 Figure 32. Stable expression of a truncated form of BERP (NBERP) in PC 12 cells. 100 Figure 33. Expression of a truncated form of BERP (NBERP) in PC 12 cells. 101 Figure 34. A truncated form of BERP inhibits NGF-induced neurite outgrowth in PC 12 cells. 103 Figure 35. Staining of NGF-treated PC 12 cells expressing full length as well as a truncated form of BERP. 104 Figure 36. Schematic representaion of various constructs used as baits for the yeast two hybrid assay. 105 Figure 37. Positives identified using the yeast two-hybrid system: alpha actinin and a class V myosin (myr 6). 113 Figure 38. Yeast two hybrid analysis shows that the N-terminal region ix of BERP interacts with alpha actinin. 114 Figure 39. Specifc interactions of various domains of BERP with alpha actinin and a class V myosin (myr 6) as determined by yeast two-hybrid analysis. 115 Figure 40. Yeast two-hybrid analysis shows that the N-terminal region of BERP is involved in protein oligomerization. 119 Figure 41. In vitro translation products of alpha actinin, BERP and myosin (myr 6). 125 Figure 42. Co-immunoprecipitation of BERP and alpha actinin using anti-BERP antibodies. 127 Figure 43. Co-immunoprecipitation of BERP and actinin using anti-FLAG antibodies. 128 Figure 44. Co-immunoprecipitation of BERP and myr 6 C-terminal region using anti-FLAG antibodies. 130 Figure 45. Co-immunoprecipitation of endogenous BERP and dilute from rat brain using anti-dilute antibodies. 132 Figure 46. Co-immunoprecipitation of endogenous BERP with dilute from rat brain using anti-dilute antibodies. 135 Figure 47. Southern blot analysis of the human BERP PAC clone. 137 Figure 48. Chromosomal mapping of human bERP gene to chromosome 1 lpl5. 138 Figure 49. Summary of some of the proposed functions for BERP. 172 X List of Tables Table 1. Constructs used for the yeast two-hybrid assay. Table 2. Control plasmids (supplied with the yeast two hybrid kit; MATCHMAKER. 40 41 Table 3. Summary of the results obtained from co-transformation experiments of various baits and targets in a yeast two-hybrid assay. 118 Table 4. Summary of the results obtained from P gal filter lift assay for interactions with Tat. 122 xi List of Abbreviations 3-AT 3-aminotriazole aa amino acids AT ataxia telangiectasia ATDC ataxia telangiectasia group D complementing gene ABP-120 actin binding protein-120 ABP-280 actin binding protein-280 BES N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid BERP brain expressed RING finger protein P-gal P-galactosidase bp base pair BSA bovine serum albumin CAK cyclin activating kinase cAMP cyclic adenosine monophosphate cGMP cyclic guanosine monophosphate cbl cas-Br-lymphoma CDK cyclin dependent protein kinase cDNA complementary DNA cGKI cGMP dependent protein kinase type I cGKII cGMP dependent protein kinase type II CNS central nervous system DAB diaminobenzidine DAPI 4',6-diaminidino-2-phenylindole 2 HC1 DMEM Dulbecco's-minimmum essential medium DNA Deoxyribonucleic acid dNTP deoxynucleotide triphosphate DTT dithiothreitol EDTA ethelynediaminetetaacetic acid EGF epidermal growth factor EtBr ethidium bromide FBS fetal bovine serum FISH fluoresence in situ hybridization fos FBJ murine osteosarcoma virus g gram h hour HEK-293 human embryonic kidney-293 cells IP immunoprecipitation kb kilobase kDa kilodaltons KLH keyhole limpet hemocyanin jun ju-nana, Japanese avian sarcoma virus L liter LOH loss of heterozygosity M molar min minute mM millimolar xii MMLV Murine mouse leukaemia virus mRNA messenger RNA NaCl sodium chloride NaOH sodium hydroxide NP-40 nonidet P40 NGF nerve growth factor NGS normal goat serum nM nanomolar PAC PI-derived artificial chromosome PBS phosphate buffered saline PC12 pheochromocytoma-12 cells PCR polymerase chain reaction PFA paraformaldehyde PKC Protein kinase C PML promyelocytic leukemia ras rat sarcoma virus RBCC RING finger-B box-coiled coil RING really interesting new gene RNA ribonucleic acid rpm revolutions per minutes RT reverse transcription SDS sodium doecyle sulfate Taq thermus aquaticus TE tris-EDTA TNF tumor necrosis factor TNFR tumor necrosis factor receptor TRAF tumor necrosis factor associated factor pi microliter pM micromolar UV ultraviolet UTR untranslated region vol volume xiii A C K N O W L E D G E M E N T M4 greatest thanks goes to mij supervisor, D r . S t e v e n X / i n c e n t , f o r providing unconditi guidance throughout this degree while a t the same time giving me endless support to pursue projects in an independent manner. lonal I am verij g ra te fu l to have had such enormous support f rom various members o f the d epartment. I thank mi) committee members, \Z)v. | qnn fTaLjmond, D f - S t e v e P e l e c h , and D f 1 - Ter rL j 5 n u " t ch fo r the i r valuable suggestions and overall support. | thank D " 1 " - P e t e r R e i n e r , \7)v. " f im M u i "ph i j , and D"1"- C h r i s E ' b i g e r f o r insightful discussions and encouragement throughout this degree. I thank D o r o t a , P a s c a l e , C ^ t ^ o n a , Chu -see f o r the i r technical assistance and friendship. | thank J u l i e \x/i l l iams, / \ n d q | aLjcock, {^ruce, ^ J J i a , C t e f a n , M ^ t t , P a u l and C7<^ro\\ne, C a ' L / \ l i / I—lossein, P u 7 b e h , M e l i n d a , f o r the i r fr iendship. C p e c i ^ l thanks f o r | i-z \)</ong fo r her unlimited help with paper work. I thank mL) sister / \ i d a and her famili) f o r being so supportive and understanding to me and mij uncle N u s a fThal idi f o r his encouregment and support throughout mL) scientif ic career . I thank eli?abeth f o r countless lengthq discussions, good advice and f o r believing in B E R P . xiv Introduction I. RING finger family The RING family of proteins contains a novel zinc finger-binding motif (Freemont '93.) which folds uniquely forming a sphere-like structure (Reddy et al, '92; Freemont, '93; Lovering et al, '93; Barlow et al, '94; Borden et al, '95; Klug and Schwabe, '95). The RING finger motif was first noted in the "Really Interesting New Gene 1" (RING1; Freemont et al, '91). Later on, this motif was found to be present in several other proteins (Lovering et al, '93). At present, there are at least 80 putative RING finger family members that have extraordinarily diverse functions (Saurin et al, '96). Various members of this novel family play crucial regulatory roles and contain unique structural motifs that are tightly conserved throughout evolution. I have cloned a new member of the RING finger family called Brain Expressed RING Protein (BERP). While BERP is highly expressed in specific regions of the rat central nervous system, other tissues possess very low (lung) to undetectable levels of BERP mRNA (heart testis and liver). The aim of this thesis is to document the cloning and characterization of BERP. Different members of the RING family are involved in signal transduction pathways ~ from cell surface receptors to cell cycle regulation and gene expression (Freemont '93; Fisher et al, '95; Saurin et al, '96). At the cell membrane, members of this family have diverse functions which include interactions with growth factor receptor tyrosine kinases {(Sli; Yoon et al, '95); (c-Cbl; Tanaka et al, '95; Galisteo et al, '95; Bowtell and Langdon '95)}, and cytokine receptors {TRAF2; (Rothe et al, '94 & '95) ; CRAF1; (Cheng et al, '95) ; LAP-1 {Mosialos et al, '95) and CAP-1 (Sato et al, '95a). Others are found associated with postsynaptic l membranes and involved in clustering of acetylcholine receptors (43 K also known as PMSP, Krikorian and Bloch '92; Bloch et al, '94). Other members are cytoplasmic proteins found associated with endosomes (EEA1; Mu et al, '95; Stenmark et al, '96) or involved in vacuolar/peroxisome biogenesis {PEP-5 (Woolford et al, '90), Vpsl8p (Robinson et al, '91), PAF-1 (Tsukamoto et al, '94), PAS7p; (Kalish et al, '95}, and post-Golgi transport (VAC1; Burd et al, '97). Others are GTP-binding proteins; such as ARD-1 (Mishima et al, '93), an ADP-ribosylation factor which is a member of the ras GTPase superfamily (ARF; Kahn et al, '93; Moss et al, '95) and FGD1, a guanine nucleotide exchange factor and a member of the Rho/Rac family (Pasteris et al, '94; Vojtek and Cooper, '95; Zheng etal, 96). Still other RING finger members are nuclear proteins that interact with nuclear steroid receptors (TIF 1; Le Douarin et al, '95) or associate with cyclin activating proteins (p-36; Fisher et al, '95, MAT1; Devault et al, '95). Some members can interact with transcription complexes at the active transcription sites and are involved in regulating gene expression in males (Msl-2; Kelly et al, '95) and females (PwA33; Bellini et al, '93&'95) in early development. If mutations in RING finger proteins exist then abnormal cell growth can result in hepatoma, leukemia and breast cancer can occur (Reddy et al, '92; Freemont '93; Miki et al, '94). Thus, the R I N G finger motif is present in proteins of diverse functions with significant roles in development, normal growth and differentiation of various types of cells. The presence of this motif in functionally unrelated proteins makes it difficult to define the molecular function of the R I N G finger, although considering the functions of 2 some of the known molecules containing this motif may indicate a common role for it as a protein-protein interaction domain rather than a D N A binding motif. The thesis will first describe the structure of the RING finger followed by a discussion of signals mediated by several known members of the RING finger family, and then speculate upon the possible role indicated for the RING finger domain. The last part of this introduction will focus on a subgroup of this family that BERP belongs to called the RING finger-B box-coiled-coil (RBCC) subgroup. In addition to the RING finger, this subgroup contains a poorly characterized new type of zinc binding motif known as the B box, and a coiled-coil domain, a structure that is usually involved in protein oligomerization. II. Structure of the R I N G finger 1. The R I N G finger is a unique zinc binding motif. The RING finger is a zinc-binding motif that is present in organisms ranging from viruses to mammals (Saurin et al, '96). This motif is distinct from other known zinc finger motifs in its sequence homology, zinc binding and folding. The RING finger was first observed in a new gene, called "Really Interesting New Gene 1" (RING 1; Freemont et al, '91). Database searches using the newly identified RING finger sequence showed that this motif is present in other known proteins, which led to the identification of a new class of zinc finger proteins, now known as the "RING finger" family. The RING finger motif can be described as C-X 2 -C- x ( 9 . 3 9 ) -C-X ( 1 . 3 ) -H-X ( 2 . 3 ) -C-X 2 -C- X ( 4. 4 g ) -C-X 2 -C, where C is cysteine, H is histidine and X can be any amino acid (Lovering et al, '93; Borden et al, '95; Saurin et al, '96). Thus, the RING finger can be abbreviated as C3HC4. The length of the domain ranges from 42 to 107 amino acids. The large 3 variation in the lengths of the loops forming the RING finger indicate a wide functional diversity between members of the family. Studies on chemically synthesized RING finger domains of the RTNG1 protein indicated a stoichiometry of two bound zinc with tetrahedral metal co-ordination and formation of metal-sulfur bonds (Lovering et al, '93). Recently, the solution structure of the RING fingers of two known proteins, the herpes virus protein, VmwllO (also known as IE110, IPCO and IEEHV; Barlow et al, '94;) and the mammalian protein PML (Borden et al, '95) have been determined. These studies have demonstrated the requirement of zinc binding for proper folding of this motif and showed that the RING finger is nearly spherical in shape forming a cross-brace zinc binding arrangement. Borden et al, '95 showed that a 56 amino acid peptide that contained the full length RING finger motif of PML could fold in the presence of zinc, indicating that the RING finger is an autonomously folded unit, and its folding does not require other domains present in the native protein. 2. R I N G finger variants A variant of the RING finger (C3H2C3), also known as the RING-H2 variant, contains a His residue in place of Cys-4. The RLNG-H2 variant is present in the 43K protein (also known as PSMP, RAPSYN) that is involved in acetylcholine receptor clustering (Bloch et al, '94) as well as a class of proteins that are involved in vacuole biogenesis and peroxisome assembly (Saurin et al, '96). Two other RING finger variants are present in the yeast protein VAC1, which is involved in vesicular transport from Golgi to the yeast vacuole (Burd et al, '97). The first VAC1 RING finger is composed of C-X 2 -C-X 1 2 -C-X 2 -C-X 4 -C-X 2 -H-X 2 2 -C-X 2 -C (C5HC2). In the second VAC1 RING finger, histidine 1 is replaced by cysteine, and thus the RING finger motif of this variant contains 8 cysteines as zinc binding ligands (RTNG-C8). The second RING 4 finger motif of VAC 1 is also present in the mammalian endosomal protein EEA1 (Mu et al, '95b; Stenmark et al, '96), the filamentous fungus Podospora anserina perixosomal protein, carl (Berteaux-Lecellier et al, '95), the vesicular sorting protein Vpsl8p (Robinson et al, '91) and the yeast endosomal protein, FAB1 (Yamamoto et al, '95). This particular RING finger variant has been called FYVE, because of the high conservation of this motif in four known vesicular transport proteins (Stenmark et al, '96). Other RING finger variants include the mouse double minute protein (MDM2; Boddy et al, '94) where Cys-3 is replaced by a Thr, and the one present in a member of the TRAF family, CART1 (Regenier et al, '95), and the GTPase ARD1 (Mishima et al, '93) where Cys-7 is replaced by Asp. Another variant of the RING finger is present in the retinoblastoma binding protein (RBQ-1) where His-1 is replaced by Asn (Saurin et al, '96). These substitutions may influence zinc binding while the overall three dimensional structure of the RING finger domain in these proteins is conserved. 3. Location of the RING finger motif In general, RING fingers are found near the N-terminus of most of the RING family members (Saurin et al, '96). Members of the TRAF family are cytoplasmic proteins with an N-terminus RING finger. Several nuclear proteins such as PML and BRCA1 also contain an N-terminus RING finger motif (Saurin et al, '96). Other RING finger proteins such as the mammalian homologues of the baculovirus inhibitors of apoptosis (IAPs) (Crooke et al, '93; Rothe e al., 95b; Hey et al, '95; Uren et al, '96), and the yeast RING B protein contain the RING finger at the C-terminus end (Kawata et al, '96). Also, members of the RING finger variant RING-H2 as well as the RING-C8 variant (FYVE; Stenmark et al, '96) contain the 5 RING finger domain at the C-terminus. A central RING finger domain is present in c-Cbl (Blake et al, '91), a signaling protein that is involved in EGF pathways (Galisto and Bowtell, '95; Soltoff and Cantley, '96; Salgia et al, '96; Wang et al, '96). To date, there is no correlation between function and location of the RING finger. 4. Structural comparison of the RING finger motif with other known zinc fingers At least 80 RING finger proteins have been identified from different species (Saurin et al, '96). The RING finger binds two zinc atoms with each zinc atom ligated tetrahedrally by either four cysteines or three cysteines and one histidine (Lovering et al, '93; Borden et al, '95). This structure results in a unique cross-brace folding which is different from other known zinc finger motifs like the GAL4-type, TFIIIA-type and the steroid/nuclear receptor family of proteins (Barlow et al, '94; Borden, et al, '95). While the RING finger shows no similarity to the GAL4 consensus sequence, similarities can be observed between the RING finger domain and the nuclear receptor domain (Tsai and O'mally, '94; Klug and Schwabe, '95). In addition, consensus sequences of both the RING finger and the TFIIIA motifs are strikingly similar, particularly when the RING is seen as a bi-partite motif consisting of C-X 2-C-X ( 9. 3 9 )-C-X (j. 3 )-H and C-X 2-C-X (4_ 4 8 )-C-X 2-C. However, recent NMR studies showed that all RING finger cysteines are involved in forming one uniquely folded zinc finger. This means the RING finger cannot be viewed as two tandem repeats, each is a TFIIIA-like zinc finger (Barlow et al, '95) The LIM domain is another cysteine-rich motif similar to the RING finger (Schmeichel and Berckerle, '94; Dawid et al, '95; Gill, '95; Schmeichel and Berckerle, '97). Both motifs have very similar spacing between the cysteines and in the variability of the length of loops. However, there is no sequence homology between the two domains apart from the zinc binding ligands. Interestingly, like BERP the LIM domain is also found in a functionally diverse family of proteins that are involved in the regulation of cell surface receptors and gene expression (Gill, '95). III. Signals mediated by RING finger proteins Cell proliferation, differentiation and apoptosis are processes tightly regulated by various signal transduction pathways (Briscoe et al, '94; Cooper, '94; Darnell et al, '94; Ihle et al, '95; Ito et al, '95; Lipton, '96; Leaman et al, '96). Members of the RING finger family of signal transduction proteins are involved in regulating cell fate by interacting with growth factor, cytokine and steroid hormone receptors. For example, TRAF-2 (Rothe et al, '94; Song and Donner '95; ), CAP-1 (Sato et al, '95), CART1 (Regnier et al, '95), LAP1 (Mosialos et al, '95), CRAF1 (Cheng et al, '95) and CD-40 binding protein (Hu et al, '94) are RING finger proteins that directly interact with members of the tumor necrosis factor (TNF) receptor family to activate target proteins like N F K B , a transcription factor mediating TNF responses (Rothe et al, '94&'95). Another RING finger protein, Sli-1 (Yoon et al, '95), is the c.elegans homologue of the mammalian proto-oncogene c-Cbl (Blake et al, '91) and acts as a negative regulator of the epidermal growth factor (EGF) receptor tyrosine kinase pathway (Yoon et al, '95). Recent studies by Galisteo et al ('95) and Bowtell and Langdon ('95) showed that the mammalian c-Cbl interacts directly with EGF receptor and other signal transduction proteins such as the adapter protein Grb2. Steroids and steroid receptor signal transduction pathways also involve RING finger proteins (Le Douarin et al, '95; Inoue et al, '93; Orimo et al, '95). For example, TIF1 (Le Douarin et al, '95) directly interacts with the activation domain present in several members of the steroid receptor superfamily. Also, estrogen responsive RING finger proteins have been identified (Inoue et al, '93; Orimo et al, '95). 7 A more direct role for cell cycle regulation is indicated by two RING finger proteins that associate with cyclin activating kinases (CAKs) (p36; Fisher et al, '95; MAT-1, Devault et al, '95). Also, the tumor suppressor gene product, p53, associates with MDM2, a RING finger protein (Boddy et al, '94). Moreover, some members of the RING family can be oncogenic which indicates the importance of these proteins in maintaining normal cell growth. These oncogenes result from mutations (BRCA1 gene mutations detected in breast tumors; Futreal et al, '94; Miki et al, '94), chromosomal translocations {T-18 - B-Raf fusion protein in hepatoma (Miki et al, '91); PML-retinoic acid receptor fusion protein in promyelocytic leukemia (Kakizuka et al, '91; Kastner et al, '92; Gillard and Solomon '93; Liu et al, '95); and rfp-tyrosine kinase (RET) fusion protein; Takahashi et al, '88}. The transforming viral protein, v-Cbl, is a truncated form of c-Cbl, which is missing the RING finger domain (Langdon et al, '89; Blake etal, '91). Cellular RING finger proteins, such as LAP-1 (Mosialos et al, '95) and HT2A (Fridell et al, '95) may mediate viral protein actions. LAP-1, a TRAF domain-containing RING finger protein, associates with the Epstein-Barr virus transforming protein LMP1 and activates N F K B , a transcription factor normally activated by members of the TNF family (Mosialos et al, '95). Using the yeast two-hybrid system, HT2A has been identified as a protein that associates with the HIV-1 virus regulatory protein Tat, and may mediate HIV-1 virus activity (Fridell et al, '95). In contrast, Staf 50 is an interferon-induced RING finger protein that down regulates transcription directed by the long terminal repeat promoter region of human immunodeficiency virus type 1, indicating that this RING finger protein may mediate interferon antiviral interaction (Tissot and Mechti, '95). 8 Interestingly, some viral proteins also contain RING fingers (Crook et al, '93; Moriuchi et al, '94; Everett&Mual, '94; Everett et al, '95; Kawase et al, '95, Lee and Hruby '95). In sum, these data indicate that cellular proteins containing the RING finger motif are important in regulating cell cycle and growth. Consequently, mutations in these RING finger proteins alter cell cycle regulation and promote oncogenesis. In addition, cells may recruit RING finger proteins as a defense mechanism against viral infections. Moreover, viruses may manipulate RING finger proteins to gain control over their own replication and activity. IV. Proteins containing the RING finger motif 1. Viral RING finger proteins A number of viral proteins contain a RING finger domain (Saurin et al, '96). The LCMV (lymphocytic choriomeningitis virus) protein consists of a single RING finger domain, and thus this viral protein may represent the primordial RING finger protein (Freemont, '93). Unfortunately, no known function has been described for LCMV protein, but it has been suggested that this protein may be involved in RNA binding (Saurin et al., '96). The immediate early herpesvirus proteins IEBHV (bovine virus) and IE110 are putative transcription factors but no direct DNA interaction has been demonstrated for the RING finger motif in these proteins (Saurin et al, 96). Mutation of the IE110 RING finger domain results in a loss of viral infectivity (Everett&Mual, '94; Saurin et al, '96). Baculovirus inhibitors of apoptosis (IAPs) are RING finger proteins, which act in insect cells to prevent cell death (Crook et al, '93; Rothe et al, '95b; Uren et al, '96). Mammalian homologues of IAPs have also been identified (Uren et al, '96). The RING finger domain has been shown to be essential for the function of the 9 baculovirus inhibitor of apoptosis (IAP) protein, and mutations in the RING finger domain of IAP abolishes its ability to inhibit apoptosis (Rothe et al, '94; Uren et al, '96; Saurin et al, '96). 2. Oncogenic RING finger proteins. Several RING finger proteins have roles in cellular transformation. Moreover, fusion proteins formed by chromosomal translocations of the RET finger protein (Rfp;), PML and T18 are oncogenic (Saurin et al, '96). In RET, the RING finger motif of Rfp is fused to a tyrosine kinase domain (Takahashi et al, '88; Saurin et al, '96). T18 is a fusion between the N-terminal region of the transcriptional intermediary factor (TIF1) and the kinase domain of B-Raf (Miki et al, '91). A translocation that involves chromosomes 15 and 17 is associated with acute promyelocytic leukemia and results in the fusion of the RBCC domain of PML with retinoic acid receptor. Other RING finger proteins with oncogenic potential include the breast cancer susceptibility gene BRCA1, c-Cbl, MEL18 and BMI-1 (Saurin et al, '96). Point mutations or deletion of the RING finger domain of the BRCA1 gene product were detected in breast cancer (Futreal et al, '94). c-Cbl becomes oncogenic after deletion of the RING finger domain (Blake et al, '91). MEL18 is a nuclear protein that is upregulated in melanoma cell lines (Tagawa et al, '89), whereas BMI-1 acts as a cooperating oncogene with myc in E(j,-myc transgenic mice (Van Lohuizen et al, '91). Endogenous BRCA1, PML, MEL 18 and BMI function as tumor suppressors (Tagawa et al, '89; Van Lohuizen et al, '91; Gillard&Solomon, '93; Futreal et al, '94; Liu, '95; Mu et al, '95; Wang et al, '97; Scully, '97). 10 3. R I N G finger proteins in transcription regulation Several RING finger proteins are thought to be involved in regulating gene transcription despite the fact that the RING finger domain has never been shown to specifically bind DNA. RTNG1, the first member of the family to be identified, has been recently shown to associate with the polycomb group protein complex and acts as a transcription repressor (Satijn et al., '97). SS-A/Ro is a human autoantigen and forms part of a ribonucleoprotein complex, although no direct interaction between SS-A/Ro and RNA has been demonstrated (Chan et al, '91). XNF7 is a maternally expressed Xenopus protein that is present in the cytosol and translocates to the nucleus during the mid blastula transition (Li et al, .'94). Male-specific lethal-2 (msl-2) is a RING finger protein that is required for X chromosome dosage compensation in Drosophila (Kelley et al, '95). PwA33 is a newt RING finger protein that is closely related to the Xenopus XNF7. PwA33 is expressed in the loop matrix of lampbrush chromosomes (Bellini et al, '93&'95), an area which contains nascent RNA transcripts, associated hnRNP proteins and RNA splicing factors. The RING finger protein BRCA1 localizes to meiotic chromosomes where it associates with Rad51, a regulator of DNA recombination (Scully et al, '97). RPT-1 has been implicated in the down-regulation of the interleukin-2 receptor (Patarca et al, '88). RAG1 is involved in the activation of the human V(D)J recombination (Schatz et al, '89). RAD 18 is required for postreplicative repair of UV damaged DNA in yeast (Jones et al, '88). KAP-1 associates with the KRAB domain and enhances KRAB-mediated repression, and the KAP1 RING finger is required for KRAB binding and co-repression (Friedman et al, '96). Staf 50 is an interferon-induced RING finger protein that down regulates transcription directed by the long terminal 11 repeat promoter region of human immunodeficiency virus type 1 indicating a role for it in mediating interferon antiviral action (Tissot and Mechti, '95). 4. RING finger proteins in vesicular transport RTNG-H2 and RING-C8 variants of the RING finger motif are involved in protein clustering and regulation of vesicular transport and vacuole/peroxisome proliferation (Saurin et al, '96). The yeast proteins PEP5 and Vpsl8p (Woodford et al, '90; Robinson et al, '91) have been implicated in vacuole biogenesis. The yeast protein, Vps8p, has been recently shown to facilitate soluble vacuolar protein localization (Horazdovsky et al, '96). Others are involved in peroxisome proliferation including PAS7p (Kalish et al, '91), PAF1 (Woodford et al, '90) and carl (Berteaux-Lecellier et al., '95). The neuronal RING finger protein Neurodapl (contains the RING-H2 motif) has been localized to the endoplasmic reticulum and Golgi apparatus as well as axosomal postsynaptic densities and may play a role in the regulation of protein sorting machinery (Nakayama et al., '95). Similarly, a novel yeast RING finger protein, VAC1, has been identified and shown to be involved in vacuolar protein sorting (Burd et al, '97). EEA1 is another mammalian protein that contains the RING-C8 variant and is associated with early endosomes (Mu et al, '95). It is noteworthy that the human perixosome proliferator factor, PAF1, contains the classical RING finger whereas its fungus homologue carl contains the RING finger variant RTNG-C8 (Berteaux-Lecellier et al, '95). Both PAF1 and carl have been shown to be localized to peroxisomes, indicating that the classical RING finger and its variants can serve the same function (Berteaux-Lecellier et al., '95). 12 Although most of the RING finger proteins involved in vesicular transport contain a C-terminus RING finger, exceptions to this rule do exist. For example, ARD-1, a member of the RBCC family, contains a N-terminus RING finger and belongs to the ADP-ribosylation factor (ARF) family of proteins that are involved in the regulation intracellular vesicular transport (Mishima et al., '93; Peters et al., '95; Moss, '95). Thus, it does not seem that the location or the presence of a specific type of RING finger variant is crucial for defining the protein site of action. 5. RING finger proteins in signal transduction pathways. There is evidence indicating that RING finger proteins play active roles in the regulation of signal transduction pathways governing cell cycle, differentiation and apoptosis. The RING finger protein MAT1 (menage-a-trois) has been found as a part of the cyclin dependent kinase7/cyclin H complex (Devault et al, '95). This complex is involved in the regulation of CAK (CDK activating kinase) phosphorylation, and a role for these complexes in the regulation of cell cycle has been demonstrated (Sherr, '93). Recent data have shown that several cytokine receptor signal transduction pathways are regulated by a growing group of RING finger proteins named TRAFs (TNF receptor associated factors) (Baker and Reddy, '96). TRAF2, the first of the TRAF proteins to be identified, was found to be associated with the tumor necrosis factor (TNF) receptors (TNFR-1 and TNFR-2) and was required for activation of the cytoplasmic transcription factor NFKB (Rothe et al, '94; Rothe et al, '95a&b; Hsu et al, '96; Cheng et al, '96). Later, five novel TRAF proteins have been isolated and were shown to be involved in the regulation of TNF and other cytokine receptors including CD-40 receptor and interleukin-1 receptor and in the activation of NF-KB (Rothe etal, '94&95a; Baker and Reddy, '96). 13 The cellular protein c-Cbl is a cytoplasmic protein that is involved in the EGF receptor signaling pathway and interacts with the adapter protein Grb2 and with the SH3 domain of p47«c& (Rjvero-Lezcano et al, '94). C-Cbl associates with the tyrosine-phophorylated She and has been implicated in the EGF-dependent activation of phosphatidylinositol-3-kinase (Soltoff&Cantley, '96; Wang et al, '96). 14 V. Putative role(s) of the RING finger domain 1. The RING finger is a putative protein-protein interaction motif The RING finger domain is present in several proteins of diverse functions (Saurin et al, '96). Some of these RING finger proteins are nuclear but several others are cytoplasmic. The identification of RING finger proteins with roles in clustering of receptors (such as the role of TRAFs in cytokine receptor clustering (Baker and Reddy, '96), and the role of postsynaptic density protein, 43K, in acetylcholine receptors; Bloch et al, '94) as well as in processes that do not involve DNA interactions (such as those involved in peroxisome formation and vacuole assembly) strongly indicate that the RING finger motif, at least in these proteins, is not a DNA binding domain (Freemont et al, '93). Accumulating evidence indicates that the RING finger domain is a protein-protein interaction motif. Mutations of the PML RING finger domain results in a diffused nuclear staining and loss of the nuclear bodies observed with wild type protein. TRAF2 lacking the RING finger motif fails to activate latent cytoplasmic transcription factor NFKB (Rothe et al., '95a). Mutations of the two RING finger motifs of the yeast protein VAC1 result in protein missorting (Burd et al, '97). Mutations of the RING finger of EEA1 leads to a loss of localization to endosomes (Stenmark et al, '96). KAP-1 lacking the RING finger and the B box domain fails to interact with its partner KRAB (Friedmann et al., '96). Although all of the above described observations support a crucial role for the RING finger motif in protein-protein interactions no direct association of the RING finger motif with other proteins has been shown. The only common feature of most of the known RING finger proteins is that they exist as part of large protein complexes. Immunolocalization of several RING finger proteins showed a punctate distribution that varied in size from large nuclear bodies as the case of PML 15 and RAG1/RAG2 complexes (Leu&Schatz, '95) or as speckles as seen in the case of BRCA1 (Wang et al, '97), carl (Berteaux-Lecellier, '95) , VAC1 (Burd et al, '97) and Neurodapl (Nakayama et al, '95) indicating the presence of proteins in large complexes. Members of the TRAF family form homo- and heterodimers, interact with several cytoplasmic proteins including IAPs, TRADD, TANK and are involved in clustering of various cytokine receptors (Rothe et al., '95b; Baker&Reddy, '96; Uren et al, '96; Hsu et al, '96; Cheng&Baltimore, '96). The post synaptic density protein 43K is involved in clustering acetylcholine receptors (Krikorian&Bloch, '92). TIF1 is a part of the nuclear steroid receptor complex (Le Douarin et al, '95), whereas XNF7 (Etkin, '91), and BRCA1 (Wang et al, 97) are part of large nuclear complexes found associated with the chromatin. The cytoplasmic protein c-Cbl is associated with several signaling proteins including Grb2, p47"c^, She and PI3-kinase (Rivero-Lezcano et al, '94; Wang et al, '96; Soltoff&Cantley, '96). All of these observations indicate that RING finger proteins are required for the formation of large protein complexes, perhaps acting as scaffolding elements, and indicate that the RING finger motif may be involved in stabilizing of protein aggregation through homo- and heteromeric interactions. In support of such a role for the RING finger in protein oligomerization, is the presence of a coiled-coil domain in association with the RING finger motif in several RING finger proteins. Coiled-coil domains are common motifs for protein-protein interactions which together with the RING finger may allow several protein-protein interactions resulting in the formation of large protein complexes. 16 V I . The R B C C group of R I N G finger proteins Members of an important subgroup of RING finger proteins, the RTNG-B box-Coiled-Coil" (RBCC) group (Saurin et al, '96) have an N-terminal RING finger followed by one or two additional zinc-binding domains (B box motifs), followed by a leucine coiled-coil region and a variable C-terminal domain (Reddy & Etkin, '91; Reddy et al, '92; Freemont '93). RBCC proteins include ARD1 (Mishima et al, '93), PML (Mu et al, '91; Koken et al, '95) TIF1 (Le Douarin et al, '95), KAP-1 (Friedman et al, '96) and EFP (Inoue et al, '93) which possess two B-boxes between the RING finger and coiled-coil. BERP together with XNF7 (Reddy & Etkin, '91), RFP (Takahashi et al, '88), SS-A/Ro (Chan et al, '91), Rpt-1 (Patarca et al, '88), Staf 50 (Tissot and Mechti, '95), PwA33 (Bellini et al, '93) and HT2A (Fridell et al, '95) form a subgroup possessing a single B-Box between the RING and coiled-coil domains. The RBCC family appears to play key roles in regulating gene expression and cell proliferation. KAP-1 may be a corepressor of KRAB-domain-containing transcription factors (Kim et al, '96; Friedman et al, '96). The fusion of the PML RBCC domain with retinoic acid receptor alpha underlies acute promyelocytic leukemia (Kakizuki et al, '91; Kastner et al, '92) and PML itself appears to be a growth suppressor (Mu et al, '94; Liu et al, '95; Le et al, '96). The oncogenic protein T18 arises from fusion of the RBCC domain of TIF 1, a putative mediator of ligand dependent transcriptional activation by nuclear receptors, with the B-Raf protein kinase domain (Miki et al, '91; Kastner et al, '92; Le Douarin et al, '95). Fusion of the RBCC domain of the nuclear matrix protein RFP to the GDNF receptor Ret tyrosine kinase domain is also oncogenic (Takahashi et al, '88; Isomura et al, '92). RFP, the human auto-antigen SS-A/Ro and the amphibian protein PwA33 may form part of ribonucleoprotein complexes (Chan et al, '91; Bellini et al, '93). The Xenopus nuclear factor 17 XNF-7 is a maternally expressed cytoplasmic protein which moves to the nucleus during mid-blastula transition, where it may regulate mitosis and dorsal ventral patterning of the embryo (Reddy and Etkin, '91). Rpt-1 may also be a transcription factor, responsible down regulating IL-2 gene and HIV-1 transcription (Patarca et al, '88). The similar protein Staf-50 also represses HIV-1 long terminal repeat expression (Tissot and Mechti, '95). HT2A, cloned from a Hela library, associates with Tat, the HIV-1 virus regulatory protein and may also regulate transcription directed by the long terminal repeat promoter (Fridell et al, '95). However, the cellular molecules that interact with the RBCC domain have not been identified. The candidate ataxia telangiectasia group D complementing gene (ATDC) has a B-Box and coiled-coil, but no RING finger domain (Brzoska et al, '95). A similar RBCC-like domain is also present in members of the TNF receptor associated factor (TRAF) family. In these proteins, the B-box is replaced by a cluster of zinc binding domains which are predicted to form up to five zinc fingers. The RBCC-like domain is present in TRAF2 (Rothe et al, '94), CRAF1 (Cheng et al, '95), and LAP 1 (Mosialos et al, '95). A few RING finger proteins such as c-Cbl (Blake et al, 91) and VAC1 (Burd et al, '97) do not contain a B-box but have a RING finger and a coiled-coil domain. In this thesis I w i l l describe the identification of the novel R B C C protein B E R P . The results of experiments designed to elucidate the function of this protein w i l l also be presented. This information w i l l be discussed in the context of the role of other R I N G finger proteins. 18 Materials and Methods I. Reverse transcription (RT) reactions A procedure similar to the one described in El-Husseini et al, (1994) was used. Briefly, total RNA (0.5 (ig) was incubated with 200 units of MMLV reverse transcriptase (Rtase; BRL) in a buffer containing a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KC1, 3 mM MgCl2, 10 mM DTT, 5% DMSO, 19 units of RNase Inhibitor (Pharmacia), 0.01% BSA, 0.25 (ig of RT primer (AGCTAC AGCTGAGCTGAGCTC AGT 2 0), and 0.5 mM of each deoxy nucleotide triphosphate (dNTP; Pharmacia) in a final volume of 10 ul. The reaction mixture was incubated for 2 h at 37 °C, and then stored at -20°C. II. Polymerase chain reaction ( P C R ) Originally, I was interested in cloning novel members of a family of G-protein coupled receptors, known as cAMP receptors (CARs) (Insall et al, '94; Johnson et al, '93). Based on the published CAR sequences (Insall et al, '94; Johnson et al, '93), degenerate PCR primers were used to amplify a 394 bp PCR product from rat brain RNA. Degenerate PCR primers were as follows: Forward primer 5'TTI GCI TGT/C TGG T/CTI TGG AC 3', Reverse primer 5' AAI ACC CAG/A CAT/A AC/AI AG/AG/A AA 3'. For amplification, 1/20 of the RT reaction was used in a buffer containing 200 uM of each dNTP (Pharmacia), 50 pmol of each primer, 2 units of Taq polymerase, 10 mM Tris (pH 8.4), 1.5 mM Mg Cl 2 > 50 mM KC1 and 0.02% gelatin. The mixture was overlayed with two drops of mineral oil and then incubated in a GTC-1 genetic thermal cycler (Scientific Precision) using the following profile : an initial denaturation step at 94 °C for 7 min, then 2 cycles of 94 °C for 45 sec (denaturation), 40 °C for 45 sec (annealing), and 72 °C for 90 sec (elongation). The two cycle profile was repeated 4 times but each time the 19 annealing temperature was raised by 2 degrees. This was followed by 30 cycles of the following profile: 94 °C for 45 sec, 48 °C for 45 sec, and 72 °C for 90 sec. Samples were finally incubated at 72 °C for 7 min and then stored at 4 °C. The resulting PCR product was subcloned into the TA cloning vector pCR II (Invitrogen). The subcloned PCR product was then sequenced using the dideoxy sequencing method. III. Library screening 1. Cloning of the rat BERP The subcloned PCR product was used to generate a 3 2P labeled probe using the random priming kit (BRL) and used to screen a lambda zap rat brain cDNA library. Library screening was as described by protocols from Stratagene for lambda zap library screening. Lambda zap rat brain cDNA library was obtained from Dr. Terry Snutch. Screening of about 5 x 105 library clones with the 3 2P labeled 394 bp PCR product resulted in twenty positives, one of which (clone 18) contained a large insert of about 2.8 kb in size. Clone 18 was sequenced using the dideoxy termination method (Sanger et al, 1977). Sequence analysis showed that clone 18, contained an open reading frame that generates 744 amino acids. The first ATG (position 221) contained a Kozack consensus sequence (ACCATGG) and it represent the first ATG in the coding sequence. Analysis of the sequence of the cDNA showed that clone 18 also contained a 220 bp GC-rich 5'-untranslated region (5'-UTR) and 400 bp 3'-untranslated region (3'-UTR). A potential poly A signal (ATAAA) was present in the 3'-UTR indicating that this clone contains most of the rat BERP cDNA. 20 2. C lon ing of the human B E R P a. C lon ing of the 3' end of human B E R P c D N A The full length rat brain cDNA was digested with EcoRI into two fragments: one is 1600 bp in size (contains the 5' end) and the other is 1200 (contains the 3' end) in size. Both fragments were 32P-labeled with the random priming kit (BRL) and used to screen a lambda phage human hippocampal cDNA library (Stratagene). After screening about 1X106 phage plaques, one positive was obtained and contained a 2200 bp insert. Sequence analysis of this cDNA insert showed that this clone contained sequences that encode the last 384 amino acids of the coding region of BERP. b. C lon ing of the 5' end of human B E R P c D N A Genbank searches of the human EST database using the rat BERP cDNA as the query sequence identified a 300 bp EST fragment with 95% homology to a 300 bp region of the rat BERP cDNA. This region contained an ATG that corresponds to the first ATG of the rat BERP cDNA. Thus based on the information obtained from the human EST fragment and the obtained sequence of the 3'end of the human BERP cDNA, PCR primers were used to amplify a 1356 bp PCR product. RT-PCR was performed as described earlier except that amplification was carried out with the following profile: an initial denaturation step at 94 °C for 7 min, then 35 cycles of 94 °C for 45 sec (denaturation), 50 °C for 45 sec (annealing), and 72 °C for 90 sec (elongation). Human cerebellum total RNA was used for RT- PCR amplification. The obtained PCR product was subcloned into the TA cloning vector (pCR II; Invitrogen) and sequenced using the dideoxy termination method. The last 300 bp at the 3' end of the human BERP PCR product overlapped 21 with the sequence obtained from the 3'-end clone of human BERP,confirming amplification of BERP cDNA. IV. RNA extraction and Northern blotting Total RNA was extracted using the single step RNA extraction method as described by Chomczynski et al, (1987). Tissues were homogenized in 1 ml of solution A (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 5% sarcosyl, 0.1 M 2-mercaptoethanol) at room temperature. After tissue homogenization, 0.1 ml of 2 M sodium acetate (pH 4.0), 1 ml of phenol (water saturated) and 0.2 ml of chloroform/isoamyl alcohol mixture (49:1) were added to the homogenate, and mixed by vortexing for 10 sec The mixture was cooled on ice for 15 min., then centrifuged at 10,000 rpm for 20 min. at 4 °C. The aqueous phase (upper layer) was transferred to a fresh tube, mixed with 1 ml of isopropanol and placed at -70 °C for 30 min. Samples were centrifuged at 10,000 rpm at 4 °C for 20 min, and the precipitated RNA pellet was resuspended in 0.3 ml of solution A, and RNA was precipitated with 1 volume of isopropanol . RNA pellet was collected by centrifugation at 4 °C for 20 min. RNA pellet was washed with 70% ethanol, vacuum dried and resuspended in water. Northern analysis was carried out as described in Maniatis et al, '82. Total RNA (20 ug) was denatured in 33% formamide and 2.2 M formaldehyde at 65 °C for 15 min. The RNA was then electrophoresed in a 1% (w/v) agarose gel containing 2.2 M formaldehyde, 40 mM MOPS (pH 7.0), 10 mM sodium acetate (pH 5.2), 1 mM EDTA (pH 8.0), and 0.06 ug/ml ethidium bromide (EtBr). The RNA was transferred to Hybond-C nitrocellulose membranes (Amersham). Filters were baked for 1 h at 80°C, and then placed into Seal-o-Meal plastic bags and incubated for 1 hour at 42 °C with prehybridization buffer (40% formamide, 5X Denhardt's solution (1 X 22 Denhardt's= 0.02% each of BSA, Ficoll and polyvinylpyrrolidine), 5X SSPE (IX SSPE=1.15 M NaCl, 0.01 M NaH2P04, 1 mM EDTA), 250 pg/ml denatured salmon sperm DNA and 0.1% sodium dodecyl sulfate (SDS)). DNA probes were labeled with 32P-dCTP and 32P-dATP (3000 Ci/mmol) by the use of a random priming kit (BRL). After prehybridization, DNA probes were boiled for 5 min, cooled on ice for 5 min and then added to the bags containing the RNA blots. Blots were hybridized overnight at 42 °C. Blots were then washed one time in 2X SAC/0.1% SDS (IX SAC= 0.15 M NaCl, 0.015 sodium citrate) at RT for 15 min, followed by 2 washes, 15 min each, in 0.1X SAC/0.1% SDS at 55 °C. Blots were then air dried, and then exposed to X-ray films (XAR5 films, Kodak) at -70 °C. For rat probes: EcoR I digestion of the rat BERP cDNA generated two fragments: a 1600 bp that represented the 5' end and a 1200 bp fragment that contained the 3' end of the rat BERP cDNA. The 1200 bp EcoR I fragment was used to generate 32P-labeled probes (generated by random priming, as described by manufacturer; BRL). For human probes the subcloned 1600 bp PCR product was used to generate 3 2P labeled probes as described earlier. V. In situ Hybridization Procedure for in situ hybridization was carried as described in El-Husseini et al, '95. Briefly, brain sections were obtained from male Sprague-Dawley rats perfused with 4% paraformaldehyde in 0.1 M phosphate buffered saline (pH 7.4). All solutions were treated with diethyl pyrocarbonate (DEPC) and then sterlized by autoclaving. Brains were immersed overnight at 4 °C in 4% paraformaldehye containing 15% sucrose. Before sectioning, tissues were mounted on cryostat stages using Tissue Tek (Fisher) and quickly frozen using dry ice. Coronal and sagittal sections (10 um thick) were mounted on 3-23 aminopropyltriethoxysilane/chrome-alum coated slides and stored at -20 °C. For in situ hybridization analysis, sections were incubated with 1 ug/ml of proteinase K for 10 min, and then treated with 0.25% acetic anhydride/1.5% triethanolamine. Sections were sequentially dehydrated in 50, 70 90 and 95% ethanol. Sections were incubated for 3 h at 54 °C with prehybridization solution containing a final concentration of 50% formamide, 600 mM NaCl, 10 mM Tris (pH 7.5), 0.02% each of Ficoll, bovine serum albumin and polyvinylpyrolidone, 1 mM EDTA, 0.05% salmon sperm DNA and 0.005% yeast transfer RNA. The original 394 bp fragment of the rat BERP obtained by PCR product was subcloned into TA cloning vector (pCR II; Invitogen). The subcloned PCR product was used to generate riboprobes as described in El-Husseini et al, '95. Antisense riboprobes were generated using the T7 RNA polymerase after linearization of plasmids with BamH I. Sense probes (control probes) were generated using the sp6 RNA polymerase after linearization of plasmids with Not I. Probes (5X105 cpm per section) were added to sections in a hybridization solution containing a final concentration of 600 mM NaCl, 10 mM Tris (pH7.5), 0.02% each of Ficoll, BSA and polyvinylpyrolidone, 1 mM EDTA, 0.01% salmon sperm DNA, 1 mM EDTA, 10% dextran, 0.1% SDS, 0.1% sodium thiosulfate, 100 mM dithiothreitol (DTT) and 0.005% yeast transfer RNA. After 16 h, sections were washed with 2X saline sodium citrate (SSC), treated with RNase (20 pg/ml) for 30 min, then washed once with 2X SSC, at 55 °C and then with 0.1% SSC at 60 °C for 2 h. Slides were exposed to X-Omat x-ray films (Kodak) for 2-3 weeks. 24 VI. Fluorescence in situ hybridization FISH analysis The 1356 bp PCR product of the 5' end region of the human BERP cDNA was used to screen a human P-l derived Artificial Chromosome (PAC) library in the Canadian Genome Analysis and Technology Program physical mapping resource facility at Hospital for sick children; Toronto. One genomic PAC clone was obtained. Southern blot analysis using the 1356 bp PCR product as a probe confirmed that the obtained PAC clone contains sequences of the human BERP gene. Mapping of the genomic PAC clone was performed by fluorescence in situ hybridization (FISH) to normal human lymphocyte chromosomes counterstained with propidium iodide and DAPI. Biotinylated probe was detected with avidin-fluorescein isothionate (FITC). The human BERP PAC clone was then used to generate labeled probes and used for FISH analysis. Images of metaphase preparations were captured by a thermoelectrically cooled charge coupled camera. The presence of the gene in the genomic clone was confirmed by Southern blotting of the PAC clone using the 5' end region of the human BERP cDNA (1356 bp PCR product) as a probe. VII. Yeast two hybrid analysis The yeast two hybrid system is a sensitive in vivo technique for identifying proteins that interact with a protein of interest (Fields and Song '89, Chien et al., '91; Guarente '93, Luban and Goff '95). This screening procedure uses growth selection based on the reconstitution of a functional GAL4 transcriptional activator of HIS3, a nutritional reporter gene controlled by a GAL4 response element. One vector encoding GAL4 DNA binding domain (BD) fused to protein X and another vector consisting of the GAL4 activation domain (AD) fused to protein Y are co-transformed into yeast. Interaction between proteins X and Y permits transcriptional 25 activation of two integrated copies of two genes; the reporter gene, LacZ, allowed detection of the clones expressing interacting proteins while the HIS3 gene allowed yeast to grow on media lacking histidine (Guarente '93 Fields and Sternglanz '94, Luban and Goff'95). We used the yeast two hybrid system to screen a rat brain cDNA library subcloned into pGADIO vector that contains GAL4 AD (Clontech; see Guarente '93; Fields and Sternglanz '94; Luban and Goff '95; Rothe et al.; '94; Song et al.; '94 and '95). All the materials required for this procedure including yeast strains, cloning vectors, and a rat brain cDNA library were obtained from Clontech (MATCHMAKER system). This kit has been used successfully in various studies utilizing the yeast two hybrid system (Rothe et al, '94, Song et al, '94, Boldin et al, '95). To identify proteins that may interact with the different domains of BERP, various constructs that contain different regions of the BERP protein were generated either by PCR or by digesting the original cDNA with restriction enzymes (see Table 1). Yeast transformation to screen for proteins interacting with various BERP domains were carried out as described in the Clontech MATCHMAKER procedure. Vector pVA3-l encoding GAL4 DNA BD/murine p53 protein in pAS2-l as well as vector pCLl encoding the full length wild-type GAL4 protein was used as positive controls in the screening procedure. Procedures for yeast transformation, growth, his3 selection, and [5-gal filter lift assay are described in Clontech MATCHMAKER protocol; Rothe et al, '94, Pandey et al,'95; Song and Dormer '95; Mosialos etal, '95; Song etal, '94). 1. Construct preparation, l ibrary amplification, and yeast strain phenotype verification Inserts for yeast two-hybrid baits were either generated by PCR or excised from the original BERP cDNA. To generate baits, inserts that represent different regions of BERP were 26 subcloned into pAS2-l vector in frame with the GAL4-DNA binding domain. Insertion of the correct products was confirmed by sequencing. For protein-protein interaction assays, target' sequences were subcloned into the pACT2 vector inframe with the GAL4 activation domain. Baits used for library screening and protein-protein interaction assays are summarized in Table 1. The rat brain cDNA library used for screening was obtained from Clontech. cDNA inserts were subcloned into the EcoR I site in pGADIO vector that contains the GAL4 activation domain. The titer of the plasmid library was determined as described in the Clontech protocols for yeast two-hybrid plasmid library titering. The library was amplified as recommended by the manufacturer (Clontech) and plasmid DNA was purified using large MAXI prep columns (Qiagen). About 10 mg of plasmid DNA was obtained and the same stock DNA was used for all the screening performed with different BERP baits. For yeast two-hybrid assays, the yeast strain Y190 (Clontech) was used. Yeast cells were streaked on YPD plates and incubated at 30 °C for 3 days. Colonies grew up to 1-2 mm in size and appear slightly pink or red in color. As expected, yeast cells (Y190 strain) failed to grow when streaked on plates lacking Leu, or Trp). 2. Yeast transformation and l ibrary screening a. Competent cell preparation: The lithium acetate (LiAc) method for preparing competent yeast cells is described in the Clontech protocols. Small scale transformation was used for studying interactions of two known proteins and for verification of positives obtained from yeast two-hybrid library screening. The large scale transformation method was used for library screening. Small scale and large scale 27 transformation methods are described in detail in Clontech protocols provided for the yeast two-hybrid MATCHMAKER kit. b. Smal l scale transformations: For small and large scale transformations yeast Y190 strain cells were freshly streaked, and 10-15 colonies were inoculated into 1 ml of YPD. Cells were vortexed and transferred to a flask containing 50 mis of YPD. Cells were incubated for 16-18 h at 30 °C with shaking at 250 rpm. Cells were then transferred to a fresh 300 mis of YPD to produce an OD600=0.2-0.3. Cells were incubated for 3 h at 30 °C with shaking at 230 rpm. Cells were then centrifuged at lOOOxg for 5 min, resuspended in 50 mis of sterile water and then centrifuged again at lOOOxg for 5 min. Cells were then resuspended in freshly prepared IX TE/LiAc and used immediately for transformations. For small scale transformations, 0.1 \ig of plasmids containing DNA binding domain were mixed in a 15 ml tube with 0.2 u,g of plasmids containing activation domain, 0.1 mg of denatured salmon sperm DNA, 100 ul of competent cells and 0.6 ml of PEG/LiAc solution. The mixture components were mixed by vortexing, incubated at 30°C for 30 min with shaking at 230 rpm. Seventy ul of DMSO were added, and mixed by inversion. Cells were heat shocked at 42°C for 15 min, cooled on ice and then centrifuged at lOOOxg for 5 min. Cells were resuspended in 0.5 ml of IX TE and 100 ul were spread on the appropriate plates. c. Large scale transformations Large scale transformations were only used for library screening. For large scale transformation, 10 times the amounts described for small scale transformations were used. Small 28 scale transformation method was used first to introduce the plasmid containing insert\DNA binding domain (bait). The obtained transformants were then used for generation of competent cells and for introduction of the library plasmids. Fifty u.g of library DNA was used for each transformation. After transformation, cells were resuspended in 10 ml of 1 X TE and 300 ul aliquots were plated on 40 large-plates (150 mm) lacking leucine /tryptophan/histidine (-leu/-trp/-his) + 25 mM 3-amino triazol (3-AT). Plates were incubated for 7-9 days and then assayed for activation of the reporter gene, LacZ, using the P-galactosidase (P gal) filter lift assay. d. P-galactosidase filter lift assay The protocol used for p-galactosidase filter assay is described in the Clontech yeast two-hybrid MATCHMAKER manual. Briefly, Whatman filters (Whatman#5; VWR) were placed over the surface of the agar plates containing yeast transformants. Filters were lifted and transfered to a pool of liquid nitrogen, thawed at room temperature and placed on the top of a presoaked filter containing Z buffer/X-gal solution. Filters were then incubated at RT for 2-6 h, and checked for the appearance of blue colonies. e. Yeast two hybr id analysis of true and false positives. The procedure described in the Clontech MATCHMAKER protocols was used. Positive colonies were restreaked on plates ( leu/-trp/-his) + 25 mM 3-AT. Colonies were then grown 16-18 h in 5 ml liquid media (-leu/-trp/-his) + 25 mM 3-AT. Plasmids were purified as described in the Clontech protocols. Plasmids were then introduced into E.coli and extracted using the miniprep RPM KIT. Purified plasmids were then introduced to yeast cells either containing the bait or control plasmids, using the small scale yeast transformation protocol. Transformed yeasts 29 were then plated on (-leu/-trp/-his) + 25 mM 3-AT plates. After 3-7 days of growth at 30 °C, plates were assayed with the filter lift method. True positives are those that interacted specifically with the bait but not with control plasmids either containing the GAL4 DNA binding domain alone or fused to non-specific proteins such as the one supplied with the MATCHMAKER kit (described in Table 1). Plasmids from positive clones expressing proteins that interact with BERP were then identified by sequencing using the dideoxy sequencing method V I I I . C e l l transfection 1. Transient Transfection of human embryonic kidney cells ( H E K ) 293 HEK-293 (HEK) cells were obtained from American Type Culture Collection (CRL 1573). HEK cells were maintained in an incubator containing 5% C0 2 at 37°C in minimum essential medium (MEM) containing Earle's salts and supplemented with penicillin/streptomycin (100 U/ml) and 10% fetal bovine serum (FBS). Cells were passaged every three to four days. For passaging cells, the medium was aspirated, and cells were washed with pre-warmed (37°C) phosphate-buffered saline (PBS), and incubated with one ml of trypsin (0.25% in PBS) at 37°C for 2 min. Five ml of fresh medium were added, and cells were agitated until they completely detached from the plate. Cells were then transferred into dishes to obtain a density of about 4xl05 per plate. a. Transfection procedure: Calcium phosphate method was used for transient transfection of HEK cells as well as PC 12 cells as described in Chen and Okayama, (1987). Cells were plated 12-24 h prior to 30 transfections. All of the different cDNA constructs used for transfection were subcloned into the mammalian expression vector pcDNA3 (Invitrogen). A total of 10-15 pg of plasmid DNA were used for transfection of 10 cm plates of cells. In co-transfection experiments, equal amounts of plasmids were used. For transfection, 10-15 pg of plasmid DNA is precipitated with 0.3 M sodium acetate and 2.5 volumes of 100% ethanol. The DNA pellet was resuspended in 450 pi of 0.1 x TE (0.045 M Tris (pH 7.5), 0.001M EDTA) buffer. Fifty pi of 2.5 M calcium chloride was added. Samples were mixed thoroughly, and 500 pi of 2 x BES (50 mM BES, 280 mM NaCl, 1.5 mM Na2HP04, pH 6.96) were added. The contents were mixed and incubated at room temperature for 20 min. DNA samples were added to cultured cells preincubated for 30 min prior to DNA sample preparation at 37°C in a 3% C0 2 incubator. Cultured cells were incubated 12-14 h at 37°C in 3% C0 2 . Cells were washed twice with pre-warmed PBS. Fresh media was then added and cells were incubated for another 24 h before harvesting. Transfected cells were either fixed with a solution containing 4% paraformaldehye (PFA) for immunocytochemical staining or lysed with cell lysis buffer for immunoprecipitation experiments and Western blot analysis. 2. Transient and stable transfection of rat pheochromocytoma (PC12) cells PC 12 cells were cultured on poly-D-lysine coated 10 cm plates in Dulbecco's minimum essential medium (DMEM) supplemented with 10% horse serum and 5% fetal calf serum. Cells were incubated at 37 °C in 5% C02, and 95% air. One day prior to transfection cells were plated at about 30-50% confleuncy. Calcium phosphate method was used for transfection of PC 12 cells, as described for HEK cell transfections. In case of transient transfection, cells were fixed with a solution containing 4% paraformaldehye (PFA) for immunocytochemical analysis. 31 The pcDNA3 vector (Invitrogen) used for mammalian transfection experiments also contains the neomyocin resistance gene which allows for the selection of stably transfected cDNAs. Geneticin, also known as G-418, can be used for selection of neomyocin resistant clones. For stable transfection experiments, three days post-transfection, media was changed and fresh media containing 500 u,g/ml G-418 (BRL) was added for selection of stably transfected cells. All media changes were carried every 3-4 days and contained 500 u,g/ml G-418. Two weeks later, the remaining individual clones were collected and plated separately and maintained in media containing 200 \xg/ml G-418. When cells reached -80% confluency, cells were divided into several plates and used to either generate freezer stocks of these clones, or for immunocytochemistry and Western blot analysis to confirm expression of the correct protein product. Transfected cells were either fixed with a solution containing 4% paraformaldehye (PFA) for immunocytochemical analysis or lysed with cell lysis buffer containing 1% NP-40 for Western blot analysis. I X . Immunocytochemistry analysis of transfected cells Forty eight h post transfection, cultured cells were washed once with PBS, and fixed for 1 h with a solution containing 4% paraformaldehyde and 0.1M potassium phosphate (pH 7.3). Cells were rinsed with PBS and incubated overnight at 4 °C with primary antibodies in PBS solution containing 0.3% Triton-X and 2% normal goat serum (NGS). Cells were washed 3 times, 10 min each, with PBS-T (PBS + 0.3% Triton X) at room temperature. Cells were incubated with secondary antibodies (biotinylated antibodies in case of DAB/ABC staining, and Texas red conjugated antibodies for fluorescent staining) for 1 h. Cells were washed 3 times, 10 32 min each in PBS-T. For fluorescent staining, antifade solution was added (Molecular Probes) and plated cells were stored in the dark until analysis. For immunoperoxidase staining, the A B C method was used using Vectastain A B C kit (Vector Laboratories, Inc., Burlingame, CA). Cells were incubated for one hour at RT in PBS-T solution containing avidin-biotinylated horseradish peroxidase complex. Following a final series of rinses in PBS, the immunoreactivity was revealed using a nickel-enhanced D A B reaction, as previously described (Robertson et al, '90, '92). After staining cells were preserved in glycerol and examined and photographed under phase and brightfield optics. X . Immunohistochemistry. Similar procedure to the one described in Vincent et al, '94 as well as the one described under D A B staining of cultured cells was used. Briefly, brain sections were obtained from male Sprague-Dawley rats perfused with 4% paraformaldehyde in 0.1 M phosphate buffered saline (pH 7.4). Brains were rinsed in sucrose solution, cut into 30 pm thick sections, washed in 0.02 M phosphate-buffered saline (PBS), and then processed for BERP immunoreactivity using standard methods (Vincent et al., '94; Robertson et al, '90, '92). Affinity purified BERP antibodies were diluted 1:200 in PBS containing 0.3% Triton X-100 (PBS-T), 2% normal goat serum (NGS) for 24 h at 4°C. The sections were then rinsed 3 X 20 min in PBS-T, and then incubated with biotinylated rabbit anti-sheep secondary antibody (Vector Laboratories) diluted 1:1000 in PBS-T contatining 2% NGS, for 1 hour at room temperature. The sections were again washed 3 X 20 min in PBS-T, and incubated for 1 hour with an avidin-biotinylated horseradish peroxidase complex (ABC Elite, Vector Laboratories, Burlingame, CA) in PBS-T. Following a 33 final series of rinses in PBS-T, the immunoreactivity was revealed using diaminobenzidine (DAB) plus nickel ammonium sulfate as chromogen. XI. Western blotting analysis and Immunoprecipitations 1. In vitro translation The TnT-T7 coupled reticulocyte lysate system (Promega) was used for in vitro transcription/ translation of constructs subcloned into the mammalian expression vector pcDNA3. This procedure was used to detect the in vitro translated products of BERP, actinin and myr 6. In each in vitro translation reaction 1 u,g of plasmid DNA was used in the presence of T7 RNA polymerase and 35S-labeled methionine for labeling in vitro translated products. Procedure for coupled in vitro transcription-translation was as described by the manufacturer (Promega). In vitro translated products were run on 8% polyacrylamide gels, dried and exposed to X-ray film to visualize bands. 2. Generation of antibodies against BERP A peptide that contains the last 9 amino acids of BERP was synthesized (UBC facilities) and conjugated to the carrier protein keyhole limpet hemocyanin (KLH). Prior to immunization, blood samples (30 mis) were collected from the assigned rabbits and serum (pre-immune serum) was separated and stored at -20°C. Three rabbits were immunized by subcutaneous injections of the KLH-conjugated peptide in complete Freund's adiuvent. Five injections (boosts) of the conjugated peptide were done once a month in a solution containing incomplete Freund's adjuvent. Blood samples were collected 10 days after each immunization (boost). Blood samples were left at room temperature for 2 h to allow blood clotting and serum was collected by 34 centrifugation of blood samples at 5000 rpm for 30 min. After the fifth boost, rabbits were sacrificed and the serum was collected as described earlier and stored at -20°C. 3. Western blotting Polyacrylamide gels (8.5%) were prepared as described in the Bio Rad manual. Proteins were transferred to nitrocellulose membranes (Amersham) using the wet transfer method and the Bio Rad transfer system. After completion, membranes were washed twice with TBS (25 mM Tris, 0.8% NaCl, 0.02% KC1, pH 7.6). Membranes were then incubated for 2 h at room temperature in blocking solution (TBS containing 0.05% TWEEN-20 and 5% milk). Membranes were then rinsed 3 times in TBST (TBS containing 0.05% TWEEN 20), and incubated with protein specific primary antibodies over night at 4°C in TBST containing 1% bovine serum albumin (BSA). Membranes were then rinsed 3 times in TBS, followed by 6 times washes, 10 min. each in TBST solution. Membranes were incubated with species specific horse-radish peroxidase-linked secondary antibodies (1/5000 dilution; Amersham) for 1 h at RT in TBST containing 0.05% BSA. Membranes were washed 6 times, 10 min. in TBST at RT. Membranes were then incubated for 1 min with ECL reagents (Amersham) and exposed to X-ray films (Kodak) to visualize protein bands. 4. Immunoprecipitat ion (IP) Cells were lysed in 500 pi lysis buffer (50 mM Tris, 250 mM NaCl, ImM EDTA, 10 pg/ml aprotinin, 1 pg/ml leupeptin, lpg/ml pepstatin and 10 mM PMSF containing 0.1% NP-40 unless indicated otherwise) for 20 min at 4 °C with agitation. Cell lysates were collected and 35 centrifuged at 10,000 rpm for 5 min. Supernatant was transfered to a fresh tube and samples were either used for IP or frozen immediately in 100 ul aliquots at -70 °C. a. Preparat ion of Protein A-Sepharose beads ( C L 4 B , Pharmacia) for IP Two ml of beads slurry were suspended in 10 ml of 50 mM HEPES (pH 7.6) by rotating 15 min. at 4 °C. Beads were collected by centrifugation at 10,000 rpm for 2 min. Beads were resuspended in 50 mM HEPES by rotation for 15 min at 4°C. Two mg/ml of crystallized BSA were added, and rotation was continued at 4°C for another 30 min. Beads were collected by centrifugation at 1000 rpm for 2 min., washed once for 10 min. at 4°C with 50 mM HEPES and then twice with lysis buffer. Beads were then resuspended in 1 volume of lysis buffer and stored at 4°C until usage. b. Immunoprecipitat ion procedure For immunoprecipitation experiments 200 ul of cell lysate were precleared for 15 min. at 4°C by incubation with 20 ul of protein A-Sepharose slurry. Samples were centrifuged at 10,000 rpm for 2 min. and supernatant was transfered to a fresh tube. Cell lysates were then incubated with antibodies (6 \ig in case of monoclonal antibodies or 10 ul of crude or affinity purified polyclonal antibodies for 1-2 h on ice. Eighty ul of beads slurry were added to each tube and incubation was continued for 1 more h at 4 °C with rotation. Beads were then collected by centrifugation at 1000 rpm for 2 min at 4 °C, washed twice in 1 ml of detergent containing lysis buffer, 10 min each with rotation at 4 °C, then twice , 10 min. each in a detergent free lysis buffer. Beads were collected between washes by centrifugation at 1000 rpm for 2 min at 4 °C. 36 After the last wash beads were collected by centrifugation (1000 rpm, 4 °C), boiled in 40 pi of SDS-sample buffer to extract immunoprecipitated proteins. X I I . Constructs generated for transfection of mammalian cells. The mammalian expression vector pcDNA3 was used for subcloning of all of the constructs used for expression in mammalian cells. 1. F u l l length B E R P ( F B E R P ) Full length BERP cDNA was subcloned into the XhoI/BamHI sites of pcDNA3. To insert the FLAG sequence at the N-terminal end of BERP, the original BERP cDNA (in bluescript) was digested with BstE II/ PpuM I. PCR primers were designed to amplify sequences that start at the BstE II site (before the first ATG) and include the PpuM I site in the reverse primer (831 bp PCR product). Forward primer contained BstEII site followed by a FLAG sequence and 18 bp of the BERP sequence that contain the first ATG; 5' AACTGGTCACCGCCACCATGGACTACAAGGACGACGATGACAAGATGGCAAAGAGG GAGGAC 3'. Reverse primer: 5' AGCCCAGTCTCAGTGCCT 3'. PCR product was subcloned into the BstE II/PpuM I site of the digested BERP cDNA. The BERP cDNA containing the FLAG sequence, was then subcloned into XhoI/BamHI sites of pcDNA3 2. N- terminal region of B E R P ( N B E R P ) FLAG-tagged full length BERP cDNA/pcDNA3 was digested with EcoRI and then religated. The generated construct coded for N-terminus FLAG followed by the first 485 amino acids of BERP. 37 3. C- terminal region of B E R P ( C B E R P ) A PCR product was generated using an upper primer that contains an Ncol site followed by a FLAG sequence and sequences of BERP that encodes amino acids 285-291 and a lower primer to amplify sequences past the Ndel site. Forward primer: 5' TGGTCACC ATGGACTACAAGGACGACGATGACAAGGCTCTGGCAGCTC AGGCC 3'. Reverse primer 5' GCTGTACATGGAGCTCGG 3' The generated PCR product was subcloned by TA cloning into the TA cloning vector (pCR II; Invitrogen), and then digested with Ncol/ Nde I enzymes. The full length BERP cDNA in bluescript was digested with Ncol/ Ndel enzymes and then ligated to the digested PCR product. The generated construct was subcloned into the XhoI/BamHI sites of pcDNA3. Thus the generated construct contained a FLAG sequence followed by sequences of BERP that encoded amino acids 285-744. 38 4. Actinin Full length actinin cDNA (-3.2 kb) was first digested with Hindlll, and converted into Xbal site by filling with Klenow fragment. The insert was then digested with Xhol and subcloned into Xbal/ Xhol sites of pcDNA3 vector. 5. His-myr 6 A histidine (HIS) tagged-myr 6 C-terminal region was amplified by PCR using the C-terminal region of myr 6 obtained from the yeast two-hybrid screening as a template. The following primers were used to generate a 1889 bp PCR product: Forward primer: 5' ACCATGCATCACCATCACCATCACGACCAAGCCATGCAGGAT 3'. Reverse primer 5' GAAACAACGCATCCTCA 3'. 39 Name Region forward pr imer reverse pr imer A B P L 280-459 a.a. 5' C C A T G G C T C T G G C A G C T C A G G C C T 3' 5' G C T G T A C A T G G A G C T C G G 3' C B E R P 2 459-744a.a. 5' C C A T G G G T G G C A A A C G G A A A G A C A 3 5' G G C C C T T G T G C A G C T A C T 3' C B E R P 3 620-744 a.a. 5' C C A T G G G G C C A A C T G A C C G C C A C T 3' 5' G G C C C T T G T G C A G C T A C T 3' R B C C 1 1-372 a.a. 5' G C T G G T G T T A G T G G C A G G 3' 5' G C C A T C G G G G C C A G T G A T 3' R B C C 2 1-262 a.a. 5' G C T G G T G T T A G T G G C A G G 3' 5' G C C C A G T C T C A G T G C C T G 3' CBERP1 383-744 a.a. _ _ Table 1. Constructs used for the yeast two-hybrid assay. Constructs RBCC1, RBCC2, CBERP3 and ABPL were subcloned into the Nco II EcoR I sites of both pAS2-l and pACT2 vectors. CBERP2 was subcloned into the Nco II BamHI sites of both pAS2-l andpACT2. CBERP 1 was subcloned into pAS2-l vector by digesting the original BERP cDNA/bluescript with Kpn I. BERP cDNA was digested with kpn I, blunt ended with T4 DNA polymerase. pAS2-l vector was digested with Ncol and then blunt ended with T4 DNA polymerase. The BERP cDNA (Kpn I fragment) was then inserted into the blunt ended pAS2-l vector. 40 Plasmid description pAS2-l contains GAL4 DNA binding domain pACT2 contains GAL4 activation domain pVA3-l contains GAL4 DNA binding domain/ p53 pLAM5 contains GAL4 DNA binding domain/ laminC pTDl contains GAL4 activation domain/ SV40 large T antigen Table 2. Con t ro l plasmids (supplied wi th yeast two hybr id ki t ; M A T C H M A K E R ) 41 Results I. C lon ing of a novel R I N G finger protein: 1. Molecu la r cloning of rat B E R P . Degenerate PCR primers were designed to identify mammalian homologues of Dictyostelium G protein-coupled receptors that are activated by extracellular cAMP (Johnson et al, '93; Insall et al, '94). A PCR product (394 bp) of the predicted size was obtained from rat cerebellum mRNA. Sequence analysis showed that this PCR product had an open reading frame, with 30% homology to the Dictyostelium cAMP receptors. The PCR product was then used as a probe to screen a rat brain cDNA library (obtained from Dr.Terry P. Snutch). One of the obtained positives contained a 2870 bp cDNA insert. Figure 1 shows the nucleotide sequence of this cDNA. Genbank searches showed that the sequence of this clone represented a novel gene. The first ATG (nucleotides 221-223; underlined in Figure 1) in the sequence was contained in a consensus sequence (ACCATGG) known as the Kozak consensus sequence for translation initiation (Kozak, '89 & '91). Translation of the cDNA (using DNA program) predicted a single open reading frame that starts at the first ATG (nucleotides 221-223; Fig. 1) and ends with a stop codon (TAG; nucleotides 2453-2456; underlined in Fig. 1). Also, a putative poly-A signal sequence (ATAAA) was present at position 2842-2846, indicating that a full length 3' untranslated region (3' UTR) was present in the sequence. Thus, this cDNA clone most likely represented the full length cDNA. The novel cDNA contained a 220 bp sequence of 5'-untranslated region (5' UTR, nucleotides 1-220), a 2232 bp coding region (nucleotides 221-2452), and a 414 bp 3' UTR sequence and its open reading frame encoded a predicted protein of 744 amino acids with a predicted size of -82 kDA (Figure 2). 42 The full length protein did not contain any significant homology to the known cAMP receptors, and lacked transmembrane domains predicted for the G protein-coupled receptors (Johnson et al, '93; Insall et al, '94). Rather, the cDNA represented a novel protein that contain a unique zinc finger binding motif known as the RING finger that is present in family of proteins known as the "RING finger" family. Several members of the RING finger family have been shown to be involved in various signal transduction pathways from cell surface receptors to regulation of cell growth and differentiation. Because the cloned cDNA was obtained from rat brain, and because it represents a novel member of the RING finger family, the novel protein encoded by this cDNA was named "Brain Expressed Ring finger Protein" (BERP). Because of the varied and interesting roles documented for members of the R I N G finger family in the regulation of several signaling pathways that involve cell cycle, growth, proliferation, apoptosis and differentiation we sought to characterize and determine the possible role(s) of this novel protein i n the nervous system. 43 CGCGGCGGCGGCGCCGGCGCGGGGACAGACGGAGGCGCGGGACGTGGGGCGGCGCCGCGGGCAGGGCCGG 70 G C G G A C G A G C G C T G G C A G C G G G G G C A G C G C T G C G G G C C G C T C C G A G T G G C T G C A G T C C G G G C C T T G C A C T 140 C C C A T C C C G C T C C C A C G C C G T G A G T G C G C G G C G C G G G C C C C A A G G G C T G G T G T T A G T G G C A G G A A C C A T C 2 1 0 T G T G G T C A C C S U G C A A A G A G G G A G G A C A G C C C T G G C C C G G A G G T G C A G C C A A T G G A C A A G C A G T T T C T G 2 8 0 G T A T G C A G T A T C T G C C T G G A T C G G T A C C G G T G C C C C A A A G T T C T G C C T T G T C T A C A C A C C T T C T G C G A A A 350 G A T G T C T C C A G A A C T A C A T C C C T C C T C A G A G C C T G A C A C T C T C G T G T C C A G T A T G C C G G C A G A C A T C C A T 420 C C T C C C A G A A C A G G G T G T C T C A G C C C T A C A A A A C A A C T T C T T C A T C A G C A G C C T C A T G G A G G C C A T G C A G 4 9 0 C A G G C A C C T G A T G G G G C C C A C G A C C C T G A A G A C C C C C A C C C C C T C A G C G C A G T G G C T G G C C G C C C T C T C T 5 6 0 C C T G C C C C A A C C A T G A A G G C A A G A C G A T G G A G T T T T A C T G T G A G G C C T G T G A G A C T G C C A T G T G T G G T G A 630 G T G C C G C G C A G G G G A G C A C C G T G A A C A C G G C A C A G T G C T G T T G C G G G A C G T G G T G G A G C A G C A C A A G G C A 7 0 0 G C T C T A C A G C G C C A G C T T G A A G C T G T G C G T G G C C G A T T G C C A C A G C T G T C T G C A G C T A T C G C C T T G G T T G 7 7 0 G G G ' G C A T C A G C C A G C A G C T G C A A G A G C G C A A G G C A G A G G C C C T G G C C C A G A T A A G T G C A G C C T T T G A G G A 840~ C C T G G A G C A A G C T C T G C A G C A G C G C A A G C A G G C T C T G G T C A G C G A C T T G G A G A G C A T T T G T G G G G C C A A G - 910 C A G A A G G T G T T G C A G A C A C A G T T A G A C A C A C T G C G C C A G G G T C A G G A A C A C A T T G G C A G T A G C T G C A G C T 980 T T G C A G A G C A G G C A C T G A G A C T G G G C T C T G C C C C T G A G G T T T T G C T A G T A A G A A A G C A C A T G C G A G A G A G 1, 050 G C T G G C T G C T C T G G C A G C T C A G G C C T T C C C A G A A A G G C C C C A T G A G A A T G C A C A G C T G G A A C T G G T C C T T 1, 120 G A A G T A G A T G G G T T G C G G A G A T C G G T G C T C A A T C T G G G T G C A C T G C T C A C C A C C A G T G C T G C T G C A C A C G 1, 190 A G A C A G T G G C C A C C G G C G A G G G C C T G C G C C A G G C A C T G G T T G G C C A G C C T G C C T C A C T C A C T G T C A C C A C 1, 260 C A A A G A C A A A G A T G G G C G G C T G G T G C G C A C G G G C A G C G C A G A G C T G T G T G C A G A G A T C A C T G G C C C C G A T 1, 3 3 0 G G C A T G C G C C T T G C G G T A C C G G T G G T G G A C C A C A A A A A T G G C A C A T A T G A G C T G G T G T A C A C A G C A C G T A 1, 400 C A G A A G G C G A C C T G C T C C T C T C A G T G C T G C T C T A T G G A C A G C C G G T G C G T G G C A G C C C T T T C C G T G T G C G 1, 470 T G C C C T G A G A C C C G G A G A C T T G C C A C C T T C C C C A G A T G A T G T G A A G C G C C G G G T C A A G T C T C C C G G T G G T 1, 5 4 0 C C T G G C A G T C A T G T G C G C C A G A A G G C A G T G C G T A G G C C G A G C T C C A T G T A C A G C A C C G G T G G C A A A C G G A 1, 610 A A G A C A A T C C A A T T G T G G A T G A A C T C G T C T T T C G T G T T G G C A G T C G T G G A A G G G A G A A A G G T G A A T T C A C 1, 680 C A A T T T A C A C C C G C T G T C C G C T G C T A G C A G T G G C C G A A T A G T G G T A G C A G A C A G C A A C A A C C A A T G T A T T 1, 7 5 0 C A G G T T T T C T C C A A T G A G G G C C A G T T C A A G T T C C G A T T T G G A G T C C G T G G G C G C T C G C C T G G G C A A C T A C 1, 8 2 0 A G C G T C C T A C G G G T G T G G C A G T G G A C A C C A A T G G A G A C A T T A T T G T G G C A G A C T A T G A C A A C C G T T G G G T 1, 890 C A G C A T C T T C T C T C C T G A G G G C A A G T T C A A G A C C A A A A T T G G A G C T G G C C G C C T C A T G G G G C C C A A G G G A 1, 960 G T G G C T G T G G A C C G G A A T G G G C A T A T C A T T G T G G T G G A T A A C A A G T C T T G C T G T G T C T T C A C C T T C C A G C 2 , 0 3 0 C C A A T G G C A A G C T T G T T G G G C G T T T T G G A G G C C G T G G G G C C A C T G A C C G C C A C T T C G C A G G G C C C C A C T T 2 , 100 T G T G G C T G T G A A C A A C A A G A A T G A G A T T G T A G T A A C G G A T T T C C A C A A C C A T T C A G T G A A G G T G T A C A G T 2 , 170 G C T G A T G G A G A G T T C C T C T T C A A A T T T G G C T C A C A T G G C G A G G G C A A T G G A C A G T T C A A T G C C C C C A C G G 2 , 2 4 0 G A G T A G C T G T G G A T T C C A A T G G A A A C A T C A T C G T G G C T G A C T G G G G C A A T A G T C G C A T A C A G G T A T T C G A 2 , 3 1 0 C A G C T C T G G T T C C T T C C T G T C C T A T A T C A A C A C G T C T G C A G A G C C A C T G T A T G G C C C A C A G G G C T T G G C A 2, 3 8 0 T T G A C C T C G G A T G G C C A C G T A G T A G T G G C T G A T G C T G G C A A C C A C T G C T T T A A G G C C T A T C G A T A C C T C C 2, 450 A G l l C T G C A C A A G G G C C C T G C T T G G C T C A T G G A G G G A C G G A C A T A G G G G T G A T T G G A C A A G A G G G T C T G 2, 5 2 0 G C T A A G A G G T G G G C C A G A C C C G G C A G C A C T G A A T G T G G G C G T G G G C A T G G A T G T G C C C A A T G C C C T T C C C 2, 5 9 0 T T C C T C C C C A A C C C C A C G G T T G C A C T T T A T T T A T T C G G T T C T T G C T T T G G T G A C T G G G T G G G C C T G G A C T 2 , 660 G G T C C C A A G G A T G T G T G C A G A G C T T C A C C C T A A C T C C T T T A C A C A C C C T C C C T T A C A C C C T C A G T C T G C T 2 , 7 3 0 C T C T G A C C C C C A G C C T G G G C C A G A A C A G C C T C T T A C T T C A G A A G T C C C T C T G G T T G G C T C C C T A A C A C C C 2 , 8 0 0 C A T A C A C A C T G A C A G A G A C A G C A A T A C C C C A C C C C A T A T T A A A T A A A T G T G T T C G C C A A G G T G G G A A T T C 2 , 8 7 0 Figure 1. Nucleotide sequence of the rat BERP. The A T G codon representing the putative translation start site as well as the inframe stop codn (TAG) are underlined. Putative poly-A signal (ATAAA) is double undelined. 44 MAKREDSPGP EVQPMDKQFL VCSICLDRYR CPKVLPCLHT FCERCLQNYI PPQSLTLSCP 60 VCRQTSILPE QGVSALQNNF FISSLMEAMQ QAPDGAHDPE DPHPLSAVAG RPLSCPNHEG 120 KTMEFYCEAC ETAMCGECRA GEHREHGTVL LRDVVEQHKA ALQRQLEAVR GRLPQLSAAI 180 ALVGGISQQL QERKAEALAQ ISAAFEDLEQ ALQQRKQALV SDLESICGAK QKVLQTQLDT 240 LRQGQEHIGS SCSFAEQALR LGSAPEVLLV RKHMRERLAA LAAQAFPERP HENAQLELVL 300 EVDGLRRSVL NLGALLTTSA AAHETVATGE GLRQALVGQP ASLTVTTKDK DGRLVRTGSA 360 ELCAEITGPD GMRLAVPVVD HKNGTYELVY TARTEGDLLL SVLLYGQPVR GSPFRVRALR 420 PGDLPPSPDD VKRRVKSPGG PGSHVRQKAV RRPSSMYSTG GKRKDNPIVD ELVFRVGSRG 480 REKGEFTNLH PLSAASSGRI VVADSNNQCI QVFSNQGQFK FRFGVRGRSP GQLQRPTGVA 540 VDTNGDIIVA DYDNRWVSIF SPEGKFKTKI GAGRLMGPKG VAVDRNGHII VVDNKSCCVF 600 TFQPNGKLVG RFGGRGATDR HFAGPHFVAV NNKNEIWTD FHNHSVKVYS ADGEFLFKFA 660 GSHGEGNGQF NAPTGVAVDS NGNIIVADWG NSRIQVFDSS GSFLSYINTS AEPLYGPQGL 720 LTSDGHVVVA DAGNHCFKAY RYLQ 744 Figure2. Amino acid sequence of rat BERP. 45 2. Molecu la r cloning of the H u m a n B E R P . The full length rat BERP cDNA was used to screen a human hippocampal lambda Zap cDNA library (Stratagene). Screening 1 x 106 plaques with the 32P-labeled probe resulted in 2 positive clones. Sequence analysis of these clones showed that they contained sequences that correspond to positions 360-744 of the rat BERP protein sequence. Genbank searches of the human EST data base identified a fragment of the human BERP cDNA that contains the first 350 bp of the human BERP cDNA (Genbank accession # H30611). Sequence comparison showed that the human EST clone overlaps with sequences of the rat BERP cDNA that contain the first inframe ATG. Thus, based on this information and the sequence of the 3'end of the human BERP, PCR primers were designed to obtain the 5' half of human BERP cDNA. Using human cerebellar total RNA, RT-PCR was used for amplification of the 5'end region of human BERP. A PCR product of the predicted size (1356 bp) was obtained. The PCR product was then subcloned into the TA cloning vector (pCR II; Invitrogen), and sequenced using the dideoxy termination method. Figure 3 shows an alignment of the full length rat and human BERP cDNAs. Sequence analysis showed that the rat BERP cDNA shares 90% identity with the human BERP cDNA. Figure 4 shows the alignment of the predicted amino acid sequence of the rat and human BERPs, and indicates that they are 97% identical. 46 Figure 3. Alignments of the nucleotide sequence of the human and rat BERP. The start codon (ATG) a well as the stop codon (TAG) are underlined. Coding sequences of the rat BERP (rBERP) cDNA share 90% homology with its human homologue (hBERP). 47 rBERP TGTGGTCACCATGGCAAAGAGG6AGGACAGCCCTGGCCCGGAGGTGCAGCCAATGGACAAGCAGTTTCTG 280 hBERP A C CT— 60 rBERP GTATGCAGTATCTGCCTGGATCGGTACCGGTGCCCCAAAGTTCTGCCTTGTCTACACACCTTCTGCGAAA 350 hBERP C A G T C—G T—G- 130 rBERP GATGTCTCCAGAACTACATCCCTCCTCAGAGCCTGACACTCTCGTGTCCAGTATGCCGGCAGACATCCAT 4 20 hBERP A T G-C G--A--C G 200 rBERP CCTCCCAGAACAGGGTGTCTCAGCCCTACAAAACAACTTCTTCATCAGCAGCCTCATGGAGGCCATGCAG 4 90 hBERP G C G—A--CG-G A 270 rBERP CAGGCACCTGATGGGGCCCACGACCCTGAAGACCCCCACCCCCTCAGCGCAGTGGCTGGCCGCCCTCTCT 560 hBERP G T-T T 340 rBERP CCTGCCCCAACCATGAAGGCAAGACGATGGAGTTTTACTGTGAGGCCTGTGAGACTGCCATGTGTGGTGA 630 hBERP G 410 rBERP GTGCCGCGCAGGGGAGCACCGTGAACACGGCACAGTGCTGTTGCGGGACGTGGTGGAGCAGCACAAGGCA 700 hBERP C T G--T C—A T G 480 rBERP GCTCTACAGCGCCAGCTTGAAGCTGTGCGTGGCCGATTGCCACAGCTGTCTGCAGCTATCGCCTTGGTTG 770 hBERP —C--G C—G C A—T A--C- 550 rBERP GGGGCATCAGCCAGCAGCTGCAAGAGCGCAAGGCAGAGGCCCTGGCCCAGATAAGTGCAGCCTTTGAGGA 840 hBERP G C G—C 620 rBERP CCTGGAGCAAGCTCTGCAGCAGCGCAAGCAGGCTCTGGTCAGCGACTTGGAGAGCATTTGTGGGGCCAAG 910 hBERP A C C A 690 rBERP CAGAAGGTGTTGCAGACACAGTTAGACACACTGCGCCAGGGTCAGGAACACATTGGCAGTAGCTGCAGCT 980 hBERP A — C C-G C 7 60 rBERP TTGCAGAGCAGGCACTGAGACTGGGCTCTGCCCCTGAGGTTTTGCTAGTAAGAAAGCACATGCGAGAGAG 1, 050 hBERP C-C G G G G—GC-C C- 830 rBERP GCTGGCTGCTCTGGCAGCTCAGGCCTTCCCAGAAAGGCCCCATGAGAATGCACAGCTGGAACTGGTCCTT 1, 120 hBERP AT G—A G—GC A 900 rBERP GAAGTAGATGGGTTGCGGAGATCGGTGCTCAATCTGGGTGCACTGCTCACCACCAGTGCTGCTGCACACG 1, 190 hBERP — G — G — C — T C C G—C—CA 970 rBERP AGACAGTGGCCACCGGCGAGGGCCTGCGCCAGGCACTGGTTGGCCAGCCTGCCTCACTCACTGTCACCAC 1,2 60 hBERP -A--G G—A G—A—G G TG- 1,040 rBERP CAAAGACAAAGATGGGCGGCTGGTGCGCACGGGCAGCGCAGAGCTGTGTGCAGAGATCACTGGCCCCGAT 1,330 hBERP G—C T A T C C G--C 1,110 rBERP GGCATGCGCCTTGCGGTACCGGTGGTGGACCACAAAAATGGCACATATGAGCTGGTGTACACAGCACGTA 1, 4 00 hBERP C C G—A G A G--C- 1,180 rBERP CAGAAGGCGACCTGCTCCTCTCAGTGCTGCTCTATGGACAGCCGGTGCGTGGCAGCCCTTTCCGTGTGCG 1,470 hBERP -G G G C A C C C 1,250 rBERP TGCCCTGAGACCCGGAGACTTGCCACCTTCCCCAGATGATGTGAAGCGCCGGGTCAAGTCTCCCGGTGGT 1,54 0 hBERP C-T—G--G C G—C T C—T--C—C 1,320 rBERP CCTGGCAGTCATGTGCGCCAGAAGGCAGTGCGTAGGCCGAGCTCCATGTACAGCACCGGTGGCAAACGGA 1, 610 hBERP — C C C G A — C A- 1,390 rBERP AAGACAATCCAATTGTGGATGAACTCGTCTTTCGTGTTGGCAGTCGTGGAAGGGAGAAAGGTGAATTCAC 1, 680 hBERP -G C A G C 1,4 60 rBERP CAATTTACACCCGCTGTCCGCTGCTAGCAGTGGCCGAATAGTGGTAGCAGACAGCAACAACCAATGTATT 1, 750 hBERP AGGTG A--C C C--C G 1,530 rBERP CAGGTTTTCTCCAATGAGGGCCAGTTCAAGTTCCGATTTGGAGTCCGTGGGCGCTCGCCTGGGCAACTAC 1,820 hBERP T G A — A A G—G- 1,600 rBERP AGCGTCCTACGGGTGTGGCAGTGGACACCAATGGAGACATTATTGTGGCAGACTATGACAACCGTTGGGT 1,890 hBERP C — C — A A 1,670 rBERP CAGCATCTTCTCTCCTGAGGGCAAGTTCAAGACCAAAATTGGAGCTGGCCGCCTCATGGGGCCCAAGGGA 1, 960 hBERP C G C 1,740 rBERP GTGGCTGTGGACCGGAATGGGCATATCATTGTGGTGGATAACAAGTCTTGCTGTGTCTTCACCTTCCAGC 2, 030 hBERP C—A A C—C C T 1,810 rBERP CCAATGGCAAGCTTGTTGGGCGTTTTGGAGGCCGTGGGGCCACTGACCGCCACTTCGCAGGGCCCCACTT 2, 100 hBERP A—G C G T T— 1,880 rBERP TGTGGCTGTGAACAACAAGAATGAGATTGTAGTAACGGATTTCCACAACCATTCAGTGAAGGTGTACAGT 2, 170 hBERP A-G AG C T A 1,950 rBERP GCTGATGGAGAGTTCCTCTTCAAATTTGGCTCACATGGCGAGGGCAATGGACAGTTCAATGCCCCCACGG 2,24 0 hBERP --C G C G A- 2,020 rBERP GAGTAGCTGTGGATTCCAATGGAAACATCATCGTGGCTGACTGGGGCAATAGTCGCATACAGGTATTCGA 2, 310 hBERP C T C—C C 2,090 rBERP CAGCTCTGGTTCCTTCCTGTCCTATATCAACACGTCTGCAGAGCCACTGTATGGCCCACAGGGCTTGGCA 2, 380 hBERP C A A T C GC 2,160 rBERP TTGACCTCGGATGGCCACGTAGTAGTGGCTGATGCTGGCAACCACTGCTTTAAGGCCTATCGATACCTCC 2, 450 hBERP A T--G--G A GCC 2,230 rBERP AGTAGCTGCACAAGGGCCCTGCTTGGCTCATGGAGGGACGGACATAGGGGTGATTGGACAAGAGGGTCTG 2,520 hBERP T—G C TG 2,2 60 rBERP CATACACACT^ACAGAGAC ,^^ "^^ 0 48 1 HBERP MAKREDSPGPEVQPMDKQFLVCSICLDRYHCPKVLPCLHTFCERCLQNYIPgQSLTLSCP RBERP M A K R E D S P G P E V Q P M D K Q F L V C S I C L D R Y j g C P K V L P C L H T F C E R C L Q N Y I P g Q S L T L S C P HBERP V C R Q T S I L P E Q G H S A L H N N F F I S S L M E A M Q Q A P D G A H D P E D P H P L S f f l V A G R P J S S C P N H E G RBERP V C R Q T S I L P E Q G H S A L @ N N F F I S S L M E A M Q Q A P D G A H D P E D P H P L S 0 V A G R P 3 S C P N H E G HBERP K T M E F Y C E A C E T A M C G E C R A G E H R E H G T V L L R D V V E Q H K A A L Q R Q L E A V R G R L P Q L S A A I RBERP K T M E F Y C E A C E T A M C G E C R A G E H R E H G T V L L R D V V E Q H K A A L Q R Q L E A V R G R L P Q L S A A I HBERP A L V G G I S Q Q L Q E R K A E A L A Q I S A A F E D L E Q A L Q Q R K Q A L V S D L E n i C G A K Q K V L Q T Q L D T RBERP A L V G G I S Q Q L Q E R K A E A L A Q I S A A F E D L E Q A L Q Q R K Q A L V S D L E g l C G A K Q K V L Q T Q L D T HBERP L R Q G Q E H I G S S C S F A E Q A L R L G S A P E V L L V R K H M R E R L A A L A A Q A F P E R P H E N A Q L E L V L RBERP L R Q G Q E H I G S S C S F A E Q A L R L G S A P E V L L V R K H M R E R L A A L A A Q A F P E R P H E N A Q L E L V L HBERP E V D G L R R S V L N L G A L L T T S A n A H E T V A T G E G L R Q A L V G Q P A S L T V T S K D K D G R L V R T G S A RBERP E V D G L R R S V L N L G A L L T T S A N A H E T V A T G E G L R Q A L V G Q P A S L T V T 0 K D K D G R L V R T G S A HBERP E L ^ A E I T G P D G H R L H V P V V D H K N G T Y E L V Y T A R T E G Q L L L S V L L Y G Q P V R G S P F R V R A L R RBERP E L S A E I T G P D G S R L M V P V V D H K N G T Y E L V Y T A R T E G S L L L S V L L Y G Q P V R G S P F R V R A L R HBERP P G D L P P S P D D V K R R V K S P G G P G S H V R Q K A V R R P S S H Y S T G G K R K D N P I H D E L V F R V G S R G RBERP P G D L P P S P D D V K R R V K S P G G P G S H V R Q K A V R R P S S S J Y S T G G K R K D N P I H D E L V F R V G S R G HBERP R E K G E F T N L R R S S S A A S S G R I V V A D S N N Q C I Q V F S N E G Q F K F R F G V R G R S P G Q L Q R P T G V A RBERP R E K G E F T N L B ^ B S A A S S G R I V V A D S N N Q C I Q V F S N E G Q F K F R F G V R G R S P G Q L Q R P T G V A HBERP V D T N G D I I V A D Y D N R W V S I F S P E G K F K T K I G A G R L M G P K G V A V D R N G H I I V V D N K S C C V F RBERP V D T N G D I I V A D Y D N R W V S I F S P E G K F K T K I G A G R L M G P K G V A V D R N G H I I V V D N K S C C V F HBERP T F Q P N G K L V G R F G G R G A T D R H F A G P H F V A V G N K N E G V V T D F H N H S G K V Y S A D G E F L F K F G RBERP T F Q P N G K L V G R F G G R G A T D R H F A G P H F V A V S N K N E H V V T D F H N H S H K V Y S A D G E F L F K F G HBERP S H G E G N G Q F N A P T G V A V D S N G N I I V A D W G N S R I Q V F D S S G S F L S Y I N T S A E P L Y G P Q G L H RBERP S H G E G N G Q F N A P T G V A V D S N G N I I V A D W G N S R I Q V F D S S G S F L S Y I N T S A E P L Y G P Q G L G 744 HBERP B J T S D G H V W A D A G N H C F K A Y g Y L Q RBERP H T S D G H V W A D A G N H C F K A Y 0 Y L Q Figure 4. Sequence alignments of the deduced amino acids for human and rat BERP. Different amino acids are highlighted. Sequence comparison shows that the rat BERP shares 97% identity with its human homologue. 49 3. Sequence analysis of B E R P Genbank searches showed that BERP contains several known protein motifs and belongs to a family of proteins known as the RING finger family. BERP shares three motifs present in a subgroup of RING finger proteins known as the "RING finger-B box-Coiled-coil" (RBCC) subgroup (Reddy et al, '92; Freemont, '93). Figure 5 is a diagramatic representation of the various motifs present in BERP. HT2A, a member of the RBCC group, is the only mammalian protein with homology to the C-terminal region of BERP (Fig. 6). a. B E R P contains the R I N G finger-B box-Coi led-Coi l ( R B C C ) domain (a.a. 1-285). i . R I N G finger motif (amino acids 22-65) Genbank searches showed that BERP contains several known domains. The N-terminal amino acids 22-62 contain a novel type of zinc finger, the RING finger (Fig. 7). Sequence alignments of various RING fingers showed the conserved zinc-binding ligands (Fig. 7). The BERP RING finger is of the type C3HC4 (Freemont, '93; Saurin et al, '96). BERP RING finger contains the conserved zinc-binding ligands and some of the hydrophobic amino acids and prolines implicated in proper folding of this motif (Fig. 7b) (Borden et al, '95). On the other hand, the amino acid sequence of the RING of BERP resembled the ones present in PML and TIF1 (38%), as well as PAS4 (36%) (Saurin et al, '96). On the other hand, spacing between zinc-binding ligands in the BERP RING finger was most similar to that present in TRAF2, and c-Cbl (Lovering et al, '93; Saurin et al, '96). These similarities may indicate similar folding and function of the RING finger motif in these proteins. Based on the known structure of the RING finger of PML (Borden et al, '96) and the proposed structure for the CRAF1 RING finger (Cheng et al, '95), a prediction of the folding of the BERP RING finger is presented in Figure 7C. 50 RING finger Coiled-ooil ^22 62*115 146 285 i i i unique C -tern jnus 1 i ~ iml B^DOX •RBCC 620 744 P sheet ^BP-ldkeiepeat) P sheet (VAD lepeats) Figure 5. Schematic representation of the various structural motis present in BERP. Two zinc finger motifs, the RING finger and the B box are indicated. A coiled-coil motif immediately follows the B box. The first 285 amino acids form the RING finger-B box-Coiled-coil (RBCC) domain which is characteristic of members of the RBCC family. The RBCC region is followed by a -100 amino acid domain that shares ~45%homology with a domain that is present as several repeats in two actin binding proteins the yeast ABP 120 and the human protein ABP280. This repeat consists of 7 p stands and forms a p sheet structure. The C-terminal region of BERP is unique, also with a predicted P sheet structure and shares ~35%homology with the C-terminal region of HT2A, a member of the RBCC family. 51 H B E R P H T 2 A H B E R P H T 2 A 1 B g K R E DHPGPEVQPMDJKQ FfflVHS Q3^ RYQCG 1 B E U A A A H H L N - - L D A L B E V W E W P I B B S I TEEOLRO * * * * Q N Y i P A Q S L T E K L l A S S I N G 57 B s B I ^ H R O T a i l P E O G f f l g A J i E N i a F F l S S O S i A M Q Q A P D G A H D P E D P H P B B V V A B R P F S B P 59 i R f f l F S s K l i R l T S - - B l o B T D i g L T i L K t l ^ T A G E E A V S - L L M S R H B E R P H T 2 A 117 NHEGHTMEFPHEAHETA^HGEBBA^EHR—E^GBPLER^WHQH^AALQRQHEA^SGRSP 102 S CGRgLPRQigRSHGLvIaE PWaEBSHoP PGl3cHUp|yKE'AAHER^R!RD FGE KaTRLgE LjJG H B E R P H T 2 A 175 QHSAAigLMGBlBQQJjRlEigKAEAHAQISAAFEDgEQABQQRPOALVSDaETgCGAKQKBi 162 EHQRRK^JEBl^KDi8gA^KAVH0EYGHEERRlQDEBARsiKFFTGsE^EyEKSNS0E H B E R P 2 3 5 Q TfflLD T2RQBQ E H I G H S HS H A E Q AE8LSS A P §fflLffl||RK H M R H R L A A H A B Q A F PQRP H E NA H T 2 A 2 2 2 E E § S Y L 3 N I § E V Q A V § R B D ^ F L A K ^ § Q § B f f l A a g E E T A D ^ E E P E H T g S L P R g L T L Q D V H B E R P H T 2 A HBERP H T 2 A 95 Q f f l E L H L E g D G L R R S V f f l N L g A L L T T S A T A H E 7 9 EQLKQGHQG P B I I H Q ^ K K -35 4 - L f f l R T G S A E L R g E I T Q ^ G T R L P V P V V D H K N G T Y E L V Y T A R T E G E L L L S V L L Y G Q P V R G S 330 E E B V A S P R A S P " K O R M 3 E 1 HBERP 413 P F R V R A L R P G D L P P S P D D V K R R V K S P G G P G S H V R Q K A V R R P S S V Y STGGKRKDNPHEDEffl HT2A 34 8 AASN0QQCH HBERP 473 | V F H V G S R G R E K G E F T J j r a Q G ^ A A - p g - g R p | f i HT2A 357 i F L B K M G A K G S T P G M F f i n - p f f i l L Y V i l i l o l a E S 2gSNiJ]QCF^ra3SNEgQraKFRFGVgG 33RGB1YRB5S1TRK@-BLKE I - -gj-HBERP H T 2 A 531 GQLQRraTG^gVDTNHDMlMAraYDNRWjViSiIFS P E W K F K T K I G A G R R 412 S G I D S F V L S F L G A D L P N L T l a L S ^ ^ N c E l a L H G B T l H S Y D N s d K V Y T L b H k ' c V A C H R S - O B l S HBERP 577 GHKH^AVDRNHHlBSvnNKSCCSSTHQPNGKLVGRFGGRGAiTDRHF A HT2A 4 71 KBwSiTALPsSQFfaTaVEGGKlgcrjTVDRGSGVVKYSCLcWAVfePKFVTCDAEGTVYFT HBERP 624 G P H F ^ t e S g ^ E W V T E J F H N H S E K ^ S A ^ E F J ^ K F G ^ G E - G i ^ C ^ A P T g V j A ^ ! HT2A 531 O G L G e N § E l 8 R @ N E H H L B G G F S I G s B G - p l M o - f f l G K o i ^ F F S E l 8 E D ( a R C I A W c B a HBERP 683 II HT2A 58 9 I f JWGNSRl J s S R K E .nQVgDSSgSFLSniNTSAgpnYGATgPgHDgDgH 0LH[ |PKG@ GQ S V L I RgJGgJT C P V g I |JL T g K g Q L yvfflAjaAGNggFjaAnAnTI H B E R P 743 L Q HT2A 646 H L R R Y S T P Figure 6. Sequence comparison of the human BERP with HT2A. Both BERP and HT2A are members of the RBCC group which belongs to the RING finger family. Identical amino acids are highlihted, whereas conserved ones are shaded. Spaces were introduced to optimize alignment. BERP and HT2A share four conserved motifs. The first is the RING finger motif (underlined with a solid line and putative zinc-binding ligands are indicated by stars). The second is the B box motif (underlined with a dotted line and putative zinc binding ligands are indicated by a solid circle). The third is the coiled-coil motif (underlined with a double line). Bothe BERP and HT2A share a unique C-terminal region (included in brackets). 52 RING finger a . B E R P L V C S I C L D R Y R C P K V L P C L H T F C E R C L Q N Y I P P Q S L T L S C P V C R Q T C A P - 1 Y K C E K C H L V L C S P K Q T F C G H R F C E S CMAAI, 1 , S S 3 S P K C T A C Q E P M L L R C Q Q C O A E A X C P K L L P C L H T L C ; JCLEASGMQ C P I C Q A P b . CONSENSUS BERP C A P - 1 T R A F 2 C - C B L PML T I F 1 PAS 4 MEL 1 8 A R D 1 I V C S I C L D R Y R C Y K C E K C H L V L C S Y L C S A C K N I L R R Q L C K I C A E N D K D LRCQQCQAEAKC DTGAVCKQNIQS i < :<C 1 LCLMNMS D LMGALGGGYFJD I E 0 3 V C E D V E S L ^CERCl P K V L P I L I T F | E R | L Q N Y I P P Q S L T L S F5 K Q T E C G H R F C E S C M A A I . L S S S S P K P F Q A Q C G 1 R Y C S F C L T S I L S S G P Q N V K I E P C G H L M C T S C L T S W Q E S E G Q G P K L L P | L | T L | S G | L E A S G M Q R V P K L L P G L H S F C Q R C L ? ? F A T Q V G V 1 R P S C A P C G H L F C W S C L M S W C K E R P E A T T I I c : H C C I V R Y L E T S K Y Q G D K V ? R L L L C G H " V C : R ) C L T R L P L H G R A T R CPVCRQ':1 C C Q E C A A G V Y -G P F C R C E G P I G Q A I G P V C Q E C G P L G R Q H CP ICDVQ C P J R Q V S 1 L P E Q T G Q V R S C S L T L A V _ „ L V p -Q ; & f ^ Q C-terminal P C~S P L H I V T Y K Y v Z " R D I y \ C I S C V L F N-terminal Figure 7. Sequence of RING finger motif. The RING finger is a uniquely folded novel type of zinc finger present in a group of proteins known as the RING finger family. A. Sequence alignments of the RING finger motif of BERP with the two closest RING fingers that are present in the cytoplasmic protein CAP-1, a member of TRAF family and PML, a nuclear protein and a member of the RBCC family. CAP1 and PML amino acids identical to BERP are highlighted (purple), b. is sequence alignments of the RING finger of several known RING finger proteins. Conserved zinc-binding ligands that are thought to form the RING finger are highlighted (purple), c. Predicted structure of BERP RING finger. 53 i i . B box (amino acids 115-146) The RING finger is followed by a putative zinc finger binding motif known as the B box. The B box in BERP is closest in structure to the ones present in TIF1 (Le Douarin et al, '95), KRIP-1 (Kim et al, '96) and PML (Freemont, '93) (Fig. 8). The B box of these proteins contains an additional potential zinc binding ligand (Fig. 8). Interestingly the B box is almost always found in association with the RING finger motif and is followed by a coiled-coil domain (Reddy et al, '92). Also, Genbank searches have identified a novel protein, known as ataxia-telangiectasia group D complementing gene (ATDC; accession# 1082717), with sequences homologous to the B box domain present in BERP. Further sequence analysis showed that ATDC does not contain the RING finger motif, but it contains a B box and a coiled-coil domain similar to the ones present in BERP. The exact function of ATDC is not known yet, but recent data showed that mutations in ATDC gene may be the leading factor for the manifestation of atataxia-telengiectasia (AT), a human disease characterized by immunological, neurological and developmental defects and an increased risk of cancer (Leonhardt et al, '94). The role and the exact structure of the B box is not determined yet, but its association with the RING finger and the coiled-coil motif in the RBCC domain indicate that these 3 motifs may form a unit that functions similarly in various members of the RBCC group. 54 B E R P : T I F 1 ( B 2 ) : A D T C : P H P L S A V A G R P L S C P N H E G K T M E F Y C E A C E T A M C G E C R A G E H R E H P E A V G V T S Q R P V t ' C P I - ' H K K E Q L K L Y C E T C D K L T C R D C Q L L E H K E H P I R D F E A R K C P V H GKTMELFCQTDQTCICYLCMFQEHKNH b. C O N S E N S U S B E R P : T I F 1 ( B 2 ) : K R I P - 1 : R P T - 1 x n f 7 PML ( B 2 ) : A R D 1 E G K T M E F K K E Q L K L K H E P L V L G E K L R L D E R L K L Y C E A C E T A M Y C E T C D K L T F C E S C D T L T F | R K B M M V I Y I K D D G T L S S N P N H R T P T L T S I Y C R G C 5 K P L 1 1 h 1 C G E C K A G E H R E H C R D C Q L L E H K E H C K D C Q L N A H K D H C W L C E R S Q E H R G H C V I C R D S L K H A S f i C C S C A L L D S S H SQ H Q V H A I E F V C L E E G C Q T S P L M C C V ' C K E Y G K H Q G H Figure 5. Schematic representation of the various structural motis present in BERP. Two zinc finger motifs, the RING finger and the B box are indicated. A coiled-coil motif immediately follows the B box. The first 285 amino acids form the RING finger-B box-Coiled-coil (RBCC) domain which is characteristic of members of the R B C C family. The R B C C region is followed by a -100 amino acid domain that shares -45% identity with a domain that is present as several repeats in two actin binding proteins the yeast ABP120 and the human protein ABP280. This repeat consists of 7 [5 strands and forms a p sheet structure. The C-terminal region of BERP is unique, also with a predicted P sheet structure and shares -35% identity with the C-terminal region of HT2A, a member of the R B C C family. 55 i i i . Coi led coi l region (amino acids 147-285) Members of the RBCC group are characterized by the presence of a predicted coiled-coil domain following the B box. In general, a coiled-coil is a bundle of a-helices that are wound into a super helix (Cohen and Parry, '86; Lupas, '91&96; Brown et al, '96). Coiled-coils serve as protein-protein interaction motifs in cytoskeletal proteins and some transcription factors. In some cytoskeletal proteins, they form large rigid structures. In others they provide a scaffold for regulatory complexes. In several families of transcription factors, coiled-coil motifs are much shorter and they serve as a dimerization motif and position the DNA-binding region on the DNA (Lupas, '96). In fact, the serum response factor (SRF) and the yeast transcription factor GAL4, coiled-coils can serve in protein dimerization as well as interactions with DNA (Lupas, '96). Coiled-coils are characterized by the presence of heptad repeats, (7 amino acids), and each repeat is characterized by the presence of hydrophobic amino acids at positions 1 and 4. Hydrophobic amino acids such as Leu, He, Ala, and Val are the most common amino acids at these positions (Cohen&Parry, '86; Lupas, '91). Amino acids that are involved in turn and loop formation such as Pro and Gly are usually rare in coiled-coil structures. In BERP, the region that contains amino acids 147-285 possesses several heptad repeats that are rich in Leu, He, Ala, and Val (Fig. 9). The spacing between the B box and the coiled-coil motif is highly conserved among members of the RBCC group. As expected, the B box in BERP is immediately followed by a coiled-coil motif (amino acids 147-285; Fig. 9a). The coiled-coil region in BERP shares homology with the coiled-coil region of members of the RBCC family. Figure 9a shows the alignment of the BERP coiled-coil domain with two other members of the RBCC family such as TIF1 (accession# X97548). Genbank searches also showed that the putative coiled-coil motif shares homology with coiled-coil domains present in a cytoskeletal 56 protein (e.g. myosin heavy chain II; MHC2; accession# Ml4628) (Fig. 9) and a putative transcription factor (SMC1; accession# 1652449). Secondary structure analysis of BERP sequence using the nn predict program available on the server from the NCBI database, showed that amino acids 147-285 of BERP has a predicted helical structure. The coiled-coil motif prediction program (also available from NCBI database search facility), also showed amino acids 147-285 is the only region in BERP with high probability of coiled-coil formation (P values: 0.6-0.9). Figure 9 is a diagramatic representation of the predicted coiled-coil region using the helical wheel model. The first (helical wheel 1) contains 10 heptad repeats, where as the second contains 4 heptad repeats. Although the RBCC domain has been shown to be very crucial for the function of proteins bearing it, the exact function of this domain as well as the roles of the three motifs forming it are not known yet. Overall, recent data showed that several of the RBCC-containing proteins have transformation potential (Saurin et al, '96). Translocation of the RBCC domain of some of the known members of the RBCC group has been reported in various types of cancer (Saurin et al, '96). These observations show the important role for the RBCC domain in maintenance of normal cell function and indicate that the RING finger, the B box and the coiled-coil motifs can function as one unit. 57 B E R P : L R D W E Q H K A A L Q R Q L E A V R G R L P Q L S A A I A I A / G G I S Q Q L Q E R K A E A L A Q I S A A F E D L E Q . T I F 1 : L E D A V R N ( ) R K L I j A S I , V K R L G D K H A T t Q K S T K E V R S S l R Q V S D V Q K R V Q V D V K M S l I i Q I M K . PML ALQEQDSAtx;AVIU\Ci4HAAVGQLGRARAEr:rE !EI / l ]^E] .<VRQVVAHVkAv)ER SMC1 ftEQSQQIQQALDQRSQ»IQTOOTE&^ a. MHC2 : . E E EQEA.KAE I J Q RAMS K A N S E V AQ B E R P : A L Q Q R K Q A L V S D L E S I C G A K Q K V L Q T Q L D T L R Q G Q E H I G S S C S F A E Q A L R L G S A P E V L L V R K T I F 1 : ELSKBGRVl»TODAQKVTEGQQERLERQHWTMTKiQKHQSH:LRFASWALESL:NNTALLLPKK PML E L L E A V D & R Y Q R D Y E E M A S R L C IRj(,DAVLQR T . R T G S A L V Q R M K C Y A S D Q SMC1 R Y N D G D R Q I T N H O Q O V G Q I Q A E I S r j S Q O Q F I j ] 11 QQEKS F H N T Q T L P Q L E A A V Q T S Q Q Q L E Q L MHC2 W R T K Y E T D A I Q R T E E L E E A K K i K L A Q R I X ) b A S E , H V E A V N A K C A S L E K T K Q R L O N T ; v ^ D p > I I Figure 9. The coiled-coil motif a. Sequence alignments of the coiled-coil motif present in BERP as well as members of the RBCC family such as TIF1. Similar motif is also present in myosin heavy chain II (MHC2) and a putative transcription factor SMC1. Identical as well as conserved amino acids are higlighted. b. Presentation of two predicted coiled-coil structures in BERP using the helical wheel model. 58 iv. B E R P contains a domain that is present as several repeats i n A B P - 1 2 0 and ABP-280 ( A B P - l i k e repeat) Following the RBCC domain, amino acids 285-430 share 45% homology with a domain present as several repeats in the yeast ABP-120 (Genbank accession# XI5430) and the mammalian protein ABP-280, two actin crosslinking proteins (also known as the gelation factor; accession# A54771), and hence will be called ABP-like repeat (Noegel et al, '89; Bresnick et al, '90; Sharma et al, '95; Stossel, '93). Along with alpha actinin, these proteins are the most abundant F-actin crosslinking proteins. ABP-120 and ABP-280 are rod-shaped homodimers (Noegel et al, '89; Sharma et al, '95). Each consists of an N-terminus actin binding domain and a rod-like C-terminal region (Noegel et al, '89; Bresnick et al, '90). The rod domain consists of several repeats each about 100 amino acids in length. The rod domain consists of 6 repeats in the yeast ABP-120 and 24 repeats in ABP-280. Recently, the solution structure of repeat 4 of the rod domain of ABP-120 has been determined (Fucini et al, '97). These studies showed that this repeat consists of seven beta-sheets arranged in an immunoglobulin-like (Ig) domain. Because of the high similarity present among different repeats, the authors predict a similar folding for all of the other repeats (Fucini et al, '97). These data indicate a role for ABP-120 and ABP-280 repeats in the dimerization of the whole protein and in the formation of the rod tail. Besides a role for these repeats in homodimer formation, recent data showed that these repeats also serve as protein-protein interaction domains. In platelets, repeats 17-19 of ABP-280 interact with the cytoplasmic tail of the Willebrand factor receptor, the glycoprotein (GP) Ib-IX complex (Meyer et al, '97). ABP-280 cross links actin filaments and anchors the membrane skeleton to the plasma membrane by its interaction with (GP) Ib-IX. In other cells that do not express GP Ib-IX, ABP-280 binds the high affinity IgG receptor (FcyRI) and the (32 integrin (Ohta et al, '91; 59 Sharma et al, '95). Other studies have shown that the C-terminal region of ABP-280 interacts with stress activated protein kinase (SEK)(Martin et al, '97). Figure 10 shows that amino acids 285-430 of BERP share -45% homology with one of the repeats present in ABP-120 (Fig. 10a) and in ABP-280 (Fig. 10b). Figure 10c is a comparison between some of the highly conserved areas in these repeats with the one present in BERP. These figures show that BERP contains a single full length ABP-like repeat, indicating that this region of BERP may function as an independent domain. These observations indicate that this domain may serve as a homodimerization motif as well as a protein-protein interaction motif. Because of the high similarity between the ABP-like repeat present in BERP and those present in ABP-280, one would predict that BERP ABP-like may be involved in interactions similar to the ones reported for the C-terminal domain of ABP-280. 60 Full repeat of ABP-120 (amino acids 658.752) 3 E R P : PHENAQLELVLEVDGLRRSVLNLGALLTTSAAAHETVATGEGLRQALVGQPASLTVTTKD ABP 120: 619 BKJ^GDYVIN^L^DNVNGFPKTVTVKPAPS(EHSY(E||^VBCVFDNAJJJ|EF|J|IFAV( B E R P : KDGRLVRTGSAELCAEITGPDGMRLAVPVVDHKNGTYELVYTARTEGDLLLSVLLYGOPV ABP 120: 739 TK§VARTD|GDPFEVA±N^]^LWDAK|T|NNDGTyG^[)APVEGNYNVNVTLR6N|l '. BERP: RGSPFRVRAL ABP 120: 739 KNM|lD§KCI 743 b. Full repeat of ABP-280 (amino acids 2550.2647) BERP: AAAHETVATGEGLRQALVGQPASLTVTTK ABP 280: 2555 |D|SK\^K|LMSK|Y^KS|FHDCS B E R P : RTGSAELCAEITGPDGMRLAVPVVDHKNGTYELVYTARTEGDLLLSVLLYGQPVRGSPFRV ABP 280: 2584 KA|NNM1LVGVHBRTPCEEIL|KHVGSRL|SVSYLLKDK|EYT|V|KWGHEHIp||yl 2644 C. BERP: ABP 280: 703 |K|P|RVQVQDNE|CPVEAL ABP 280: 1285 |GPHVK|RVANPS|NLTETY ABP 280: 1403 ABP 280: 2096 GSAELCAEITGPDGMRLAVPWDHKNGTYELVYTARTEGDLLLSVLLYGQPVRGSPFRV | K | P | R V Q V Q D N E | C P V E A l J K | S N C - N G i y ^ C r Y V P | K P V K H T A t « S W ^ v - - J l ' N S P F R V | G P H V ? i R V A N P s | N L T E T Y | Q | R G D M K V E f c p Y E f c H S V I ^ T y D | s P V P s | p F R V 1HQVPGSPFKV IKVPGSPFSV Figure 10. BERP contains a domain that is present as several repeats in two actin binding proteins, the yeast ABP 120, and the human ABP 280. Each repeat consists of 7 P strands and forms a P sheet structure. Sequence alignments of an ABP-like repeat present in BERP (amino acids 285-459) with one of repeats present in ABP 120 (a) and ABP 280 (b). Sequence alignments of BERP ABP-like repeat with 4 of the repeats present in ABP 280 (c). Identical amino acids are highlighted (red) and conserved ones are shaded (yellow). Repeats are highly conserved and share -45% sequence identity. This domain is present as one repeat in BERP, 6 repeats in ABP 120 and 24 repeats in ABP 280. 61 v. B E R P contains a putative nuclear localization signal. Signals that are involved in nuclear targeting are usually short sequences that contain a high proportion of positively charged (basic) amino acids (Dingwall and Laskey, '91). One potential nuclear localization signal in BERP is present following the ABP-like repeat which contains the following amino acids: ''KRRKSPGGPGSHVRQKAVRRPSSMYSTGGKRKDNP". This region contains basic amino acids (underlined) followed by a spacer of 11 amino acids and a basic cluster (bolded) characteristic of the proposed bi-partite nuclear localization signal (NLS; Dingwall and Laskey, '91). Similar potential bi-partite nuclear targeting signals are present in transcription factors such as c-Fos and c-Jun (Dingwall and Laskey, '91). Whether the presence of the predicted bi-partite nuclear localization signal is enough for nuclear targeting is not known yet. Thus, the presence of such potential nuclear localization signals can only indicate the potential targeting of a protein to the nucleus. Localization experiments will be required to determine if BERP is indeed a nuclear protein. v i . B E R P contains a unique C-terminus As shown in Figure 6, the last 279 amino acids of BERP share -35% identity with the C-terminal region of the RING finger protein HT2A (Fridell et al, '95). HT2A is the only mammalian RING finger protein with homology to the BERP C-terminal region. The only other protein with similarity to this region of BERP is the bacterial protein Mbk, a putative protein serine threonine kinase (Peirs et al, '97). HT2A has been identified in a yeast two-hybrid screen 62 as an interactor with the HIV regulatory protein Tat. The cellular functions of HT2A and the bacterial protein, Mbk, are still unknown. Interestingly, the C-terminal region of BERP has a predicted beta sheet structure (as determined by the nn predict program), and contains six repeats of a novel domain that is rich in Val Ala and Asp (Fig. 11). More significantly, secondary structure analysis showed that members of the TRAF family have an overall structure similar to the one predicted for BERP. In addition, the C-terminal region of BERP as well as the TRAF domain found at the C-terminus of TRAF proteins have a predicted beta strand formation. Figure 12 is a comparison between the secondary structure of BERP and a member of the TRAF family, CRAF1 (Cheng et al, '95). Members of the TRAF family contain an RBCC-like motif at their N-terminal region but differ from the RBCC family in that the B box is replaced by a cluster of approximately five zinc fingers (Baker and Reddy, '96; Saurin et al, '96). The C-terminal TRAF domain is involved in interactions with cytokine receptors as well as dimerization with other members of the TRAF family. Thus, this overall protein conformation may be required for the multiple protein-protein interactions reported for the TRAF proteins, including protein homo- and heterodimerization as well as clustering of cytokine receptors. Although there is no significant sequence homology between the C-terminal region of BERP and the TRAF domain, the overall structural similarity indicates that the BERP protein may also be involved in multi-protein complexes including homo- and heterodimer formation. 63 BERP ELVFRVGSRGREKGEFTNLHPLSAASSGRIWADSNNQCIQVFSNQG QFKFRFGVRGRSPGQLQRPTGVAVDTNGDIIVADYDNRWVSIFSP EGKFKTKIGAGRLMGPKGVAVDRNGHIIWDNKSCCVFTFNPN LVGRFGGRGADT DRH FAGPH FVAVNNKNEIWTDFHNH SVKVYSA EFLFKFGSHGEGNGQFNQPTGVAVDSNGNIIVADWGNSRIQVFDSS GSFLSYINTSAEPLYGPQGLALTSDGHVWADAGNHCFKAYRYLQ 74 5 HT2A LFLKKMGAKGSTPGMFNLPVSLYVTSQGEVLVADR-NYRIQVFTRKGFLKEIRRSPS GIDSFVLSFLGADLPNLTPLSVAMNCQGLIGVTDSYDNSLKVYTL DGHCVACHRSQLSKPWGITALPSGQFWTDVEGGKLWCFTVDR GSGVVKYSCLCSAVRPKFVTCDAEGTVYFTQGLGLNLENRQNEHHLEGGFSIGSVGPDG QLGRQISHFFSENEDFRCIAGMCVDARGDLIVADSSRKEILHFP KGGGYSVLIREGLTCPVGIALTPKGQLLVLDCWDHCIKIYSYHLRRYSTP 653 Mbk QASGQTVLPFTGIDFRLSPSGVAVDSAGNVYVTSEGMYGRVVKL ATGSTGTT YQPQGIiAVDG AGTVYVTD FNN- RWTL AAGSNNQTVLPFDGLNYPEGLAVDTQGAVYVADRGNNRWKL AAGSKTQTVLRFTGLNDPDGVAVDNSGNVYVTDTDN-RWKL E AE SNNQVVLP FT DIT APWGIAVDEAGTVYVTE HNTNQWKL LAGSTTSTVLPFTGLNTPLAVAVDSDRTVYVADRGNDRWKLTS 662 Figure 11. The C-terminal region of BERP contains repeats with a (3 strand structure. The C-terminal region of BERP has a predicted (3 strand structure and shares homology with sequences present in the mammalian RBCC protein, HT2A and the bacterial protein Mbk. This domain consists of 6 conserved repeats rich in valine (V), alanine (A) and aspartic acid (D). Similar repeats are present in HT2A and Mbk 64 Figure 12. Secondary structure comparison of BERP and CRAF1. Secondary structure prediction was determined using the nn predict program available on the server. H=helix, E=stand, - = no prediction. Ligands predicted to form the RING finger are highlighted. TRAF domain in CRAF1 as well as the unique C-terminal region of BERP are underlined. Both proteins contains a central coiled-coil motif followed by a C-terminal region rich in (3 strand conformation. 65 Secondary structure prediction (using nnpredict program) (H = h e l i x , E = s t r a n d , - = no p r e d i c t i o n ) C r a f l Craf1 M E S S K K M D A A G T L Q P N P P L K L Q P D R G A G S V L V P E Q G G Y K E K F V K T V E D K Y E ( | E K | R L V L C N P K Q T E | G | R F i nn p r e d i c t H E E E H - E H H H H c r a f i E S ( M A A L L S S S S P K | T A | Q E S I I K D K V F K D N C C K R E I L A L Q V Y C R N E G R G C A E Q L T L G H L L V H L K N E C Q F E E nn p r e d i c t HHHHHHE HHHHHHHEH HHHHHHHHHHHH C r a f l L P C L R A D C K E K V L R K D L R D H V E K A C K Y R E A T C S H C K S Q V P M I K L Q K H E D T D C P C V W S C P H K C S V Q T L L R S E nn p r e d i c t H H H H H - H - - H H H H H H H E H E E HEHHHH C r a f l L S A H L S E C V N A P S T C S F K R Y G C V F Q G T N Q Q I K A H E A S S A V Q H V N L L K E W S N S L E K K V S L L Q N E S V E K N K S I Q nn p r e d i c t H H - H E E E H H E H H - H - H H H H H H H H H H H H H H H H H H H H H H - H H H C r a f l S L H N Q I C S F E I E I E R Q K E M L R N N E S K I L H L Q R V I D S Q A E K L K E L D K E I R P F R Q N W E E A D S M K S S V E S L Q N R V nn p r e d i c t -HH HHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHH HH H-HH-H C r a f l TELESVDKSAGQAARNTGLLESQLSRHDQTLSVHDIRLADMDLRFQVLETASYNGVLIWKIRDYKRRKQEAV nn p r e d i c t -HH—HHHHH-HHH HHHHHH H—H—HHHHHEEE EEEEEH H HH C r a f l MGKTLSLYSQPFYTGYFGYKMCARVYLNGDGMGKGTHLSLFFVIMRGEYUALLPWPFKQKVTLMLMDQGSSR nn p r e d i c t HH E EE EHHHEEE E-EEEEEEEH HHEEHH C r a f l RHLGDAFKPDPNSSSFKKPTGEMNIASGCPVFVAQTVLENGTYIKDDTIFIKVIVDTSDLPDP nn p r e d i c t EE EEE—EEHH EEEEEEE rBERP rBERP MAKREDSPGPEVQPMDKQFLV|Sl|LDRYRCPKVLP|L|TFiERiLQNYIPPQSLTLS|PV|RQTSILPEQG nn p r e d i c t EEEE H-HH rBERP VSALQNNFFISSLMEAMQQAPDGAHDPEDPHPLSAVAGRPLSCPNHEGKTMEFYCEACETAMCGECRAGEHR nnpredict -HHH EEHHHHHHHHH HHHEHHHH H--H rBERP EHGTVLLRDWEQHKAALQRQLEAVRGRLPQLSAAIALVGGISQQLQERKAEALAQISAAFEDLEQALQQRK nn p r e d i c t HEEHHHHHHHHHHHHHHHHHHHH HHHHEEE HHHHHHHHHHHHHHHHHHHHHHHHHHHH rBERP QALVSDLESICGAKQKVLQTQLDTLRQGQEHIGSSCSFAEQALRLGSAPEVLLVRKHMRERLAALAAQAFPE nn p r e d i c t H-E HHHHH-H HHHHHHHHHH EE HHHHHHH HHHHHHH-HHHHHHHHHH rBERP RPHENAQLELVLEVDGLRRSVLNLGALLTTSAAAHETVATGEGLRQALVGQPASLTVTTKDKDGRLVRTGSA nn p r e d i c t HHHHHHHHH—HHHHHHHHHHHHHHHHHHHEHH—HHHHHHE EEE EEH rBERP ELCAEITGPDGMRLAVPWDHKNGTYELVYTARTEGDLLLSVLLYGQPVRGSPFRVRALRPGDLPPSPDDVK nn p r e d i c t -EEE EE EEEEEE HHHHHEH EEE rBERP RRVKSPGGPGSHVRQKAVRRPSSMYSTGGKRKDNPIVDELVFRVGSRGREKGEFTNLHPLSAASSGRIVVAD nn p r e d i c t HHEEEEE EEEE— rBERP SNNQCIQVFSNEGQFKFRFGVRGRSPGQLQRPTGVAVDTNGDIIVADYDNRWVSIFSPEGKFKTKIGAGRLM nn p r e d i c t EEEE EEEEE EE EEEE EEEE EEE rBERP GPKGVAVDRNGHIIWDNKSCCVFTFQPNGKLVGRFGGRGATDRHFAGPHFVAVNNKNEIVVTDFHNHSVKV nn p r e d i c t EE EEEE EEEE EEEEE EE EEEE EE rBERP YSADGEFLFKFGSHGEGNGQFNAPTGVAVDSNGNIIVADWGNSRIQVFDSSGSFLSYINTSAEPLYGPQGLA nn p r e d i c t E HEEEEH EE EEEE EEEE EEEEE E rBERP LTSDGHVWADAGNHCFKAYRYLQ nn p r e d i c t E EEE HHHHHHH 66 II. Gene expression of B E R P Northern blotting analysis was used to study the expression of BERP transcripts in the brain and in other tissues, whereas in situ hybridization was used to examine the distribution of BERP transcripts in the brain. Immunohistochemistry was used to determine the expression of BERP protein in brain sections and cultured cells, respectively. 1. Nor thern analysis a. Rat tissues: Northern blotting was used to identify tissues that express BERP mRNA. Using the rat BERP cDNA as a probe (1.6 kb EcoRl fragment; 3'end region of BERP cDNA) a major band of ~3 kb in size was detected in several of the tissues examined (Fig. 13; similar results were obtained from three independent experiments). BERP transcripts were highest in the brain (Fig 13a). Lung and placenta are the only tissues that contain moderate levels of BERP mRNA (Fig. 13a,b). Other tissues contain either very low levels of BERP transcripts (kidney, heart, testis, liver) or undetectable amounts (muscle, spleen). PC 12 cells also expressed detectable levels of BERP mRNA (Fig. 13b). The presence of other various but very weak bands was also observed (Fig 13a & b). These bands may represent alternatively spliced isoforms of BERP mRNA, or a weak cross-reaction between the BERP probe sequence and other closely related gene products. Northern blotting also showed that BERP transcripts are found in all of the brain regions examined, indicating a wide distribution of BERP mRNA in various parts of the brain (Fig. 13c & d). Athough the band for mouse BERP mRNA appears lowers than that of the rat BERP (Fig. 13c), this difference in size was not observed in Figure 13d. Thus, it is more likely that the difference in size between rat and mouse BERP mRNAs (Figure 13 c) is due to an experimental artifact due to uneven electrical current distribution during gel running. 67 b. Human tissues: A 1.4 kb fragment of the human BERP cDNA (5'end region; obtained by RT-PCR) was first used as a probe. Two major transcripts of 5 and 3kb were detected in human cerebellum (Fig 14a, similar results were obtained from two independent experiments). These transcripts were not detectable in two tumor cell lines examined, a human neuroblastoma (SK-N-MC) and astrocytoma (U-373) (Fig. 14a). Also, the human probe was able to detect the 3 kb transcript in rat cerebellum (Fig 14a). When a 400 bp probe was used (corresponding to the last 400 bp of the human BERP coding sequence), a single 3 kb band was detected in human cerebellum as well as in breast tissue (Fig. 14b). Since the two human probes used were part of the coding region, and both detected a ~3 kb band, it seems that the 5 kb transcript detected when using the 1.6 kb probe may be an artifact generated from cross-reaction of the probe with 28 S ribosomal RNA. 2. In situ hybridization Like the Northern analysis, in situ hybridization detected high levels of BERP transcripts expressed in the brain (sections from at least three different rat brains were used for in situ hybridization analysis). Riboprobes were generated using the original 394 bp PCR product for BERP subcloned into the TA cloning vector pCR II (Invitrogen). This construct product was linearized either with BamH I or Xho I and used to generate 35S-labeled antisense and sense riboprobes respectively. Figures 15A&B (upper panels) show the results obtained using the antisense riboprobe. In situ hybridization analysis showed that high expression of BERP exists in the cerebellum, cerebral cortex and hippocampus as shown in sagittal (Fig. 15a; top panel) and coronal (Fig. 15B; top panel) brain sections. No specific binding was obtained when the sense probe was used (Fig.l5A&B; lower panels). These results are consistent with the wide expression of BERP in various brain regions as observed with Northern blotting. 68 BERP 28S Figure 13. Northern blot analysis of rat BERP mRNA. Twenty micrograms of total RNA from various tissues were electrophoresed on 1% denaturing agarose gels, transferred to a nitrocellulose membrane and hybridized with a 32P-labeled 1.6 kb EcoR I fragment of the rat BERP cDNA. Panel a: a major band 3 kb (indicated by an arrow head) was detected in brain, kidney, lung and heart. As shown, highest levels of BERP transcripts are present in the brain. Panel b: BERP mRNA is detected in all of the brain regions examined. Ethidium bromide photo showing the 28S ribosomal R N A is shown to compare amounts of R N A used. Panels c&d: BERP mRNA expression was detectable in rat cerebellum (rCerebellum) mouse cerebellum (mCerebellum), PC 12 cell and rat placenta (rPlacenta). 69 a . b Figure 14. Northern blot analysis of human BERP mRNA. Twenty micrograms of total R N A from various tissues was electrophoresed on 1% denaturing agarose gels, transferred to a nitrocellulose membrane and hybridized with either a 32P-labeled 1.4 kb fragment (left panel) or 400 bp fragment (right panel). Panel a: using a 1.4 kb fragment of the human BERP cDNA as a probe. Panel b: using a 400 bp fragment of the human BERP cDNA as a probe. A major 3.0 kb band was detected in both panel a & b in human cerebellum (HUM CB), rat cerebellum (Rat CB) and human breast. An additional 5.0 kb was detected in H U M CB only when the 1.6 kb probe was used. In panel a) actin probe was used as a control. 70 Figure 15. In situ hybridization analysis of BERP mRNA expression in the brain. Sagittal (a) and coronal (b) sections were hybridized with 35S-labeled riboprobes. a & b (top panels): Results obtained using antisense riboprobes. a & b (lower panels): Results obtained using sense riboprobes. As shown, BERP mRNA is detected in various regions of the brain with highest expression in the cerebellum (CB), hippocampus (HIP), olfactory bulb (OB) and cortex (CX). 71 3. Immunohistochemical analysis. a. Generation and characterization of B E R P antibodies: i . Serum from an immunized rabbit detects an ~82 k D a protein that corresponds to B E R P . Polyclonal antibodies were raised against the last 9 amino acids of BERP. Western blotting of proteins extracted from brain tissues was used to examine the specificity of anti-BERP antibodies. Several protein bands were detected in rat cerebellum extracts when crude serum of the immunized rabbit (# A 2B 3) was used (Fig. 16, lane 1). One of the bands was ~ 82 kDa in size which corresponds to the predicted size of the BERP protein. To confirm this, BERP antibodies were pre-incubated for 2 h with 100 jig/ml of the peptide used for immunization and then used for Western blotting. Results show that the staining of 82 kDa band was blocked (Fig. 16; lane 2), confirming that this band represented BERP protein (similar results presented in Figure 16 was obtained from at least 4 independent experiments). Occasionally, a weaker but visible 59 KDa protein was detected mainly from cerebellar extracts, which was also blocked by the C-terminal peptide, indicating that the 59 kDa protein may represent a degradation product. Other bands detected by the crude serum were not blocked by BERP peptide indicating that these do not represent isoforms or degradation products of BERP protein. Because of the presence of several bands that cross reacted with the crude serum, it was necessary to purify anti-BERP antibodies. Anti-BERP antibodies were purified using the C-terminal peptide originally used for BERP antibody production. Figure 16 (Lane 3) shows that the affinity purified antibody recognized a major protein of a -82 kDa in size. Figure 17 show that affinity purified anti-BERP antibodies recognize a -82 kDa protein from rat cerebellum, hippocampus and cortex (n=3). Again detection of this protein was blocked by pre-incubating the antibodies with C-terminal BERP peptide (n=3). 72 Figure 16. Western blot analysis of BERP expression in cerebellum. Polyclonal anti-BERP antibodies were generated against the last 9 amino acids at the C-terminal region of BERP. Extracts from rat cerebellum were used for this analysis. Lane 1 shows that an ~82 kDa protein (corresponds to the predicted size for BERP) as well as others were detected when using crude serum (1/1000 dilution) of the immunized rabbit. In lane 2, the 82 kDa protein was blocked by preincubating the crude serum with the C-terminal peptide for BERP (100|ig/ml). Affinity purified BERP antibodies (1/250 dilution) detected a major -82 kDa band (indicated by an arrow). 73 i i . A n t i - B E R P antibodies recognizes B E R P in H E K cells transfected wi th B E R P c D N A . Anti-BERP antibodies were used next to test if the BERP antibodies were able to recognize the transiently expressed BERP protein in HEK cells. For this study, BERP cDNA as well as a FLAG-tagged BERP cDNA (N-terminal FLAG) were subcloned into the mammalian expression vector pcDNA3 (the generation of FLAG-tagged cDNA is described in more details under cell transfection experiments). These constructs as well as the vector (pcDNA3) alone were used for transient transfection experiments in HEK-293 cells. Figure 18 summarizes results obtained from at least 10 independent transfection experiments. FLAG-BERP as well as BERP were recognized with BERP antibodies; -82 kDA band was detected in lysates from HEK cells transfected with FLAG-BERP as well as BERP cDNA, but not in those transfected with the vector (pcDNA3) alone (Figure 18). Pre-immune serum failed to recognize any proteins in both HEK cell lysates. Also, the immunoreactivity of 82 kDA protein was blocked when pre-incubation of crude anti-BERP antibodies with 100 ug/ml of the peptide used for immunization was used. These results show that anti-BERP antibodies can recognize a major -82 kDA protein which represents the transfected BERP. i i i . Crude as wel l as affinity purified a n t i - B E R P antibodies immunoprecipitate B E R P from H E K cells transfected wi th B E R P . Anti-BERP antibodies were able to immunoprecipitate an -82 kDA protein from HEK cell lysate transfected with FLAG-BERP (Fig. 19; n=3). The same protein was immunoprecipitated with anti-FLAG antibodies, confirming that the immunoprecipitated protein was the transfected BERP (n>10). All of the above data indicate that anti-BERP antibody can specifically recognize BERP protein, and thus can be used for immunohistochemical analysis of BERP distribution in the brain. 74 BERP Abs BERP Abs+ peptide § f § § 1 & * 1 & I M K 0 o. o o o. o , v ' O If O U x O 190 -112 r " 86 — Sft' 70 59 Figure 17. Western blot analysis of BERP expression in various brain regions, affinity purified BERP antibodies (1/200 dilution)were used to examine the expression of BERP in rat cerebellum, hippocampus and cortex. Left panel shows that in all of the brain regions examined a -82 KDa was detected (arrow head). This band was blocked by preincubating BERP antibodies with the C-terminal peptide for BERP (100ug/ml) (right panel). 75 Figure 18. Western blot analysis of BERP expression in transiently transfected H E K cells. BERP antibodies (1/500 dilution) were used to examine the expression of BERP in H E K cells transiently transfected with the a FLAG-tagged full length BERP cDNA. BERP antibodies detected a major protein -82 KDa in size in H E K cells tranfected with BERP cDNA as well as a FLAG-tagged BERP cDNA. H E K cells transfected with vector alone did not contain BERP protein. Also, -82 kDa protein was blocked by preincubating BERP antibodies with the C-terminal peptide for BERP (lOOug/ml). Preimmune serum did not detected any bands of the same H E K cell lysates. Ant i -FLAG antibodies (3p.g/ml)detected -82 kDa protein only in lysates of H E K cells that express FLAG-tagged BERP. 76 Figure 16. Western blot analysis of BERP expression in cerebellum. Polyclonal anti-BERP antibodies were generated against the last 9 amino acids at the C-terminal region of BERP. Extracts from rat cerebellum were used for this analysis. Lane 1 shows that an ~82 K D a protein (corresponds to the predicted size for BERP) as well as others were detected when using crude serum (1/1000 dilution) of the immunized rabbit. In lane 2, the 82 K D a protein was blocked by preincubating the crude serum with the C-terminal peptide for BERP (lOOug/ml). Affinity purified BERP antibodies (1/250 dilution) detected a major -82 KDa band (indicated by an arrow). 77 iv. Immunocytochemical detection of B E R P protein in H E K cells transfected wi th B E R P c D N A , using the affinity purified a n t i - B E R P antibodies. Next, BERP antibodies were used for immunocytochemical analysis of HEK cells transfected with full length BERP, using the DAB/ABC method. Anti-BERP antibodies stained HEK cells transfected with BERP cDNA but not with vector alone (Fig. 20; n=5). Stained cells showed a cytoplasmic localization of BERP similar to the results obtained with the FLAG antibodies (shown under transfection experiments in HEK cells). These results demonstrated that BERP antibodies can specifically recognize the native form of the overexpressed BERP in HEK cells. All of the above mentioned experiments confirmed the specificity of the anti-BERP antibodies. Also, these data show that anti-BERP antibodies can recognize the denatured (Western blotting) and native form (immunoprecipitation) of the protein indicating that these antibodies can be useful for immunhistochemical detection of BERP in tissue sections 78 Figure 20. Irnmunodetection of BERP protein in H E K cells transiently transfected with BERP cDNA using the D A B / A B C method. Calcium phosphate method was used for transfection. 10 pg of plasmid D N A was used /large plate (10 cm). BERP antibodies (1/500 dilution) were used to stain H E K cells transfected with BERP cDNA (a) or with empty vector (pcDNA3) (b). As shown, Staining was only detected in cells transfected with BERP cDNA (a) but not with vector alone (b) 79 b. Dis t r ibut ion of B E R P protein in the bra in Affinity purified anti-BERP antibody was used for immunohistochemical analysis of BERP expression in the brain (1:200 dilution). Results presented in Figures 21-24 were obtained from at least 3 independent experiments. Results show that BERP protein was mainly expressed in neuronal cells in the brain. Several neuronal populations in various brain regions were stained positive for BERP. This staining was blocked when anti-BERP antibodies were pre-incubated with the C-terminal peptide used for immunization. Figure 21 shows an example of blocking of the strong staining for BERP in the nucleus of the trapezoid body by preincubating the antibodies with the C-terminal peptide. These results confirmed the specificity of these antibodies and that the observed staining represent BERP protein. Figure 22 shows that prominent staining was present in several neuronal populations in the brain. Strongest staining was present in the inferior colliculus and several brain stem nuclei including the nucleus of the trapezoid body, supraoptic nucleus and reticular formation. Moderate expression was detected in neurons in the cortex, olfactory bulb and hippocampus. Faint staining was present in Purkinje cells in the cerebellum. The most interesting observation was the unique punctate staining for BERP (Fig. 21a). In some brain stem nuclei, such as the nucleus of the trapezoid body (Figure 21) and giant cells of the reticular formation (Figure 22), BERP staining was mainly present as large punctae in the cell body. In most of the other brain regions including inferior colliculus, cortex, hippocampus and olfactory bulb, BERP staining was present in the neuron soma and dendrites (Fig. 23&24). In the cell soma, staining was sometimes observed concentrated around the nucleus (Fig. 24). In some neurons in deep layers of the cortex, the staining was present as punctae near the plasma membrane as well as in the dendrites. These results may supprt 80 different roles for BERP in various neuronal populations. Also, its punctate distribution may indicate that BERP is present in large protein complexes or associated with cell organelles or vesicles. Several of the newly identified RING finger proteins, especially those involved in vesicular transport, possess punctate distributions in the soma of cells (Berteaux-Lecellier et al., '95; Mu etal., '95;Nakayama et al., '95; Burd et al, 97). 81 Figure 21. Immunohistochemical detection of BERP in the nucleus of the trapezoid body. BERP antibodies were used to detect the expression of BERP in brain sections. In brain stem, neurons in the nucleus of the trapezoid body stained strongly for BERP with a punctate pattern present in the cell body (a).To examine the specificity of BERP antibodies, BERP antibodies were preincubated with the C-terminal peptide (last 9 amino acids) before incubation with brain section. As shown, no staining was observed in the same region, indicating the observed staining (b) was BERP specific. Magnification, x 40. 82 Figure 22. BERP is expressed in several neuronal populations in the brain. Several neuronal populations were stained strongly for BERP including neurons in inferior colliculus (a), medial cerebellar nucleus (b), deep layers of the cortex (c), as well as hippocampus (d). Magnification, x 10 83 Figure 23. B E R P is expressed in several neuronal populations with a unique punctate pattern. Immunohistochemical analysis o f B E R P expression using D A B / A B C method showed that B E R P expression is mainly neuronal specific. Strong statining (punctate pattern in the soma) was present in the giant cells o f reticular formation (a). Faint staining was present in Purkinge cells in the cerebellum (b). In deep layers o f the cortex, several neurons stained with a punctate pattern mainly present close to the plasma membrane as wel l as in dendrites (c). Besides the observed neuronal staining, epithelial cells forming the ventricles also stained strongly for B E R P . In the bottom right panel staining epithelial cells o f 4 t h ventricle (d). Magnification, x 40 84 Figure 24. High power magnification of some brain stem neurons stained positive for BERP. High power magnifications (x 100) shows the punctate pattern observed for BERP in most of the brain neurons including brain stem (a-e). For comparison, no punctate staining was observed in brain stem neurons stained with cGK II antibodies (f)-Staining in most of the neurons was mainly present in the soma and dendrites (a-e). In some neurons, staining was concentrated around the nucleus (b, e). 85 III. C e l l transfection experiments. Some RING finger proteins can associate with cytoplasmic proteins and thus are present in the cytosol (Freemont, '93; Saurin et al, 96) whereas others are nuclear proteins that are associated with chromatin or found in the nucleus with a punctate pattern. Still, others such as XNF7 are cytoplasmic proteins and are capable of translocating to the nucleus at certain stages of development (Li et al, '94; Shou et al, '96). BERP is a novel member of the RING finger family and is abundant in brain tissue and is expressed in several neuronal populations. BERP contains motifs that are present in cytoskeletal elements, indicating that BERP might interact with cytoskeleton-associated proteins (Sharma et al, '95; Stossel, '93). Also, BERP has a putative nuclear localization signal sequence, indicating that BERP may be capable of translocating to the nucleus. In addition, BERP is a member of the RBCC family, a group of proteins that appear to be involved in signaling pathways that regulate cell growth and division (Reddy et al, '92; Fremont, '93). Mutations or translocations that involve the RBCC domain are usually oncogenic. To gain more insight about the role of full-length BERP as well as its various domains, cultured HEK cells as well as PC 12 cells were used for transfection experiments to study the expression of BERP and truncated forms of it. First, transfection experiments were used to examine the subcellular distribution of BERP and truncated forms of it in HEK cells. Figure 25 is a diagramatic presentation of the various constructs generated for transfection experiments. To allow for the detection of the expressed proteins, all of the generated BERP constructs were tagged at their N-terminal end with a sequence that corresponds to the FLAG epitope (Met-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) (Kodak). FLAG epitope can be recognized by M5 anti-FLAG antibodies (Kodak), and thus these antibodies were used for detection of expressed proteins. 86 R1N G finger CoiJed-coil ^ 2 2 62'115 146 285 unique C -term inus / \ B^oox •RBCC 430 620 744 P sheet A BP-like repeat) P sheet (VA D repeats) FBERP 1 ct 744 oo NBERP j. I 485 CBERP 285 744 Fig.25. C onstructsuasd for rail teansuk;Uon expedm gits. FBERP containsfuiliengthBERP. NBERP containsN-terminarmotifeintrudingRBCC dom ain and A B P-Jike repeat CBERP contains C Seminal sequences and iack the RBCC dom ain. A11 constructs were tagged w ith an N -term inalFLAG tag. FLAG tag (M DYKDDDDK ) 1. Transient expression i n H E K cells. Human Embryonic kidney (HEK) cells are useful for transient expression of desired genes because of their high transfection efficiency rate. The calcium phosphate method was used for transfection with the BERP cDNA. The full length BERP was subcloned into pcDNA3 vector as described in Materials and Methods. N-terminus FLAG-tag was inserted inframe before the first ATG in the coding sequence of BERP using PCR. M5 anti-FLAG antibodies (Kodak) were used for detection of FALG-BERP. The ABC kit was used for immuno detection of transfected BERP using a basic immunocytochemical method (Vincent et al, '94). For fluorescent staining, Texas red-conjugated secondary anti-mouse antibodies were used (IBI). To stain cell nuclei, the fluorescent DAPI staining was used. Similar results were obtained for immunostaining either with the ABC/DAB method or fluorescent staining in at least 5 independent transfection experiments. a. Dis t r ibut ion of ful l length B E R P protein in H E K cells transfected wi th B E R P c D N A . Forty eight hours post-transfection with FLAG-BERP, cells were fixed and then incubated with anti-FLAG antibodies (3 pg/ml). Figure 26 shows the detection of BERP expression in HEK cells with fluorescent staining. Staining studies showed that BERP protein is expressed with a punctate pattern mainly in the cytoplasm (Fig 26; n>5) indicating that BERP may be associated with some cellular organelles. These results are consistent with the punctate staining observed for BERP in brain neurons. In some cells a peripheral distribution of the full length BERP was observed close to the plasma membrane. In all of the transfected cells, nuclei were not stained for BERP protein. These studies indicate that the primary site for BERP action is the cytoplasm. It is still possible that stimulation of specific signal transduction pathways may induce the translocation of BERP from cytoplasm to the nucleus. 88 Figure 26. Expression of the full length BERP in HEK cells. Full length FLAG-BERP was transiently expressed in HEK cells using the calcium phosphate method. Immunofluorescent detection of expressed FLAG-BERP in HEK cells using anti-FLAG antibodies, and Texas red-conjugated secondary anti-mouse antibodies. As shown in (a), BERP is expressed in the cytoplasm (a; left), (a; right) is staining of nuclei of the same cells using DAPI. Arrow heads in (a) point at a nucleus of one cell, (b) shows the punctate pattern observed for BERP in the cytoplasm, c) is a confocal microscopy image showing the cytoplasmic localization of BERP. 89 b. Subcellular distr ibution of truncated versions of B E R P protein. The expression of the full length BERP in HEK cells showed that BERP protein was -mainly present in the cytoplasm, as described earlier, BERP contains a coiled-coil motif and a P sheet domain that are present in cytoskeletal elements (Lupas, '91&'96). The presence of these domains indicate that BERP might interact with cytoskeletal proteins. Also, BERP has a putative nuclear localization signal sequence. The RBCC domain has been implicated in oncogenesis, and a role for it in regulation of gene expression was indicated (Reddy et al. '92; Freemont, '93). The effects of truncating the RBCC domain and the unique C-terminal region on BERP localization were therefore examined. For this study, two constructs were generated as described in Materials and Methods, one containing the RBCC domain, the ABP-like repeat and the putative nuclear localization signal sequence (NBERP) and the other lacking the RBCC (CBERP). Both the NBERP and the CBERP constructs were engineered to contain a FLAG tag inserted in frame with the first ATG in the sequence. Expression of the predicted size proteins was confirmed by Western blotting using anti-FLAG antibodies (Fig. 29; n=3). i . Local izat ion of a truncated form of B E R P that contains the C- terminal region ( C B E R P ) . Immunocytochemical analysis of expressed CBERP in HEK showed that CBERP was mainly present with a punctate pattern in the cytoplasm (Fig. 27). These results are consistent with a punctate cytoplasmic localization observed with the full-length protein as well as in various neuronal populations in the brain. The punctate staining was prominent especially in areas around the cell nucleus (Fig. 27) and at specific sites near the plasma membrane that appeared as an outgrowth from the cell (Fig. 27a). The prominent punctate staining obtained with CBERP indicated that the C-terminal region of BERP may be involved in interactions with 90 cytoplasmic factors and may be responsible for the cytoplasmic punctate distribution of the full length protein. i i . Local izat ion of a truncated form of B E R P ( N B E R P ) that contains the R B C C domain. Transfection experiments showed that NBERP protein was present in the cell nucleus (Fig. 28; n>3). These results are different from those observed for the full length or the truncated version that contains the C-terminal sequences of the protein. These observations are consistent with a possible role for the RBCC domain in the regulation of nuclear events (Reddy et al, '92). These results also indicate that the full length protein may be capable of translocation to the nucleus, and mediating signals that involve regulation of gene expression. Recent investigations have shown that XNF7, a member of the RBCC family, is present in the cell cytoplasm and it translocates to the nucleus at specific stages in early embryonic development (Li et al, '94; Shou et al, '96). Thus BERP may be like XNF7, may be involved in transmitting signals at specific conditions from the cytoplasm to the nucleus. Overall, cell transfection studies in HEK cells are consistent with the cytoplasmic localization of BERP in brain neurons. Punctate distribution was observed in both HEK cells as well as brain neurons indicating the presence in large protein complexes or an association with cell organelles. These data also indicate a role for the C-terminal region of BERP in the cytoplasmic retention of BERP since truncation of this domain results in protein translocation to the nucleus. It still needs to be determined whether full length BERP is capable of translocating to the nucleus. It is worth noting that full length BERP was not detected in the nucleus in CNS neurons as well as in transfected HEK cells. Thus, if translocation of full length BERP to the nucleus do occur, then this may require the activation of certain signaling pathways which at the moment are unknown 91 Figure 27. Staining of HEK cells transiently expressing a truncated BERP (CBERP). Immunofluorescent staining of HEK-293 cells transiently transfected with a FLAG-tagged truncated BERP cDNA that contains the C-terminal region (amino acids 285-744; CBERP). CBERP was detected by anti-FLAG antibodies, followed by Texas red conjugated secondary anti-mouse antibodies, (a; top) staining of CBERP was observed in the cytoplasm, (a) also shows that CBERP accumulates at specific sites near the cell membrane (punctate), (a; bottom) nuclei of the same cells stained with DAPI. (b) CBERP staining (top) was observed around the nucleus, (b; bottom) is a light phase image of the same cells, (c) is a confocal microscopy image showing the punctate pattern observed for CBERP in HEK cells. 92 Figure 28. Staining of H E K cells transiently expressing a truncated BERP (NBERP). Immunofluorescent staining of H E K cells transiently transfected with a FLAG-tagged truncated BERP cDNA that contains the N-terminal region (amino acids 1-485; NBERP). NBERP expression was detected by anti-FLAG antibodies, followed by Texas red conjugated secondary anti-mouse antibodies, (a) shows the nuclear staining observed for NBERP. (b) shows staining of the same cells with DAPI. 93 2. Stable expression of full length and truncated BERP sequences i n P C 1 2 cells Transient expression experiments in HEK cells have shown that the full-length protein was cytoplasmic, whereas a truncated version of it that contains the N-terminus RBCC domain (NBERP) is nuclear. Immunohistochemical analysis showed that BERP was expressed in the soma and dendrites of CNS neurons (Fig. 24). The distributions of full length as well as truncated versions of BERP were examined in a neuronal-like cell line. PC 12 cells, a commonly used cell line, respond to NGF treatment and differentiate to neuronal-like cells (Greene&Tichler, '82; Halegoua et al, '91; Kaplan and Stephens, '94). The use of this cell line allows the localization of BERP and its truncated forms before and after induction of differentiation of these cells to neurons. Several members of the RBCC group of RING finger proteins possess oncogenic activity (Reddy et al, '92; Body et a/.,'94; Saurin et al, '96). Mutations in the RING finger as well as translocations of the RBCC are oncogenic (Freemont, '93; Futreal et al, '94; Miki et al, '94). Chromosomal translocations that results in the fusion of RBCC domain with other proteins is are also oncogenic (Saurin et al, '96). In summary, these data indicate that mutations or translocations of the RBCC domain can be oncogenic. In the next section, the long term effects of over-expression of BERP constructs on cultured PC 12 cells were examined. a. Generation of stably transfected PC 12 cells Cultured PC 12 cells were transfected with various BERP constructs (described earlier) using the calcium phosphate method as described under Materials and Methods. Clones were selected using 500 u.g/ml G-418 (geneticin; BRL). Ten clones expressing NBERP were obtained. Only one clone for each of the full-length or CBERP constructs was obtained. Western blot 94 analysis using anti-FLAG antibodies confirmed the expression of the proteins (Fig. 29; n=2). Full length BERP expression in the one stable cell line obtained for FBERP was very low as determined by Western blotting (Fig. 29). The expressed protein was still detectable using immunocytochemical detection method, A B C / D A B . A common problem with the generation of stable cell lines is the variability in the level of expression of the transfected protein, and thus the generation and the use of several cell lines expressing the desired protein is recommended for studying the outcome of expression of a stably expressed protein . Because one cell line was only obtained for each FBEP and CBERP, changes in the cell morphology and proliferation of these two cell lines will not be taken in consideration. The generation of several independent cell lines expressing FBERP and CBERP will be required in the future to examine the consequences of overexpression of these proteins, b. Expression of C B E R P Expression of CBERP was also present in the cytoplasm (Fig. 30). Unexpectedly, cells expressing the C-terminal region of BERP (CBERP) were differentiated to neuron-like cells. Unfortunately only one clone was obtained that express CBERP. Also, the expression of this truncated form was not confirmed by Western blotting due to the lack of enough amount of cells to perform protein extraction. This is because cells expressing CBERP were differentiating rather than proliferating. Thus, at the moment it is not clear whether the observed differentiation of PC 12 cells was indeed due to the expression of CBERP or was an artifact of prolonged culturing of that clone. Generation of other clones stably expressing CBERP will be necessary. It is worth mentioning that none of the other generated cell lines expressing the full length or NBERP behaved this way. Because of the uncertainty of the results obtained with the clone expressing CBERP, the significance of these observations will not be discussed. 95 Figure 29. Western blot analysis of expression of truncated forms of BERP. Ant i -FLAG antibodies were used for detection of expression of FLAG-tagged full-length as well as truncated versions of BERP. Several G-418 resistant PC 12 stable clones expressing a truncated form of BERP that contains the N-terminal region (NBERP) were obtained: N10, N14, N16, N17, N19, N29, N38, N46, N48. Only one clone expressing the full-length BERP (F22) was obtained. Controls include cell lysates of H E K cells transiently transfected with full-length BERP (FBERP); a truncated version of BERP that contains the N-terminal region (NBERP) or the C-terminal region of BERP (CBERP). Size of the molecular weight markers are indicated on the left hand side. 96 Figure 30. Stable expression of a truncated form of BERP (CBERP) in PC12 cells. The calcium phosphate method was used for transfecting PC 12 cells with a FLAG-tagged truncated form of BERP that contains the C-terminal region (amino acids 285-744; CBERP). Only one G418-resistant clone expressing CBERP was obtained. Expressed CBERP was detected using anti-FLAG antibodies and the DAB method. Stable expression of CBERP resulted in the differentiation of PC 12 cells (neurite formation). CBERP staining was detected in the soma and neurites of PC 12 cells. Magnification x 100. 97 c. Expression of full length B E R P Immunocytochemical analysis, showed that full length BERP was expressed in the cytoplasm (Fig. 31; n=3). These results are consistent with the results obtained in transiently transfected HEK cells. Interestingly, cells expressing the full length BERP appeared larger in size, when compared to normal PC 12 cells. The significance of this change in size is not clear at the moment. No other morphological changes were observed in that cell line. d. Expression of N B E R P Ten clones expressing the N-terminal region of BERP were obtained. These cells were small and rapidly dividing but aggregating and adhering to each other. NBERP staining was restricted to nuclei. Figure 32 shows DAB staining of 5 different clones expressing NBERP. F-actin staining (using Phalloidin) showed that the cells contained a single nucleus, but were present in large aggregates forming lace-like structures (Fig. 33; n=5). These results indicate that cells expressing the truncated NBERP may have failed to separate and migrate away from each other, and thus remained in large aggregates. The reason and the mechanism of these changes are not clear, although these observations may indicate that the truncated BERP caused cell adhesion defects. This phenotype was not observed in normal PC 12 cells or in the PC 12 cell line expressing the full length BERP or CBERP. Because NBERP contains the RBCC domain, these results indicate an important role for the RBCC motif in maintaining normal BERP function. Also, these observations indicate that overexpression of the RBCC motif may be detrimental for normal cell functioning. Northern analysis showed that PC12 cells express the endogenous BERP mRNA (Fig. 13). Whether NBERP acted as a dominant negative mutant and suppressed normal function of BERP is not known. 98 Figure 31. Stable expression of full length BERP in PC 12 cells. The calcium phosphate method was used for transfecting PC 12 cells with a FLAG-tagged BERP. Expressed BERP was detected using anti-FLAG antibodies and the D A B method, (a) Only one G418-resistant clone that expresses BERP was obtained. BERP was expressed in the cell cytoplasm. Magnification, x 100. 99 Figure 32. Stable expression of a truncated form of BERP (NBERP) in PC 12 cells. The calcium phosphate method was used for transfecting PC 12 cells with a FLAG-tagged truncated form of BERP that contains the C-terminal region (amino acids 1-485; NBERP). Several G418-resistant clones expressing NBERP were obtained. Expressed NBERP was detected using anti-FLAG antibodies and the DAB method. NBERP was detetected in the nucleus as shown in NBERPclones: N10, N14, N19, N29, N35. Magnification, x 10. 100 Figure 33. Expression of a truncated form of BERP (NBERP) in PC 12 cells. (a). Staining of one of the G418-resistant clones expressing NBERP (N10). Expressed NBERP was detected using anti-FLAG antibodies and the D A B method. NBERP was detected in the nucleus as shown, (b). F-actin staining using Oregon green Phalloidin. Similar results were obtained for N19, N29 and N35. Magnification, x 100. 101 e. N G F treatment of stably transfected P C 12 cells Stably transfected PC 12 cells express NBERP in the nucleus. As described earlier, these cells form large aggregates indicating that the truncated BERP (NBERP) may interfere with the normal function of endogenous BERP. To examine if the truncated BERP (NBERP) exerts any effect on the normal growth and differentiation of PC 12 cells induced by NGF, three of the NBERP cell lines were treated with NGF for 4-7 days. Controls, including a cell line that expresses the full length, a cell line that express the neomycin resistance gene (pcDNA3 vector) as well as normal PC 12 cells were also exposed to NGF treatment. Similar results to those presented in Figure 34 were obtained from at least 5 independent experiments. Interestingly, cell lines expressing NBERP did not respond to NGF with differentiation and the production of neurites (Fig. 34). Instead, these cells continued to proliferate at the same rate as those without NGF treatment. The cell line that expressed the full length protein, or empty vector, as well as normal PC 12 cells responded to NGF treatments and developed long processes. Immunostaining of NGF treated PC 12 cells expressing full length BERP showed that BERP was present in the cell cytoplasm and in the processes. Again, NBERP was exclusively expressed in the nucleus (Fig. 35). These studies indicate that induction of neuronal differentiation does not result in the translocation of the full length protein to the nucleus. Also, NGF treatment did not change the localization of the truncated BERP (NBERP). In general, these results indicate that the full length BERP may be involved in processes that determine neuronal differentiation and neurite outgrowth and truncation of the protein (as observed with NBERP) may inhibit normal neuronal differentiation. Also, these results indicate that the truncated BERP may be acting as a dominant negative mutant competing with and knocking out wild-type BERP activity. 102 Figure 34. A truncated form of BERP inhibits NGF-induced neurite outgrowth in PC 12 cells. Results obtained seven days after NGF treatment (50ng/ml) of PC 12 cells and stable cells expressing full length BERP (F22) or a truncated form of BERP (N10, N19, N45) that contains the N-terminal RBCC domain (NBERP) (a-f). NGF-induced neurite outgrowth in untransfected PC 12 cells (a), a stable cell expressing empty vector (b), and a stable cell line expressing full length BERP (c). Neurite outgrowth was inhibited in cell lines expressing NBERP: N10 (d), N19 (e), and N45 (f). Magnification, x 10. 103 b * .1 *' * * • • * Figure 35. Staining of NGF-treated PC12 cells expressing full length as well as a truncated form of BERP. Cell were treated with 50 ng/ml N G F (7days). Expressed BERP and the truncated form, NBERP were detected using anti-FLAG antibodies and the D A B method. After NGF treatment. BERP was detected in the cytoplasm (a), whereas N B E R P was detected in the nucleus (b). High power magnification showing that lull length BERP is detected in the cytoplasm of NGF-treated cells (c). B E R P was present in the soma as well as in neurites (c). Magnification. \ 10 for (a&b): x 100 for (c). 104 I V . Yeast two-hybr id analysis: The yeast two-hybrid system is a sensitive in vivo technique for identifying proteins that interact with a protein of interest (Fields and Song '89, Chien et al, '91; Guarente '93, Luban and Goff '95). Cellular protein-protein interactions including membrane bound, cytosolic and nuclear proteins are readily detectable using this system (Vojtek et al, '93; Song et al, '94; Sato et al, '95b; Boldin et al, '95). Several members of the RING family have already been identified using the yeast two hybrid system (Rothe et al, '94, Song and Donner '95; Sato et al, '95, Le Douarin, '95, Mosialos et al, '95). Figure 36 is a diagrammatic representation of the different BERP baits used for yeast two hybrid analysis. 1. Us ing the yeast two-hybrid system to identify proteins interacting wi th the R B C C domain a. Background Members of a subgroup of RING finger proteins, the RING-B box-Coiled-Coil" (RBCC) group (Saurin et al, '96) contain an N-terminal RING finger followed by one or two additional zinc-binding domains (B box motifs), and a coiled-coil motif. In addition to the RBCC domain, members of the RBCC group contain a variable C-terminal region indicating that this region may be involved in interactions specific to each member of the group (Reddy & Etkin, '91; Reddy et al, '92; Freemont '93). Although several members of the RING finger family have been identified, the actual mechanism by which the RING finger motif functions is unknown (Freemont '93). The RING finger domain is a highly conserved region among members of the RING finger family (Freemont '93; Fig 1). Zinc fingers are identified in many DNA binding proteins such as steroid receptors, GATA and GAL4 (Tsai and O'Mally '94; Klug and Schwabe 105 '95). Recent data have also shown several signal transduction proteins with zinc fingers such as the protein kinases B-raf, protein kinase C, and LIM kinase (Schmeichel and Beckerle '94; Lehel et al, '95; Dawid et al, '95; Klug and Schwabe '95). In addition, zinc finger binding domains are present in actin binding proteins and focal adhesion molecules (Zyxin and paxillin; Dawid et al, '95). Spacing between the zinc-binding ligands in the RING finger motif is similar to the LIM domain zinc fingers, a protein-protein interaction domain (Schmeichel and Beckerle '94; Dawid et al, '95; Hildebrand et al, '95; Schaller et al, '95). Also, a group of RING finger proteins are associated with vacuoles (PEP3; Robinson et al, '91, PEP-5; Woolford et al, '90) and perixosomes (PAF-1; Tsukamoto et al, '91; PAS7p; Kalish et al, '95). In addition, mutations in the RING finger domain of PML results in changes in its distribution from specific nuclear bodies to a diffused nuclear localization (Borden et al, '95). Also, by deleting the RING finger motif of TRAF2, this protein looses its ability to activate N F K B (Rothe et al, '95). These results support a role for the RING finger in protein-protein interaction. Following the RING finger- B box motif, BERP contains a coiled-coil domain (amino acids 147-328). This coiled-coil structure is present in several members of the RING finger family as well as cytoskeletal proteins. Cytoskeletal proteins such as actinin contain coiled-coil domains. In these proteins, the coiled-coil domain is involved in homodimerization. Because the RBCC motif is present as a unit in several RING finger proteins it was interesting to identify putative function(s) for this domain. To use this domain in a yeast two hybrid screen, PCR primers with restriction sites (Ncol/ EcoRI) were designed to amplify sequences that contain a region that corresponds to the RBCC domain (amino acids 1-372). As described earlier, amino acids 1-285 contains the full RBCC domain. 106 Yeast cells (Y190 strain) were first transformed with the pAS2-l vector containing the RBCC bait. Cells were plated on agar plates containing the required amino acids for yeast growth but lacking tryptophan (-Tip) to select for those tranformed with the pAS2-l vector. Control experiments were carried out to examine if RBCC bait can autoactivate the reporter genes his and lacZ. Control experiments included co-tranforming the bait either with pACT2 vector, which contains the GAL4 activation domain (AD) alone or with pTD-1 vector, which contains GAL4 AD/SV40 large T antigen protein and were plated on -Leu/-Trp plates as well as -Leu/-Trp /-His + 25 mM 3-AT plates. After 3-4 days of incubation, the plates were examined for yeast growth and for P-gal activity to test for the activation of reporter genes. The RBCC bait alone or in the presence of pACT2 or pTD-1 failed to activate the reporter genes. These results indicate that the RBCC bait is not capable of autoactivation of the reporter genes and thus can be used for yeast two hybrid screening. Yeast colonies obtained from transformation with the pAS2-l/RBCC bait alone were grown in a liquid medium lacking Trp and used to generate yeast competent cells for cDNA library screening using the LiAc method as described in the methods. Large scale transformation experiments were used to screen a rat brain cDNA library subcloned into the pGADIO vector (Clontech). Transformed yeast cells were plated on triple dropout medium lacking Trp, Leu and His + 25 mM 3-AT to select for those transformants that contain the pAS2-l/RBCC bait vector and the library pGADIO vector containing cDNA inserts. Twenty five mM 3-AT was included in the media as recommended by the supplier (Clontech) to suppress background growth due to the leakiness of the expression of the reporter his gene in the yeast strain Y190. 107 R I N G finger Coiled-coil ^ 2 2 62 tT5 \46 2^5 / \ B-box R B C C l unique C-tenninus 430 620 P sheet p sheet ( V A D repeats) (ABP-like repeat) o CO R B C C 1 R B C C 2 A B P L C B E R P 1 C B E R P 2 C B E R P 3 Figure 36. Schematic representaion of various constructs used as baits for the yeast two hybrid assay. b. Positives obtained wi th R B C C domain as a bait Initial screening of 6 x 106 transformants generated 100 positives that were able to grow on the triple dropout media lacking Leu, Trp, and His amino acids and were able to activate the Lac Z reporter gene as determined by the p-gal filter lift assay. Upon retransforming yeast cells carrying the pAS2-l vector that contained RBCC bait, only two of the positives examined were capable to activate the reporter genes (Fig. 37; n=5). Sequence analysis of those two positives showed that they carried a library plasmid that contained a sequence identical to the full length cDNA of a non-muscle isoform of alpha actinin (3200 bp; Genbank accession# U19893). The positive that possesse the full length alpha actinin cDNA contains 36 nucleotides of the 5' untranslated region that were inserted inframe with the GAL4 activation domain. Open reading frame analysis shows that the presence of a 36 bp of the 5' UT region of alpha actinin cDNA results in the generation of an extra 12 amino acids inserted inframe with the first ATG of alpha actinin coding region. Thus, this positive results in the generation of a fusion protein containing GAL4 activation domain and a full-length alpha actinin protein. This isoform of alpha actinin has been previously identified in rat astrocytes (Geizert et al., '96). 2. Using the yeast two-hybrid system to identify proteins interacting wi th B E R P C -terminal region, a. Background As described earlier, the C-terminal region of BERP is unique. HT2A is the only mammalian protein with homology to that region of BERP (Fridell et al, '95). HT2A had been identified in a yeast two-hybrid screen as an interactor of the HIV regulatory protein Tat (Fridell et al, '95). Still, the cellular function of HT2A is not known yet. To identify proteins that may 109 interact with the C-terminal region of BERP, a bait that contains BERP amino acids 383-744 was designed and will be called CBERP 1 (see Table 1). To generate this bait, PCR was used to amplify sequences that correspond to amino acids 383-744. PCR primers were made to contain NcoI/EcoRI restriction sites to allow directional subcloning of the generated PCR product into the pAS2-l vector inframe with the GAL4 DNA BD. Similar control experiments to those described for the RBCC bait were done to test if the CBERP 1 bait can autoactivate the reporter genes. Results obtained (data not shown) showed that CBERP bait was not able to independently activate reporter genes and thus can be used for library screening. b. Positives obtained with the C-terminal bait (CBERP1). Yeast initially transformed with the pAS2-l/CBERP 1 bait were used to generate competent cells using the LiAc method as described earlier. Sequentially, the competent yeast were used for large scale transformation experiments with the rat brain cDNA library (Clontech) to screen for putative interacting proteins. After screening of about 15xl06 clones, two positives were obtained. Plasmids obtained from these positives were introduced in E. Coli and analyzed with restriction enzymes to determine insert size. Both positives contained identical insert size (2200 bp insert). The library plasmids containing the inserts were used to re-transform yeast cells that already contain the pAS2-l/CBERP 1 bait, plated on -leu/-trp plates as well as -leu/-trp /-his + 25 mM 3-AT plates to test for activation of reporter genes. Results showed that both plasmids were able to activate the yeast reporter genes in the presence of pAS2-l/CBERP 1 bait and thus confirmed that these plasmids represented the true positives obtained from library screening. The plasmid inserts were then sequenced using pGADIO forward primer supplied with the MATCHMAKER kit (Clontech). Sequence analysis showed that both positives 110 represent the same C-terminal region (encodes the last 641 amino acids; Genbank accession # RNU60416)of a recently identified class V unconventional myosin known as myr 6 (Zhao et al, '96). The sequence of myr 6 C-terminal region (c-myr 6) was inframe with GAL4 activation domain (AD), confirming that the fusion protein that would be generated from this plasmid will contain a GAL4 AD/c-myr 6 fusion protein. Figure 37 shows that using the p gal filter lift assay, alpha actinin interacted specifically with N-terminal region of BERP (RBCC2; n=5) whereas myr 6 interacts specifically with BERP C-terminal region. 3. Analysis of various domains of B E R P for interaction wi th m y r 6 and actinin For this study, beside the described RBCC bait and the C-terminal bait several other baits were generated by PCR, subcloned into pAS2-l vector and were tested in the yeast two-hybrid system to identify the regions of BERP interacting with actinin and myosin. The different baits generated are described in Table 1 and summarized in Figure 36 (n=5). a. Interaction wi th alpha actinin Among all of the various baits examined only the bait that contains the full length RBCC domain and half of the ABP-like repeat was able to interact with alpha actinin (Fig. 38; n=5). These results indicated that neither the C-terminal domain nor the ABP-like repeat alone are capable of interaction with alpha actinin. Also, a bait that contained the RBCC domain but lacked the last twenty amino acids of the coiled-coiled motif failed to interact with oc-actinin, indicating that a full length RBCC domain and the sequences in the first half of the ABP-like repeat were required for interaction with alpha actinin. i l l b. Interaction wi th myr 6 The various baits described above were also used to determine sequences of BERP that can specifically interact with myr 6. All the various C-terminal baits examined were able to interact with myr 6 C-terminal region (Fig. 39; n=5). Neither of the RBCC containing baits nor the ABP-like repeat region were able to interact with myr 6 -C-terminal domain. Interestingly, only the last 120 amino acids was sufficient for interaction with myr 6 C-terminal region. 112 Figure 37. Positives identified using the yeast two-hybrid system: alpha actinin and a class V myosin (myr 6). The yeast two-hybrid system was used to screen a rat brain cDNA library in yeast cells previously transformed with baits containing various domains of BERP. (3 galactosidase filter lift assay was used to identify yeast two hybrid interactors in yeast, (a) represents results obtained from streaking transformed cells on -leucine/-tryptophane agar plates, (c&d) represent results obtained from streaking transformed cells on -leucine/-tryptophane/-histidine +25 mM 3-AT agar plates. N-terminal region of BERP ( amino acids 1-372) does not interact with myr 6 (a), but it interacts with actinin (b). In contrast, BERP C-terminal region (383-744) does not interact with actinin but it interacts with myr 6 (d). 113 Figure 38. Yeast two hybrid analysis shows that the N-terminal region of BERP interacts with alpha actinin. The yeast two-hybrid system was used to screen a rat brain cDNA library in yeast cells previously transformed with a bait that contains BERP amino acids 1-373. P galalctosidase filter lift assay was used to identify yeast two hybrid interactors in yeast. Filters shown represent results obtained from streaking transformed cells on -leucine/-tryptophane agar plates, a-actinin specifically interacts with the N-terminal region of BERP (8). a-actinin interaction with the N-terminal region of BERP was abolished by deleting amino acids 262-372 (1). l=actinin/pGAD10+BERP aa l-262/pAS2-l 2=actinin/pGAD10+BERP aa 280-459/pAS2-l 3=actinin/pGAD10+BERP aa 383-744/pAS2-l 4=actinin/pGAD10+HT2A aa 5=actinin/pGAD 10+pAS2-1 6=actinin/pGAD10+pLAM5 (a control plasmid containing lamin C) 7=actinin/pGAD10+pVA3-l (a control plasmid containing SV40 large T antigen) 8=actinin/pGAD10+BERP aa l-372/pAS2-l 114 Figure 39. Specifc interactions of various domains of BERP with alpha actinin and a class V myosin (myr 6) as determined by yeast two-hybrid analysis. The yeast two-hybrid system was used to identify regions of BERP that interacrt with alpha actinin as well as myr 6. p galactosidase filter lift assay was used to identify yeast two-hybrid interactors in yeast. Filters shown represent results obtained from streaking transformed cells on -leucine/-tryptophane agar plates (left panel) and on leucine/-tryptophane/-histidine +25 mM 3-AT agar plates (right panel). The N-terminal region of BERP (amino acids 1-372) interacts with actinin (11). C-terminal sequences (amino acids 383-744 as well as 629-744) interact with the C-terminal region of the rat myr 6 (8&10 respectively). Results show that BERP amino acids 629-744 are involved in self oligomerization (9). Left and right Panels l=myr6/pGAD 10+pLAM5 2=BERP aa 383-744/pAS2-l+pTD-l 3=BERP aa 383-744/pAS2-l+actinin 4=myr6/pGAD 10+pAS2-1 5=BERP aa 383-744/pAS2-l+BERP aa 629-744/pACT2 6=BERP aa 383-744/pAS2-l+pTD-l 7=BERP aa 383-744/pAS2-l+actinin/pGAD10 8=BERP aa 383-744+myr6/pGAD10 9=BERP aa 629-744/pAS2-l+BERP aa 629-744/pACT2 l O B E R P aa 629-744/pAS2-l+myr6/pGAD10 11=BERP aa 1- 372/pAS2-l+actinin/pGAD10 12=BERP aa 1- 372/pAS2-l+myr6/pGAD10 13=myr6/pGAD10 alone 115 4. Us ing the yeast two hybr id to examine B E R P oligomerization a. Background i . A role for R B C C domain in protein oligomerization The function of the RBCC domain present in several RING finger proteins is not known yet. Accumulating evidence indicates that the RING finger domain is possibly a protein-protein interacting motif rather than a DNA interaction motif. For example, mutations of the PML RING finger domain result in a diffused nuclear staining and loss of the nuclear bodies observed with wild type protein (Borden et al, '95). Also, TRAF2 lacking the RING finger motif fails to activate latent cytoplasmic transcription factor NF-KB (Rothe et al, '95a). Mutations of the two RING finger motifs of the yeast protein VAC1 (Burd et al, '97) result in protein missorting and deletion of the RING finger-B box domain of KAP-1 resulted in the failure of KAP-1 to interact with its partner KRAB (Friedmann et al, '96). Altogether, these observations support a crucial role for the RING finger as well as the RBCC domain in mediating protein-protein interactions. Members of the TRAF family can form homo- and heterodimers, interact with several cytoplasmic proteins including IAPs, TRADD, TANK and are involved in clustering of various cytokine receptors (Rothe et al, '95a&b; Cheng and Baltimore, '96; Hsu et al, '96). Also, the post synaptic density protein 43K is involved in clustering acetylcholine receptors (Bloch et al., '94). The nuclear steroid receptor complex contains TIF1, whereas chromatin associated complexes contain XNF7, MSL2 and BRCA1 (Li et al, '94; Kelly et al, '95; Scully et al, '97; Wang et al, '97). A l l of these observations indicate a role for the R I N G finger as well as the R B C C domain in mediating protein-protein interactions. ii. A role for B E R P C-terminal region in protein oligomerization BERP contains a central domain that shares homology with repeats present in the actin binding proteins, ABP-120 and ABP-280 (Noegel et al, '89; Stossel, '93). Previous data showed that the C-terminal regions of ABP-120 and ABP-280 containing these repeats are involved in protein dimerization (Noegel et al, '89; Fucini et al, '97). Also, secondary structure analysis of BERP using the nn predict program predicts that the rest of BERP C-terminal region also has a beta strand conformation. A similar structure is predicted for the C-terminal region of the TRAF domain present in CRAF1 and TRAF2 (Rothe et al, '94; Cheng et al, '95). Recent investigations showed that the TRAF domain is required for protein dimerization. Thus besides the indicated role for the RBCC motif in protein oligomerization, it is possible that BERP ABP-like repeat as well as its C-terminal region are involved in protein oligomerization. F o r these reasons it was tempting to test i f various domains present in B E R P are involved in protein oligomerization. b. B E R P oligomerization as determined by the yeast two-hybrid system Various baits and their targets were designed to test this hypothesis. The PCR primers used to amplify specific BERP sequences for the generation of various baits and targets are described in Table 1 and Figure 36. PCR products were subcloned into pAS2-l and pACT2 vectors to be used as baits and targets respectively, in a yeast two-hybrid assay. Each pair of bait and target constructs were co-transformed into the yeast strain Y190 and tested for their ability to activate the reporter genes his and lacZ. Figures 39 and 40 show some of the results obtained with the P gal filter lift assay. Table 3 summarizes the results obtained from the P-gal filter lift assay of the various yeast two-hybrid interactions examined (n=5). 117 The following table summarizes the results obtained from these experiments. BaitYTarget RBCC1 RBCC2 ABPL CBERP2 CBERP3 PTD-1 RBCC1 HIS+/Blue HIS+/Blue . . . . RBCC2 HIS+/Blue HIS+/Blue . . . . ABPL . . . . . . CBERP1 . . . . . . CBERP2 . . . . . . CBERP3 . . . . HIS+/Blue PVA3-1 . . . . . HIS+/Blue PLAM5 . . . . . . Table 3. Summary of the results obtained from co-transformation experiments of various baits and targets i n a yeast two-hybrid assay. All baits were subcloned inframe with GAL4-DNA binding domain in pAS2-l, whereas all the targets were subcloned inframe with the GAL4-activation domain in pACT2. His+ indicates activation of the his gene as determined by positive growth on -leu/-trp/-his +25 mM 3-AT plates. Blue indicates activation of the lacZ gene as determined by the P-gal filter lift assay. Constructs are RBCC1 (amino acids (a.a.) 1-372, RBCC2 (a.a. 1-262), ABPL (a.a. 285-495), CBERP 1 (a.a. 383-744), CBERP2 (a.a. 459-744) and CBERP3 (a.a. 620-744). Control plasmids including pVA3-l, pTDl and pLAM5 are described in Table 1. Also, all transformation products were plated on -leu/-trp plates to confirm plasmids co-transformations. 118 Figure 40. Yeast two-hybrid analysis shows that the N-terminal region of BERP is involved in protein oligomerization. The yeast two-hybrid system was used to identify regions of BERP that possibly involved in self oligomerization. p galactosidase filter lift assay was used to identify yeast two-hybrid interactors in yeast. Filters shown represent results obtained from streaking transformed cells on -leucineA-tryptophane agar plates. Results show that the N-terminal region of BERP (amino acids 1-262) can self-associate (7&8). 1=BERP aa l-372/pACT2+aa 383-744/pAS2-l 2=BERP aa l-372/pAS2-l+pACT2 3=BERP aa l-372/pAS2-l+pTD-l 4=BERP aa l-372/pACT2+pAS2-l 5=BERP aa l-372/pACT2+pVA3-l 6=BERP aa l-372/pAS2-l+actinin/pGAD10 7=BERP aa l-372/pAS2-l+aal-372/pACT2 8=BERP aa l-372/pAS2-l+aa l-262/pACT2 119 Results show that RBCC1 Bait was able to interact with RBCC1 and RBCC2 targets (Fig. 40; n=5). Also, RBCC2 bait was able to interact with RBCC1 and RBCC2 targets. RBCC1 and RBCC2 baits and targets failed to interact with control plasmids or with other baits and targets containing other regions of BERP. Also, results show that the C-terminal region BERP (CBERP3) was able to interact specifically with the target bait CBERP3. These results indicate that the RBCC domain (a.a. 1-372) may be involved in BERP oligomerization. Actually a.a. 1-262 were enough for this interaction. Amino acids 1-262 were able to interact with constructs containing the same region but not others containing the ABP-like central region or the C-terminal region of BERP indicating that the interaction was specific to N-terminal amino acids 1-262. These results also indicate that a complete coiled-coil domain was not required for this interaction. These results support a role for the RBCC domain in protein-protein interaction and protein oligomerization. Interestingly, the last 120 amino acids of BERP C-terminal region (CBERP3) were also capable of dimerization, indicating a role for the C-terminal region of BERP in protein oligomerization (Fig. 39; n=5). Surprisingly, the larger bait CBERP 1 that also contains the last 120 amino acids, failed to interact with CBERP3 (Fig. 39). The reason for this is not clear but the presence of other C-terminal sequences in association with the last 120 amino acids of BERP inhibited the oligomerization of the C-terminal region. Thus whether the C-terminal region is indeed involved in protein oligomerization has to be confirmed by other methods. Also, although the repeats present in the C-terminal region of ABP-120 and ABP-280 have been shown to be involved in protein oligomerization, the ABP-like repeat present in BERP failed to oligomerize. It is possible that this region requires other sequences in BERP for proper 120 folding and oligomerization. Also, these results may indicate that the ABP-like repeat may be involved in heterodimer rather than homodimer formation. 5. Using the yeast two hybrid to test for interactions between BERP and the HIV regulatory protein Tat Previous research has identified a mammalian RING finger protein, HT2A, a member of the RBCC family that specifically interacted with the HIV protein, Tat. Tat interacts with the last 120 amino acids of HT2A. Interestingly, HT2A is the only mammalian RING finger protein that possess an overall 35% identity to BERP including its unique C-terminal region. Thus, it was important to determine if BERP C-terminal regions can also interact with Tat. Full length Tat in a vector that contains the GAL4 binding domain as well as the C-terminal region of HT2A in the GAL4 activation domain-containing vector were obtained from Dr. Cullen, BR (Duke University). Because the obtained Tat cDNA was in a vector that contains the his gene (Friddle et al, '95), we could not use His selection to screen for interaction with any of the various domains of BERP. We were only able to assess interactions using the B-gal filter lift assay. P-gal filter lift assays indicated that none of the BERP constructs used in yeast two-hybrid screens, including the bait containing the last 120 amino acids (CBERP3), were capable of activating the reporter gene Lac Z. On the other hand, a construct that contained the last 120 amino acids of HT2A (Fridell et al, '95) was used as a positive control and tested positive with the P-gal assay (Table 4; n=3). 121 BaitATarget HT2A Tat HT2A\AD N/D + Tat\GAL4 BD + N/D RBCC2 (a.a. 1-372) ABPL (a.a. 280-459) CBERP2 (a.a. 459-744) CBERP3 (a.a. 620-744) Table 4 summarizes the results obtained from P gal filter lift assays. 122 Thus these results indicated that although BERP shares -35% identity with HT2A, BERP failed to interact with Tat, indicating sequences for interaction of HT2A with Tat may not be conserved in BERP. These results also indicate that the C-termini of BERP and HT2A may serve different functions. V . Using immunoprecipitat ion to identify proteins interacting wi th B E R P 1. Background Although the yeast two-hybrid system is a very sensitive tool, false positives are not uncommon (Fields and Sternglanz '94; Luban and Goff '95). Therefore, another method confirming interactions is required. Immunoprecipitation is widely used to detect and confirm protein-protein interactions (Rothe et al, '94; Sato et al, '95; Sharma et al, '95; Fisher et al, '95; Song and Donner '95; Mosialos et al, '95). Tagging proteins of interest with epitope tags is a very convenient and widely used method for the localization and immunoprecipitation of various signal transduction proteins including membrane bound (Yu et al, '95; Ashworth et al, '95), cytosolic (Whiteway et al, '95; Guan and Butch '95; Yamauchi et al, '95b), vesicle (Grote et al, '95) and Golgi (Peters et al, '95; Lehel et al, '95) associated, as well as nuclear proteins (Zhu et al, '95; Zhao et al, '95). The FLAG epitope tag has been previously used to localize and immunoprecipitate FLAG-tagged proteins (Chiang et al, '93; Gerard and Gerard '94; Vigna et al, '94; Lee and Hruby '95; Lee and Culbertson '95; Shi et al, '95; Kuusinen et al, '95; Otto et al, '95; Yamauchi et al, '95; Smit et al, '95; Kawase et al, '95; Mosialos et al, '95). In our study, we have introduced a FLAG epitope to the amino terminus of BERP protein. In the following experiments we used anti-FLAG antibodies to co-immunoprecipitate the FLAG-tagged BERP and associated proteins. 123 2. Construct preparation A FLAG-tagged BERP in pcDNA3 was generated as described earlier under cell transfection experiments. Alpha actinin cDNA, obtained from yeast two-hybrid library screening, was digested with BamH I/Xho I and subcloned into the mammalian expression vector pcDNA3. PCR was used to generate C-terminal region of myr 6, (nucleotides 3830-5719; same region identified by yeast two-hybrid screen). Also, the amplified myr 6 was designed to contain an N-terminus HIS tag (6 histidines) to allow detection with anti-HIS antibodies (Clontech). The generated HIS-tagged myr 6 was then subcloned into pcDNA3. Expression of the correct size product was first examined using in vitro translation (TnT kit; Invitrogen). Using 35S-labeled methionine to visualize in vitro translated products, a ~100 kDA product that correspond to full length alpha actinin was obtained (Fig. 41; n>5). The size of the in vitro translated alpha actinin was similar to that reported previously (Geizert et al, '96). Also, the predicted size in vitro translated product was obtained for BERP (~82 kDA; Fig. 41). Three major products were generated for myr 6, the largest one (~70 kDA) corresponds to the predicted size of subcloned myr 6. The other smaller bands (65 kDA and 63 kDA) could be generated from another two inframe ATGs present downstream in the sequence. Thus, in vitro translation experiments confirmed that the constructs generated for alpha actinin and myr 6 can produce the expected size proteins. In the next section, the use of these constructs for transfection experiments and immunoprecipitation will be described. 124 190 Figure 41. In vitro translation products of alpha actinin, BERP and myosin (myr 6). TnT-coupled in vitro trancription\translation kit (Promega) was used to confirm the expression of a-actinin cDNA, BERP cDNA and myr6 C-terminal tail. All of the cDNA inserts were subcloned in pcDNA3 vector. One microgram of plasmid DNA was used for in vitro translation. 3 5 S -labeled methionine was used in the reaction to allow detection by autoradiography. In vitro translated samples were run on 8% polyacrylamide gels. Gels were dried and exposed to X-ray films. As predicted for actinin a -100 kDa band was generated. Also, BERP was expressed with a predicted size product of -82 KDa. Myr6 was expressed as 3 bands, the top one corresponds to the predicted size of myr6-C-terminal region (-69 kDa). The other two bands could be generated from 2 inframe ATGs present at the 5'end of the insert. 125 3. A l p h a actinin immunoprecipitat ion For immunoprecipitation experiments a-actinin constructs were co-transfected transiently in HEK cells with a FL AG-tagged BERP cDNA (6 pg of each plasmid/10 cm plate). Forty eight hours post-transfection HEK cells were lysed in 600 pi lysis buffer containing 0.1 or 1% NP-40. Cell lysates (300 pi)were then incubated for 1 hour with 6 pg of either anti-FLAG antibodies or 10 pi of anti-BERP antibodies. Forty pi of Sepharose A beads were then added and the mixture was incubated for another 1 h at 4°C. The beads were then washed twice with the detergent-containing buffer and then twice with detergent-free buffer. SDS-sample buffer was added to beads, boiled for 3 min, and immunoprecipitated proteins were run on SDS-PAGE gel, transferred into a nitrocellulose membrane, and then incubated with anti-FLAG antibodies to detect immunoprecipitated BERP and with alpha actinin antibodies (Sigma) to detect a-actinin. As shown in Figure 42, the available a-actinin antibodies (1:250 dilution) detected two major but weak bands in cells transfected with a-actinin cDNA. Cells transfected with BERP cDNA but without actinin cDNA, contained one single protein -110 kDa in size (n=2). When anti-BERP antibodies (Fig. 42; n=2) as well as anti-FLAG antibodies (Fig. 43; n=2) were used for immunoprecipitation, a -110 kDA protein was detected with the alpha actinin antibodies (Sigma). Several closely related a-actinin isoforms that range in size from 100-110 kDa have been previously reported (Fukarni et al, '92; Kato et al, '96; Wyszynski et al, '97; Mukai et al, '97). Thus, these results indicated that the endogenous rather than the transfected alpha actinin was co-immunoprecipitated with BERP. Several other non-specific bands were detected in all of the immunoprecipitated products including those immunoprecipitated with antibodies that do not recognize BERP (e.g. HIS antibodies). Thus, antibodies specific to the alpha actinin isoform may be required to confirm BERP interaction with alpha actinin. 126 IP C r u d e lysates (BERP Abs) (His Abs) 112 86 70 59 o O a_ z: a. 1 — d 1 + + c c c. c ti o o 0 + + Q_ Q_ I i i c; 1—1—1 CD L U CQ } m Cr: + D co Q_ c: L U CQ + CD c c o o + Q_ a. L U CQ CD c O D + CD O D D CO CO CO > >- >-y y y L U L U L U IE X X Actinin B E R P Figure 42. Co-immunoprecipitation of BERP and alpha actinin using anti-BERP antibodies. Anti-BERP antibodies (10 pi) were used to immunoprecipitate (IP) BERP from H E K cell lysates transiently expressing BERP and actinin cDNAs. IP products were detected by western blotting using actinin antibodies (top) and anti-BERP antibodies (bottom). Results show that two bands were detected for alpha actinin in crude lysates of H E K cells expressing BERP and alpha actinin. Only one band was detected for a-actinin in H E K cell lysates expressing BERP alone (top band; ~110 kDa) suggesting that the top band represents endogenous a-actinin whereas the lower band (-100 kDa) represents transfected a-actinin. IP experiments show that a -110 kDa protein that corresponds to endogenous a-actinin immunoprecipitates with BERP only in the presence of low concentration of detergent (0.1% NP40). Neither endogenous nor transfected alpha actinin were detected in IP reactions when anti-HIS antibodies were used for IP. 127 0 c bs E < E CO o / — < I o CO L l _ Q_ g _ g _ C c c o o t3 o o o + + + Q_ Q_ Q_ or o: Ci LU LU LU CD CO CO 1 2 3 112 — 86 4 5 - < 2 Actinin B E R P Figure 43. Co-immunoprecipitation of BERP and actinin using anti-FLAG antibodies. Anti-FLAG antibodies (6 pg) were used to immunoprecipitate (IP) FLAG-BERP from HEK cell lysates transiently expressing BERP and alpha actinin cDNAs. IP products were detected by western blotting using alpha actinin antibodies (top) and anti-FLAG antibodies (bottom). Results show that two bands were detected for alpha actinin in crude lysates of HEK cells expressing BERP and actinin (Extract; lane 5). IP experiments show that a ~110 kDa band that corresponds to endogenous actinin IP with BERP when low concentration of detergent (0.1% NP40; lane 1) was used. Neither endogenous nor transfected were detected in IP reactions when non-immune serum or anti-HIS antibodies were used for IP (lane2,3). No actinin was detected in IP reactions using 1% NP40 or in HEK cell lysates transfected with FLAG-BERP cDNA alone. 128 4. M y r 6 immunoprecipitat ion. Similar procedure to the one described for the immunoprecipitation of alpha actinin were followed. In transfection experiments, both FLAG-BERP and HIS-tagged C-terminal tail of myr 6 (HIS-myr 6) were expressed in HEK cells. As shown in Figure 44, myr6 co-immunoprecipitate with BERP from HEK cell lysates co-transfected with a FLAG tagged-BERP and a HIS-tagged- myr 6 (n=3). Stronger signal was detected for His-myr 6 in the presence of low concentrations of detergent (Fig. 44). The weak signal for the immunoprecipitated myr6 indicate that the interaction between BERP is probably a weak transient interaction. Another possibility is that strong interaction between BERP and myr 6 may require the formation of protein complexes that probably are not expressed in HEK cells. Anti-myr 6 specific antibodies will be required to confirm this interaction in the brain. 5. B E R P co-immunoprecipitates wi th rat dilute Our transient transfection experiments showed that myr6 c-terminal tail domain co-immunoprecipitates with BERP. Dilute, a class V myosin, is closely related to myr 6 (Merecer et al, '91). Antibodies against dilute are available (a gift from Dr. Bridgman, PC), and thus can be used to examine if BERP co-immunoprecipitates with dilute. Dilute antibodies has been used previously to detect and immunoprecipitate the dilute protein (Evans et al, '97). Figure 45 shows that BERP immunoprecipitates with dilute but not with cGMP-dependent protein kinase I (cGKI) or II (cGK II) antibodies. Also, co-immunoprecipition experiments using dilute antibodies showed that BERP co-immunoprecipitates with dilute from cortex (n=2) as well as cerebellar extracts (n>3) (Fig. 46). These results demonstrate that an interaction between a class V myosin and BERP exists. Future work should determine if BERP co-localizes with dilute in brain cells. 129 Figure 44. Co-immunoprecipitation of BERP and myr 6 C-terminal region using anti-FLAG antibodies. Anti-FLAG antibodies (6 (xg) were used to immunoprecipitate (IP) FLAG-BERP from HEK cell lysates transiently expressing FLAG-BERP and HIS-tagged myr 6 cDNAs (C-terminal region). IP products were detected by western blotting using anti-HIS antibodies (Clontech, top panel) and anti-FLAG antibodies (Kodak, lower panel). Results show that a major band (-69 KDa) was detected for myr 6 in crude lystaes of HEK cells expressing BERP and myr6 (Extract; lane 4). IP experiments show that a 69 KDa band that corresponds to Hl-myr 6 IP with BERP when low concentration of detergent (0.1% NP40; lane 1) was used. Myr6 was not detected in IP reactions when non-immune serum was used for IP (lane2). Weak myr 6 was detected in IP reactions using 1% NP40. The antibody heavy chain (IgH) band is indicated by an arrow. 130 cn o ~ —• o oo ro o o cn •o o op - O -= O ro o Z5 4fr I - BERP+MYR6 (0.1 % NP40) IP (FLAG Abs) ro BERP+MYR6 (0.1 % NP40) IP (non immune) oo BERP+MYR6 (1 % NP40) IP (FLAG Abs) Extract CQ co m TO CQ O Figure 45. Co-immunoprecipitation of endogenous BERP and dilute from rat brain using anti-dilute antibodies. Dilute (myosin V) antibodies (10 pg) were used to immunoprecipitate (IP) dilute from rat brain tissues. Western blotting was used to detect IP products using dilute antibodies (1/1000 dilution; upper panel) and BERP antibodies (1/100 dilution; lower panel). Results show that one band was only detected for dilute (-190 KDa; lane 1; top panel) and BERP (-82 KDa; lane 1; lower panel) when dilute antibodies were used to IP dilute from rat cerebellum (CB). No IP products for dilute or BERP were detected in cerebellum (CB) or thalamus (Thai) using cGMP-dependent protein kinase type I (cGKI) antibodies (lanes 3&4) or using cGKII antibodies (lanes 4&5). 132 133 Figure 46. Co-immunoprecipitation of endogenous BERP with dilute from rat brain using anti-dilute antibodies. Dilute (myosin V) antibodies (10 ug) were used to immunoprecipitate (IP) dilute from rat cerebellum (CB), cortex (CX) as well as mouse CB. IP products were detected by western blotting using dilulte antibodies (1/1000 dilution; upper panel) and BERP antibodies (1/100 dilution; lower panel). Results show that one band was only detected for dilute (-190 kDa;top panel) and BERP (-82 kDa;lower panel) in crude cerebellar lysate. Also, similar bands were detected for dilute and BERP in rat CB and CX as well as mouse CB when dilute antibodies were used for IP. 134 190 135 V I . F I S H analysis and chromosomal mapping of the human B E R P gene. The 5'end region of human BERP cDNA (1356 bp PCR product) was used to screen a human PAC library at the Human Genome FISH Mapping Resource Centre. Southern blotting, using the human BERP cDNA (5'end ) as a probe, confirmed that the isolated PAC clone contained genomic DNA sequences that represent the BERP gene (Fig. 47; n=2). The obtained PAC clone identified BERP gene on chromosome llpl5. Figure 48 shows the results obtained for mapping the human BERP gene using the PAC clone as a probe. The regional assignment of this probe was determined by the analysis of 20 well-spread metaphases. Positive hybridization signals at 1 lpl5 were noted in >95% of the cells. Signals were visualized on both homologues in >90% of the positive spreads. The band assignment was determined by measuring the fractional chromosome length and by analyzing the banding pattern generated by DAPI counterstained image. 136 1 2 3 4 5 Figure 47. Southern blot analysis of the human BERP PAC clone. A 32P-labeled human BERP cDNA probe (~1.4 kb fragment of 5' end region of human BERP) was prepared using the random priming kit (BRL). Results show that the human cDNA probe hybridized to fragments generated from digesting the human PAC clone with BamH I (lane 1), EcoR I (lane 2), and Not I (lane 3). Control lanes (4 and 5) show the hybridization of the probe with BERP cDNA either digested with BamH I (lane 4) or digested with EcoR I (lane 5). 137 Figure 48. Chromosomal mapping of human BERP gene to chromosome 1 lpl5. A human P-l derived Artificial Chromosome (PAC) genomic clone was used to map BERP gene in the Canadian Genome Analysis and Technology Program physical mapping resource facility. Mapping was performed by fluorescence in situ hybridization (FISH) to normal human lymphocyte chromosomes counterstained with propidium iodide and DAPI. Biotinylated probe was detected with avidin-fluorescein isothionate (FITC). The PAC mapped to chromosome 1 lpl5 as shown above. 138 Discussion The RING finger family includes proteins mediating signals from cell surface receptors, nuclear receptors and transcriptional regulation to others mediating vesicular transport (Saurin et al, '96). The striking feature of the RING finger family is the presence of the RING finger, a zinc binding motif, which is present in several proteins of unrelated functions (Reddy et al, '92; Freemont, '93). This has made the search for a common role for the RING finger very difficult. The vital roles of several of the known proteins containing the finger, made this motif the focus of many researchers who are trying to define its role as well as searching for other proteins containing it. In this w o r k the identification and characterization of a novel member of the R I N G finger family, in part icular the R I N G finger-B box coiled coi l subgroup ( R B C C ) is presented. Because this novel protein is highly expressed in the brain compared to other tissues, the name Brain Expressed RING finger Protein (BERP) was given to it. A region that contains the last 394 bp of BERP cDNA was first identified by serendipity from rat cerebellum using degenerate PCR primers designed for the amplification of unrelated products. This PCR product was used to screen a rat brain library and a full length BERP cDNA (2870 bp) was obtained. This cDNA has an open reading frame of 2232 bp. This open reading frame predicts a 744 amino acids protein of -82 kDA in size. The cloned cDNA also contains a 220 bp 5'-untranslated (UT) region and 414 bp 3'UT region with a potential poly A signal near its 3'-end (nucleotides 2842-2846). Next, rat BERP cDNA was used to screen a human hippocampal cDNA library, and a clone that contains sequences that correspond to amino acids 360-744 of rat BERP was obtained. The rest of the human BERP cDNA was obtained by RT-PCR using human cerebellar RNA as a 139 template. For amplification, a forward primer was designed based on the known sequence of a human EST that contains the first ATG. The reverse primer was designed based on the sequence of the cDNA fragment obtained from human hippocampal library screening. Sequence analysis of the full length cDNA showed that the human BERP also contains an open reading frame which is predicted to generate a 744 amino acid protein of -82 kDA in size. Sequence analysis showed that BERP is highly conserved, rat BERP shares 97% amino acid identity with its human homologue. Northern blot analysis showed that rat BERP (rBERP) is expressed in the brain as a major band of -3 kb in size, consistent with the size of the full length cDNA (~2870 bp) obtained from screening rat brain cDNA library. When compared to several tissues, rat brain contained the highest amounts of BERP mRNA. Placenta and lung tissues had moderate levels, while the rest of the tissues examined either contained very small or undetectable amounts of rBERP mRNA. In addition, Northern blot analysis of BERP expression in various brain regions detected a single 3 kb band in all of the regions examined. In situ hybridization analysis showed that rBERP mRNA is high in the cortex, olfactory bulb as well as cerebellum. Human BERP was first identified by screening a human hippocampal cDNA library and later by RT-PCR and Northern blotting in human cerebellum and breast tissues. The rat BERP cDNA predicts a -82 kDA protein. This was confirmed by in vitro translation experiments as well as Western blotting on HEK cell extracts transfected with a full-length BERP cDNA. Also, polyclonal anti-BERP antibodies were generated against the last 9 amino acids of BERP sequence, and used to detect BERP expression in rat brain tissues. Western blotting showed that BERP antibodies recognized the predicted size protein (-82 kDA) in cerebellum, cortex and hippocampus. This band was blocked by preincubating BERP 140 antibodies with C-terminal peptide used for antibody generation. These results confirmed the specificity of the generated BERP antibodies. Immunocytochemical analysis showed that BERP antibodies are able to stain HEK cells transfected with BERP cDNA but not those transfected with vector (pcDNA3) alone, indicating that the antibodies can recognize BERP and can be used also for immunohistochemical studies. Immunohistochemical analysis identified rBERP in several neuronal populations throughout the brain, including cerebral cortex, inferior colliculus, hippocampus and olfactory bulb. Weak staining was present in Purkinje cells in the cerebellum. In brain stem, strong signal was detectable in the giant cells of the reticular formation and in the nucleus of trapezoid body. Strong staining was also present in neurons of the inferior colliculus as well as in deep layers of the cortex. In most of brain neurons BERP possesses a punctate labeling which was observed in the soma as well as in the dendrites. In few brain stem nuclei such as the giant cells of the reticular formation as well as the nucleus of trapezoid body, a strong punctate labeling was only present in the cell body. In addition, epithelial cells lining the ventricles stained strongly for BERP. CRAF1, a member of the TRAF family shares an overall structural similarity with BERP (Cheng et al, '95), although no sequence similarities between BERP and TRAFs have been observed. TRAF proteins are thought to form homo- and heterodimers. Also, TRAFs are involved in the clustering of cytokine receptors required for their activation. These structural similarities indicate that BERP like TRAF proteins, can form homo- and heterodimers and is involved in protein clustering and the formation of large protein complexes. Localization experiments showed that BERP is expressed in a punctate pattern indicating its presence in large protein complexes. 141 The only RING finger protein that has been localized to CNS neurons is Neuropdapl (Nakayama et al, '95). This protein has been identified in the search for proteins that are down regulated after axotomy. Immunohistochemical analysis showed that Neurodapl is also expressed in several neuronal populations throughout the brain with a punctate pattern, similar to the one observed for BERP. Immunoelectron microscopic analysis showed that Neurodapl was mainly localized on the cytoplasmic side of membranes forming Golgi apparatus and endoplasmic reticulum as well as at the postsynaptic density (PSD) region of axosomatic synapses and a role for it in the regulation vesicular transport to the PSD has been indicated (Nakayama et al, '95). Whether BERP serves a similar function to Neurodapl in vesicular transport is yet to be determined. B E R P is a member of the R B C C group of the R I N G finger family Members of a subgroup of RING finger proteins, the RING-B box-Coiled-Coil" (RBCC) group (Saurin et al, '96) have an N-terminal RING finger followed by one or two additional zinc-binding domains (B box motifs), a coiled-coil region and a variable C-terminal domain (Reddy & Etkin, '91; Reddy et al, '92; Freemont '93). Genbank searches showed that BERP represents a novel member of the RBCC group, which contains an N-terminus RBCC domain. BERP contains a RING finger, followed by a B box and a coiled-coil domain. The RBCC family appears to play key roles in regulating gene expression and cell proliferation. The majority of members of the RBCC group have been shown to localize to the nucleus (PML, RFP, SS-A/Ro, PWA33) or to interact with nuclear proteins and to be involved in transcription regulation (KAP1, Rpt-1, Staf-50 and HT2A) 142 (Freemont, '93). XNF7 is another member of the RBCC family which is a cytoplasmic protein that translocates to the nucleus during mid-blastula transition (Reddy and Etkin, '91). KAP-1 is a corepressor of KRAB-domain-containing transcription factors (Friedman et al, '96). Rpt-1 has been implicated in the downregulation of interleukin-2 receptor. Staf-50, a protein that is induced by interferon, represses HIV-1 long terminal repeat expression (Tissot and Mechti, '95). HT2A, cloned from a Hela library, associates with Tat, the HIV-1 virus regulatory protein (Fridell et al, '95). Interestingly, several members of the RBCC group are oncogenic and were originally identified as fusion proteins resulted from chromosomal translocations (Reddy et al, '92; Freemont , '93). The oncogenic protein T-18 arises from the fusion of the RBCC domain of TIF1 with the kinase domain of B-Raf (Miki et al, '91). The fusion of the PML RBCC domain with retinoic acid receptor alpha has been identified in acute promyelocytic leukemia (Kakizuka et al, '91; Kastner et al, '92; Gillard and Solomon '93; Liu et al, '95; Koken et al, '95). Fusion of the RBCC domain of RFP to the tyrosine kinase (RET) is also oncogenic (Takahashi et al, '88; Isomura et al, '92). The sequence and spacing between the RING finger, the B box and the coiled-coil are highly conserved among members of the RBCC group including BERP, indicating a functional similarity for this domain in various members of this group. Based on the known functions of members of the RBCC group discussed above, a role for the RBCC domain in the regulation of processes that involve cell division is expected. The only member of the RBCC group that appears to function outside the nucleus is the GTPase ARD1, a member of the ARF family of proteins that are involved in the regulation of vesicular transport (Mishima et al, '93). Several other RING finger proteins which do not 143 belong to the RBCC family are involved in vesicular transport including EEA1 (Mu et al, '95; Stenmark et al, '96), VAC1 (Burd et al, '96), PAF-1 (Tsukamoto et al, '94) , and Neurodapl (Nakayama et al, '95). Some of them such as VAC1, also contain, besides the RING finger, a coiled-coil domain. Our studies have shown that BERP is a cytoplasmic protein, and is expressed in a punctate pattern similar to the one reported for some RING finger proteins including Neurodapl (Nakayama et al, '95), EEA1 (Mu et al, '95), carl (Berteaux-Lecellier et al, 95) and VAC1 (Burd et al, '97) indicating a role for BERP in vesicular transport. Thus, unlike most RBCC members, ARD1 and BERP seem to have functions that do not directly involve DNA regulation. It is still possible that BERP may be able to function like XNF7, a cytoplasmic protein that is capable of translocating to the nucleus and in regulating transcription only at specific stages of early development (Li et al, '94). In postmitotic cells, translocation of BERP to the nucleus may depend on the activation of certain signal transduction pathways. Recently, BRCA1, a RING finger protein, has been shown to be present in NIH3T3 cell cytoplasm in the presence of serum and it translocates to the nucleus in the absence of serum (Wang et al, '97). Thus, the extracellular environment may also play important roles in inducing protein translocation to the nucleus. Members of the TRAF family of cytoplasmic protein share an N-terminal domain very similar to the RBCC motif (Baker & Reddy, '96; Saurin et al, '96). The only difference between the N-terminal region of TRAF-containing proteins and the RBCC motif is that the B box motif is replaced by a cluster of zinc fingers. These observations indicate that RBCC and RBCC-like domain are also present in cytoplasmic proteins indicating that the RBCC domain may also serve some functions that do not involve DNA binding. 144 Interestingly, Genbank searches with the RBCC motif of BERP identified another protein which contains a B box and a coiled-coil but no RING finger. This protein is known as ataxia-telangiectasia group D complementing gene (ATDC; Leonhardt et al, '94; Brzoska et al, 95). M o r e significantly, the sequence of the B box of A T D C is the closest to the one present in B E R P (61% sequence homology). The exact function of ATDC is not known yet, but recent investigations showed that ATDC is a candidate gene for ataxia-telangiectasia (AT), a human genetic disease characterized by immunological, neurological, and developmental defects and an increased risk of cancer. Cells from AT patients show sensitivity to ionizing radiation, cell cycle abnormalities, increased DNA recombination and aberrant cytoskeletal organization (Leonhardt et al, '94; Brzoska et al, '95). Indeed, ATDC has been shown to complement the ionizing radiation of AT group D fibroblasts (Leonhardt et al, '94; Brzoska et al, '95). Recently, ATDC has been shown to interact with the intermediate filament protein vimentin as well as with an inhibitor of protein kinase C (hPKCI-1) (Brzoska et al, '95). Although ATDC has been implicated in the regulation of transcription, localization experiments showed that this protein is cytoplasmic and is localized to vimentin filaments (Brzoska et al, 95). Thus, ATDC represents a third cytoplasmic protein with motifs (B box and a coiled-coil motifs) which are implicated in the regulation of processes that involve cell growth. At present it is not clear how cytoplasmic proteins such as ATDC can regulate signals to the nucleus. In conclusion, although data indicate a strong role for the RBCC domain in the regulation of events that involve cell division, the exact function of this domain remains unknown. The R I N G finger is a putative protein-protein interaction motif 145 The RING finger is a zinc binding motif, which binds two zinc atoms and folds in a brace-like structure (Saurin et al, 96). Because it forms a zinc finger it was initially thought that this motif is involved in DNA interactions and transcriptional regulation (Freemont et al, '91; Lovering et al, 93; Freemont, '93). In support of such a role, several RING finger proteins have been reported to associate with chromatin in large protein complexes and others were required for transcription regulation (Saurin et al, 96). Another support for a vital role for the RING finger motif in transcription regulation comes from the observed cell abnormalities when the RING finger was mutated, translocated or deleted. Several RING finger proteins were identified in the search for oncogenes that triggered cell transformation (Saurin et al, 96). PML, a tumor suppressor gene, was first identified in chromosomal translocations in leukemia. BRCA1 has been identified in the search for oncogenes involved in breast cancer. Mel 18 is another tumor suppressor which was identified in melanomas (Reddy et al, '92). Bmi-1 cooperates with c-Myc in oncogenesis (Van Lohuizen et al, '91). Thus the RING finger motif was thought to be only present in transcription regulatory proteins which indicated a strong role for the RING finger as a DNA binding motif (Freemont et al, '91; Freemont, '93). The hypothesis that the RING finger was a DNA binding motif was accepted by many researchers until the discovery of two new subgroups of proteins that are involved in processes that do not involve direct DNA regulation. The first group includes members of the TRAF family (TNF Receptor associated factor) and IAPs (inhibitors of apoptosis) that have been found associated with cytokine receptors (mainly with members of the tumor necrosis factor (TNF) family) (for review see Baker and Reddy, '96; Saurin et al, '96). Although TRAF proteins have been shown to be cytoplasmic proteins, they play an important role in activating cell surface receptors and modulate signals that regulate cell growth and survival. Indirect role for these 146 proteins in transcription regulation has been shown through the activation of the transcription factor N F K B . It is not yet determined if TRAF proteins are capable of translocating to the nucleus and directly regulating gene expression. The second group includes proteins involved in the regulation of vesicular transport (reviewed in Saurin et al, '96). These include proteins involved in peroxisome proliferation (in yeast: PER8, PAS4, PAS7, PEP3 and PEP5; in fungus: carl; in human PaFl), in vesicular transport from Golgi to vacuole (in yeast; e.g. FAB1, Vpsl8p, VAC1) and endosomes (in mammals; e.g. EEA1 ). Others had less defined functions but either their structure or localization indicate a role for them in regulation of vesicular transport. For example, ARD1 is a GTPase that belongs to the ARF family of proteins which are involved in regulation of secretory pathways (Mishima et al, 93). The RING finger protein, FGD1, is a guanine nucleotide exchange factor for Rho GTPases and a role for it in the regulation of cytoskeleton as well as membrane trafficking has been indicated (Pasteris et al, 94; Zheng et al, '96). Also, Neurodapl is a neuronal RING finger protein present in membranes of Golgi and endoplasmic reticulum as well as in postsynaptic densities of axosomal synapses and a role for it in vesicular transport was indicated (Nakayama et al, 95). A role for the RING finger as a protein binding motif is supported by results obtained from mutation studies in RING fingers of cytoplasmic proteins. For example, mutating the RING finger of TRAF2 abolishes its ability to activate the latent cytoplasmic transcription factor N F K B (Rothe et al, 95). Mutations of the RING finger of Pas7p disrupt the formation of perixosome lumen (Kalish et al, '95), whereas mutations of VAC1 RING fingers leads to protein missorting (Burd et al, '97). In all of these examples, these proteins are part of large complexes and their functions do not involve DNA interactions. 147 The LIM domain is structurally the closest zinc finger motif to the RING finger (Sanchez-Gracia and Rabbits, '94; Schmeichel and Beckerle, '94 & '97). The LIM domain consists of 8 zinc binding ligands with the consensus CX2CX]g_23 HX2CX2CX2CXjg_2iCX2_3C/H/D (Schmeichel and Beckerle, '94 & '97). Similarly the RING finger consists of 8 zinc binding ligands with the consensus CX 2 CX 9 . 8 9 C X ^ t ^ . a C X ^ X ^ g C X j C . In both motifs, the number of zinc-binding ligands as well the spacing between them is conserved. Besides the conserved spacing between the zinc-binding ligands, there are no sequence similarities between the two motifs, which indicates that both motifs may have similar folding, but they may mediate different interactions. Interestingly, the LIM domain has been shown to be a protein-protein interaction motif (Schmeichel and Beckerle, '94 & '97). The LIM domain of zyxin was enough for its interaction with its partner CRP. The LIM domain is also responsible for association of LIM-kinase 1 (LIMK1) and 2 (LIMK2). Thus, the LIM domain represents a novel zinc finger that is involved in diverse protein-protein interactions (Hiraoka et al, '96). In addition, the LIM domain, like the RING finger, is present in proteins of diverse functions including those involved in mediating signals of cell surface receptors as well as those involved in transcription regulation (Sanchez-Gracia and Rabbits, '94; Schmeichel and Beckerle, '94 & '97). Thus, the RING finger is possibly another motif that serves as a protein-protein interaction domain rather than a DNA binding domain. Recent studies have shown that the RING finger of BRCA1 was enough for its interaction with the transcription factor E2F in vitro (Wang et al, '97) It still needs to be determined if BRCA1 RING finger serves the same function in vivo. It is st i l l possible that motifs l ike the R I N G finger and the L I M domain can serve dual functions, by binding to proteins as wel l as D N A . 148 R I N G finger proteins are usually part of large complexes Interestingly, one remarkable feature of members of the RING finger family is that they exist as a part of large protein complexes which indicates a role for the RING finger as a protein-protein interaction motif and indicates that the RING finger may be required for the formation and stability of protein complexes. RING finger proteins are either found associated with cell surface receptors (e.g. TRAFs with cytokine receptors; c-Cbl with EGF receptors; 43K with acetylcholine receptors), involved in vesicular transport (including endosomes, perixosomes, vacuoleMysosomal transport and post-Golgi vesicle transport) or as a part of large nuclear complexes (MAT1 in CAK; TIF1 in steroid receptor complex). In all of these pathways, the formation of large protein complexes have been described. For example, TRAF proteins are involved in homo- and heterodimeric interactions, in clustering of cytokine receptors (Baker and Reddy, '96). c-Cbl is a cytoplasmic protein that is involved in EGF signaling pathways and is involved in interactions with several adaptor proteins including Grb2 and She (Fukazawa e al., '95, Galisteo et al, '95; Tanaka et al, '95). Salgia et al, '96; Wang et al, '96; Soltoff et al, '96,). Also, several RING finger proteins are involved in vesicular transport, a process which involves protein complex formation. In addition, nuclear RING finger proteins are found in large complexes. MAT1 is part of the cyclin dependent kinase- activating kinase (CAK) complex which consists of cyclin-dependent kinase 7 (CDK7), cyclin H (CycH) and MAT1 (Devault et al, '95). Recent data have shown that MAT1 acts as an assembly factor for CDK7-CycH complex and acts to modulates CAK substrate specificity (Rossingol et al, '97). PML is found in large nuclear bodies in the nucleus and mutations in the PML RING finger results in the loss of these structures. TIF1 is a 149 part of the nuclear receptor complex and can associate directly with some members of the steroid receptor superfamily (Le Douarin et al, '95). There are two major questions left to answer: Can the RING finger motif serve as a protein-protein interaction motif in vivo? and if it does, would various members of the family mediate similar interactions through the RING finger motif? If this is the case then one possible common role for it may be in interactions with cytoskeletal elements and serving in anchoring of protein complexes to the cytoskeleton. But comparisons of the RING finger motif of various proteins show that variations are quite common in the ligands and in the length of loops forming the RING finger indicating diversity in its interactions. Thus, binding of the RING finger to a common factor may not be the case . Another possibility is that the RING finger may function as a dimerization motif, and thus linking different signaling pathways via heterodimeric interactions of proteins containing this motif. In support of this hypothesis is the multimeric interactions present among various members of the TRAF family, although a role for the RING finger in these interactions has not been demonstrated. Roles implicated for B E R P R B C C domain as determined by the yeast two hybr id assay. Protein oligomerization The N-terminal region of BERP (amino acids 1-372) that contains the RING finger (amino acids 22-65) was able to oligomerize. Deletion of amino acids 263-372 did not abolish protein oligomerization showing that the first 262 amino acids are enough for protein oligomerization. Although coiled-coils are implicated in protein homodimerization (Lupas et al, '91; Lupas, '96), deletion of one third of the BERP coiled-coil motif did not affect protein 150 oligomerization. These results support a role for the RBCC motif in protein homodimerization in vivo. Mutations in the B box and the coiled-coil regions of PML affect PML nuclear body formation in vivo (Saurin et al, '96). These results indicated that the PML RBCC motif may function as an integral unit and is possibly involved in protein oligomerization. Our data are consistent with a role for the RBCC domain in protein oligomerization. Because of the high conservation of the R B C C domain among members of the R B C C group, it is predicted that this domain may also serve as a heterodimerization domain. Consider ing the diverse roles of members of the R B C C family, this k i n d of interaction may be an important mechanism for cross talk that takes place between different signaling pathways. Interaction wi th alpha actinin Using the BERP RBCC domain as a bait in the yeast two hybrid system, we have shown that the RBCC domain in BERP interacts with a recently identified isoform of the cytoskeletal protein alpha actinin (Geizert et al, '96). This is the first demonstration of a role for the R B C C domain in protein-protein interactions. BERP-actinin interaction requires the last third of the coiled-coil motif (amino acids 263-272) indicating that this interaction may not be mediated by the RING finger (amino acids 25-65). Because the alpha actinin clone contained the full cDNA it is not clear at present which part of alpha actinin is involved in this interaction. Zyxin, a zinc finger protein of the LIM family has been shown to be part of the focal adhesion complex and it also associates with alpha actinin (Craford et al, '94). Also, as discussed earlier, ATDC, a protein that contains a B box and a coiled-coil has been found to associate with the cytoskeletal protein vimentin (Brzoska et al, '95). Thus it is possible that BERP may serve similar functions to zyxin and ATDC in regulating signals to the cytoskeleton. 151 Actinin is an actin cross-linking protein that is also involved in signals from cell surface adhesion receptors at specific sites known as focal adhesions. Also, actinin serves as a signaling molecule and is involved in interactions with integrin receptors, vinculin and PI-3K (Clark and Brugge. '95), rabphillin-3A (Kato et al, '96), protein kinases (PKN; Mukai et al, '97) and with NMDA receptors (Wyszynski et al, 97). Actinin is involved in Rho-activated pathways, as well as the regulation of functions of cell surface receptors such as the NMDA receptor. At present, the significance of the association of BERP with actinin is not clear yet. BERP may function in signaling pathways that involve transmitting signals to the cytoskeleton and in the regulation of cytoskeleton organization, although currently we have no evidence to prove or disprove this. Although the yeast two-hybrid data show that the R B C C domain interacts wi th alpha actinin, this interaction has not been confirmed wi th immunoprecipitat ion yet. The lack of antibodies specific for this isoform of actinin isoform made it difficult to confirm such interactions. Although our results indicate an association of BERP with an endogenous actinin (-110 kDA) that is recognized by the actinin antibodies (available from Sigma). This antibody also interacted with several other bands that were also present in the control samples. Thus, interaction of BERP with actinin needs to be confirmed. Either protein tagging or obtaining specific antibodies against this isoform of actinin may be required to confirm the yeast two-hybrid data. A truncated version of B E R P that contains the R B C C motif is expressed i n the nucleus in H E K cells. 152 Translocation of the RBCC motif is usually oncogenic. As described earlier, translocations that involve the RBCC domain of PML underlie acute promyelocyte leukemia (Kakizuki et al, '91; Kastner et al, '92). Also, translocations of the RBCC domain of TIF1 (Miki et al, '91; Kastner et al, '92; Le Douarin et al, '95) as well as RFP are also oncogenic (Takahashi et al, '88; Isomura et al, '92). These results indicate a significant role for the RBCC domain in the regulation of normal cell growth and division. To gain more insight about the role of the BERP RBCC motif a construct that contains the N-terminal region of BERP which includes the RBCC motif (NBERP; amino acids 1-485) was expressed transiently in HEK cells. For comparison, the full length BERP as well as another truncated version of it that lacks the RBCC domain (CBERP; amino acids 285-744) were also generated and expressed in HEK cells. All of the constructs were FLAG-tagged at their N-termini to allow detection with M5 anti-FLAG antibodies. Immunofluorescent staining was used to examine the expression of these constructs in transient transfection experiments. Immunofluorescent studies showed that the transiently expressed full length protein was distributed all over the cytoplasm with a punctate pattern. Nuclei were not stained for BERP. These results are consistent with the pattern observed for endogenous BERP in CNS neurons. The truncated version that lacks the RBCC domain (CBERP) was also distributed in the cytoplasm with a punctate pattern. A dense staining of CBERP was also observed in areas around the nucleus indicating Golgi localization. In some cells, CBERP was present in clusters close to the membrane at sites involved in membrane outgrowth. The significance of these results is not clear yet. The possible accumulation of CBERP in the Golgi indicates that loss of RBCC may have resulted in protein missorting and consequently accumulation of CBERP in the Golgi apparatus. Another possibility is that the C-terminal region is capable of interacting with 153 proteins that reside in the Golgi apparatus, and this interaction is inhibited by N-terminal sequences. In contrast, immunofluorescent staining showed that NBERP was exclusively present in the nucleus. Consequently, these results are in agreement with a possible role for the RBCC domain in transcriptional regulation. The cellular mechanisms involved in translocating the truncated version of BERP, NBERP, to the nucleus is still unclear. As described earlier under the results section, BERP contains a putative nuclear localization signal (NLS) located between the ABP-like repeat and the unique C-terminal region. This putative NLS signal is present in both of the truncated forms of BERP. Thus, the C-terminal sequences may block the NLS or interact with cytoplasmic proteins that block NLS. Truncation of that region will expose NLS and in turn translocation of N-terminal sequence to the nucleus. This hypothesis can be tested by studying the effect of deleting the NLS on the localization of the truncated form NBERP. The RBCC domain of BERP may also be responsible for translocation of NBERP to the nucleus because the expression of a truncated BERP missing the RBCC domain (CBERP) was detected in the cytoplasm. Second, C-terminal sequences seems to be involved in the retention of full length BERP in the cytoplasm because the expression of both the full length and a truncated version of BERP that contains the C-terminal region are cytoplasmic. It is possible that nuclear localization of NBERP is due to heteromeric interactions between N-terminal sequences of BERP with a nuclear protein which resulted in NBERP translocation to the nucleus. Other members of the RBCC group are good candidates for such oligomerization for two reasons, first, the RBCC domain is highly conserved among members of the RBCC family. Secondly, the RBCC is present in several nuclear proteins. In addition, our results do show that the BERP 154 RBCC domain is involved in protein oligomerization. Based on these observations, it is possible that oligomerization of BERP RBCC domain with another protein present in one of the members of the RBCC group may have resulted in its translocation to the nucleus. Examining the ability of BERP RBCC domain for dimerization with other known RBCC sequences may help us to identify possible partners that are responsible for its nuclear translocation. Finally, the cytoplasmic localization of the full length protein may be a result of interactions between its C-terminal region with cytoplasmic factors which prevented its translocation to the nucleus. Activation of specific signaling pathways may be required for BERP dissociation from cytoplasmic factors and translocation to the nucleus. Thus, these results provide evidence for a possible role for the R B C C domain of B E R P i n the regulation of nuclear events which may include transcription regulation. In fact, a member of the RBCC family, XNF7, is a cytoplasmic protein and it translocates to the nucleus at specific stages of early embryonic development (Li et al, '94). In addition, the RING finger protein BRCA1 is localized to the cytoplasm in NIH3T3 cells and translocates to the nucleus upon serum deprivation (Wang et al, '97). Furthermore, the activation of different signal transduction pathways is required for the translocation of various cytoplasmic proteins to the nucleus including NFkB (Lipton, '96), mitogen activated protein kinase (MAP kinase; Blumer and Johnson, '94; Cooper, '94; Marshal, '95), and members of the STAT-JAK pathway (Leaman et al, 96; Darnell et al, '94; Ihle and Kerr, '95. At present, the limited information about the cellular functions of BERP makes it difficult to predict the possible pathways that might be involved in stimulation of BERP translocation to the nucleus. Thus, whether or not the full length BERP can translocate to the nucleus is still to be shown. 155 A truncated B E R P containing the R B C C motif inhibited neurite outgrowth in PC12 cells after N G F treatment. The rat pheochromocytoma cell line PC 12 has been used as a model system for studying the mechanism of action of neuronal growth factors (Alema et al, '85). Treatment of PC 12 cells with nerve growth factor (NGF) or fibroblast growth factor (FGF) results in the differentiation of these cells and in the development of a phenotype similar to sympathetic neurons, including neurite extension, cessation of cell division and expression of neuronal specific genes (Halegoua et al, '91). NGF actions can be mimicked by expression of the viral src and ras in PC12 cells (Alema et al, '85; Noda et al, '85; Thomas et al, '91). Overexpression of an N-terminally truncated raf (oncogenic raj) also results in the elaboration of a neuron-like phenotype, including neurite outgrowth and induction of NGF responsive genes (Wood et al, '93). Our immunohistochemical data showed that BERP is highly expressed in adult CNS neurons. In this study, the use of PC 12 cells will help us to understand more about the possible role for BERP on the growth and differentiation of neuronal-like cells. The effect of full length as well as truncated forms of BERP were examined for this study. Stable expression of B E R P and the truncated form N B E R P in PC12 cells. PC 12 cells were used for the generation of stable cell lines to determine the long term effects of the expression of BERP and the truncated version of BERP (NBERP). For full length BERP one clone that expresses the protein was obtained. DAB staining showed that the expressed full-length BERP was also present in the cytoplasm of PC 12 cells. These results are consistent with the localization of full length BERP in the transiently transfected HEK cells. 156 There was no obvious phenotypic changes due to the expression of the full length protein and cells continued to proliferate. Ten G418-resistant clones expressing NBERP were obtained, and the expression of the predicted size protein was confirmed by Western blotting. A l l of these clones expressed NBERP in the nucleus. Again, these results are similar to the ones obtained for the localization of NBERP in transient transfection experiments in H E K cells. Unlike normal PC 12 cells or those expressing the full length protein, cell lines expressing NBERP formed large aggregates and failed to separate indicating a cell spreading defect. NBERP Staining was detected in the nuclei, and aggregated cells appeared as a continuous multinucleated cytoplasmic mass. However, F-actin staining using fluorescent Phalloidin (Molecular probes), showed that cells were distinct and uninucleated. These results indicated that the cells completed cell division. The observed abnormal clustering of cells may be due to an increased rate of cell proliferation combined with defects in cell spreading. Because neither normal PC 12 cells nor those expressing full length BERP show the abnormal phenotype observed with NBERP, the current data indicate that expression of the truncated BERP, NBERP, may have resulted in the abnormal aggregation observed for these cells. Northern blot analysis showed that normal PC 12 cells express endogenous BERP. In addition, our yeast two hybrid analysis showed that the R B C C domain of BERP may be involved in protein oligomerization. Thus, it is possible that overexpression of a truncated form of BERP that lacks C-terminal sequences but contain the R B C C domain was able to dimerize with endogenous BERP and disrupt its normal folding as well as its interactions with other proteins, thus acting as a dominant negative mutant. Consequently, the abnormalities observed with the 157 truncated BERP may have resulted from interference with the function of endogenous BERP. At present, the cellular functions of BERP are still unknown, and thus the mechanism underlying the observed abnormalities when overexpressing the truncated BERP are unclear. Truncated B E R P ( N B E R P ) inhibits N G F - i n d u c e d differentiation in P C 1 2 cells. Next, the effect of nerve growth factor (NGF) treatment on the differentiation of the transfected cells was examined. NGF is a neurotrophic factor that is known to induce the differentiation of PC 12 cells to neurons (Greene and Teschler, '82). Seven days post NGF treatment, cultured cells were examined for the induction of neurite extension, the most prominent marker of PC 12 differentiation. Cells expressing the full length as well as untransfected PC 12 cells responded to NGF treatment and developed long neurites. Next, the expression of transfected BERP was examined after NGF treatment. Immunocytochemical analysis using DAB staining showed that transfected BERP was cytoplasmic and also present in neurites similar to its distribution in adult CNS neurons. In contrast, NGF treatment of cells expressing the truncated BERP, NBERP, failed to form long neurites, instead, cells continued to divide rapidly. Using immunocytochemistry, NBERP was detected in the nucleus. Thus NGF treatment did not results in the change of localization of either the full length or the truncated version of BERP. The mechanism that underlies the inhibi t ion of P C 1 2 cell differentiation by overexpressing a truncated form of B E R P is not clear at present, but these results indicate that truncation and possibly mutations in B E R P may have detrimental effects on normal neuronal differentiation. Previous data showed that expression of a truncated version of raf (oncogenic raf) results in induction of neurite growth (Wood et al, '93). In contrast, 158 overexpression of a LIM protein, LIM kinase 1 (LIMK1) blocked the oncogenic Ras-induced differentiation of PC 12 cells, but a mutant lacking the LIM domain failed to do so, indicating a role for the LIM domain in this inhibition (Higuchi et al, '97). Mutations in the zinc binding ligands of the LIM motif also abolished the inhibitory effect confirming that blocking of Ras-induced neuronal differentiation was mediated by the LIM motif. Like the LIM motif, the BERP RBCC domain also blocked differentiation of PC 12 cells. Thus, whether BERP is involved in regulatory pathways downstream of NGF that determine cell differentiation is not known yet. Overexpression of the full length protein in PC 12 cells had no obvious effect on NGF-treated or untreated cells. Also, the expressed full length BERP was in the cell cytoplasm before and after NGF treatment, indicating that BERP does not translocate to the nucleus upon neuronal differentiation. Thus, considering the current data, it is not clear at this time if BERP has a direct effect on neuronal differentiation. Because the expression of the full length protein was not detectable in the nucleus of NGF treated (differentiated) or untreated PC 12 cells, it is more likely that the abnormalities generated from expression of the truncated form are due to its interactions with some nuclear factors that are directly involved in the regulation of cell division and differentiation rather than acting as a dominant negative mutant of BERP. In conclusion, expression of a truncated version of BERP that contain the RBCC domain inhibits NGF-induced differentiation of PC 12 cells as assessed by inhibition of neurite formation. These results indicate that the RBCC motif may have an important regulatory function in BERP Also, the expression of endogenous BERP may be essential for neuronal differentiation, and mutations or truncations of this protein may have severe implications on the normal development of a neuronal cell. 159 B E R P contains an A B P - l i k e repeat, a domain wi th unknown function in B E R P . Following the RBCC motif, BERP contains a -100 amino acid domain (amino acids 328-430) that shares 45% homology with a region that is present as several repeats in actin binding proteins known as ABP-120 and ABP-280 (Stossel, '93; Fucini et al, '97). ABP-120 and ABP-280 are rod-shaped homodimers known to cross link actin filaments. Each consists of an N-terminus actin binding domain and a rod-like C-terminal region (Stossel, '93). The rod domain consists of several repeats each about 100 amino acids in length (repeats will be called ABP repeats). Six ABP repeats are present in the rod domain of the yeast ABP-120, whereas 24 ABP repeats are present in ABP-280 C-terminal regions (Stossel, '93; Sharma et al, '95; Fucini et al, '97). NMR studies showed that repeat 4 of the rod domain of ABP-120 consists of seven beta-sheets arranged in an immunoglobulin-like (Ig) domain (Fucini et al, '97). The high similarity present among different repeats indicate a similar folding for all of the other repeats. A role for ABP-120 and ABP-280 repeats in the dimerization of the whole protein and in the formation of the rod tail has been indicated. Besides a role for these repeats in homodimer formation, recent data showed that these repeats also serve as protein-protein interaction domains. In platelets, repeats 17-19 of ABP-280 interact with the cytoplasmic tail of the Willebrand factor receptor, the glycoprotein (GP) Ib-IX complex (Meyer et al, '97). ABP-280 cross-links actin filaments and anchors the membrane skeleton to the plasma membrane by its interaction with (GP) Ib-IX. In other cells that do not express GP Ib-IX, ABP-280 binds the high affinity IgG receptor (FcyRI) and the (32 integrin (Ohta et al, '91; Sharma et al, '95). Other studies have shown that the C-terminal region of ABP-280 interacts with stress activated protein kinase activator (SEK-1) (Martin etal, '97). 160 ABP repeats have been implicated in homodimer formation. To investigate if the ABP-like repeat in BERP is involved in protein oligomerization, a yeast two-hybrid-based assay, was used. The ABP-like repeat (amino acids 280-459) was subcloned into pAS2-l as well as pACT2 vectors and used for co-transformation experiments. These results indicated that the ABP-like repeat alone does not oligomerize, indicating that either this domain is not involved in BERP oligomerization or by itself is not sufficient for protein dimerization. In conclusion, because of the high similarity between the ABP-like repeat present in BERP and those present in ABP-280, one would predict that this domain in BERP may be involved in interactions similar to the ones reported for the C-terminal domain of ABP-280. Yeast two-hybrid analysis indicated that alpha actinin, a component of cell adhesion complexes is a putative interactor with BERP. Also, BERP contains an ABP-like repeat similar to the ones present in ABP-280, which are also involved in interactions with cell-adhesion proteins including integrin receptors. Thus, it is tempting to speculate that BERP through interactions with cell adhesion and cytoskeletal molecules is also involved in the regulation of cell-cell contact as well as adhesion signals. If this is true, then this may explain the abnormal aggregation and spreading defects observed when expressing the truncated BERP in PC 12 cells. BERP C-terminal region shares homology with a Tat-interacting protein, HT2A Following the ABP-like repeat, the BERP C-terminal region (amino acids 465-744) contains a unique sequence with a predicted beta sheet conformation. HT2A is the only mammalian RING finger protein with a significant overall homology to BERP (Fridell et al, '95). BERP share -35% overall sequence homology with HT2A including C-terminal sequences. HT2A was identified in a yeast two-hybrid screen for proteins that interact with the 161 HIV viral protein HT2A (Firdell et al, '95). The last 120 amino acids of HT2A were enough for interaction with Tat (Fridell et al, '95). The significance of the interaction between HT2A and Tat is not known yet. Also, no cellular proteins have been identified that interact with HT2A. Thus, the exact cellular function of HT2A is still unknown. B E R P does not interact wi th the H I V regulatory protein Tat The last 120 amino acids of HT2A were involved in interaction with Tat. Because of the high similarity between the C-terminal regions of BERP and HT2A (-40% homology), I examined if BERP can also interact with the HIV protein Tat. Several baits that contained BERP C-terminal sequences were used (See Table 1 and 3 ). The C-terminal region of HT2A was used as a positive control to study protein interactions with Tat. All of the examined BERP baits failed to interact with Tat whereas the positive control HT2A did interact strongly with Tat as reported by Fridell et al, ('95). Thus, in spite of the overall homology between BERP and HT2A, BERP does not interact with Tat, indicating that HT2A-specific sequences are involved in the interaction of HT2A with the viral protein Tat. Whether BERP and HT2A can still serve similar cellular functions is still unknown. The C-terminal region of B E R P interacts wi th class V myosins. The yeast two-hybrid system was used to identify putative interactors with the C-terminal region of BERP that includes amino acids 383-744. Using this region as a bait to screen a rat brain cDNA library, 2 positives were identified that interacted specifically with the BERP C-terminal region and not with the ABP-like repeat or the N-terminal RBCC domain present in BERP. Sequence analysis of these positives showed that they represent the same C-terminal 162 region of a protein that belongs to class V unconventional myosins, known as myr 6 (amino acids 1206-1847; Zhao et al, '96). Further analysis showed that the last 120 amino acids of BERP were enough for this interaction. To confirm this interaction, the C-terminal region of myosin cDNA (corresponds to amino acids 1222-1847) was subcloned into the mammalian expression vector pcDNA3. A HIS tag (6 histidines) was inserted at the N-terminus of this construct to allow the detection of the expressed protein in cell transfection experiments. Next, HIS-tagged MYR6 (C-terminus domain) was transiently co-expressed in HEK cells with a FLAG-tagged full-length BERP. Western blotting was used to confirm the expression of the predicted size proteins using anti-FLAG (IBI) and anti-HIS (Clontech) antibodies to detect FLAG-tagged BERP and HIS-tagged myr 6, respectively. Immunoprecipitation experiments using anti-FLAG antibodies confirmed that HIS-myr 6 co-immunoprecipitated with BERP. These experiments confirmed the interaction between BERP and the C-terminal domain myr 6 observed in the yeast two hybrid assay. To be able to demonstrate the interaction between BERP and myr 6 in brain tissues, antibodies that can detect endogenous myr 6 will be required. At present, antibodies against myr 6 protein are not available, and thus the generation of such antibodies will be required in the future to confirm BERP association with myr 6 in brain tissues. Myr 6 has been recently cloned and identified in brain tissues (Zhao et al, '96). In situ hybridization analysis showed that myr 6 transcripts are highly expressed in various brain areas with a predicted neuronal expression. A member of class V myosins and a close relative of myr 6 is the well-characterized protein dilute (Mercer et al, '91). Dilute shares high overall sequence homology with myr 6. Dilute is mainly expressed in the brain and its expression has been detected in several neuronal 163 populations indicating that dilute is another candidate protein that may interact with BERP. To examine if BERP associate with dilute, the antibodies against dilute (Evans et al, '97) were used for co-immunoprecipitation experiments. Indeed, our results showed that BERP co-immunoprecipitated with dilute from cortex and cerebellar extracts. These data demonstrate the in vivo association of BERP with a member of the class V myosins. Dilute is expressed in several bra in neurons in a punctate pattern in the soma and dendrites and is implicated i n vesicular transport (Nascimento et al., '96; Evans et al., '97; Col ing et al., '97; Titus, '97; W u et al, 97). A similar punctate pattern was also observed for B E R P i n bra in neurons, indicating that B E R P is a partner of myosin and possibly involved i n processes s imilar to the ones predicted for class V myosins. Although the exact roles of class V myosins are not known yet, recent evidence indicates the importance of these proteins in specific protein targeting as well as organelle transport from cell soma to other regions including dendrites and synapses and a role for them in the development as well as the maintenance of neurons has been implicated (Mooseker and Cheney, '95; Larson et al, '96; Bahler, '96; Titus, '97; Prekeris et al, '97). A discussion of the some of known roles of these mysoins may help us to predict some of the potential roles for B E R P . Unconventional myosins Prototypical myosin was originally discovered as the major protein in muscle. Recent investigations have revealed a diverse family of myosins that share a common motor domain, but have variable carboxy-terminal tail regions. Muscle type myosins are known as the conventional or class II myosins. Other classes of myosins are referred to as unconventional myosins. Different myosins share a motor domain that consists of a head region of conserved amino acids 164 (-80 kDA) followed by a neck region that binds to 1-6 light chains of calmodulin/EF-hand proteins. The motor domain converts the energy released from ATP hydrolysis into a unidirectional force along actin filaments (Mooseker and Cheney, '95). The newly discovered unconventional myosins have a motor domain that is coupled to a tail region that differs between individual classes of myosins (Mooseker and Cheney, '95). Actually, this tail region contains protein-protein interaction sequence motifs that are present in non myosin proteins. Class V myosins (including dilute and myr 6) contain a carboxy terminal region with a sequence motif, known as the DIL domain, that is also present in the human AF-6 gene product and its Drosophila homologue canoe (Ponting, '95). The exact function of AF-6 and canoe is not known yet, but recent mutational studies showed that canoe is involved in genetic interactions with members of the Notch pathway and therefore indicating a role for canoe in regulating adhesive cell-cell interactions that determine cell fate (Mooseker and Cheney, '95; Bahler, '96; Spana and Doe, '96). These observations indicate that unconventional myosins are not purely involved in intracellular transport but they may represent a new class of signaling proteins that transmit and regulate signals of different cellular pathways. A key role for unconventional myosins in maintaining normal cellular function is apparent from the severe abnormalities observed when mutations in these myosins occur (Mercer et al, '91; Mooseker and cheney, '95). M y r 6 and dilute are members of class V of unconventional myosins Class V myosins include the mammalian dilute (also known as myosin V), and myr 6 as well as two yeast class V myosins known as Myo2p and Myo4p (Mooseker and Cheney, '95). 165 Dilute was first identified from brain tissues as a 190 kDA calmodulin-binding protein that has a Mg-ATPase activity and can generate directed movement along actin cables. Later studies showed that myosin V is expressed in most tissues but it is most abundant in nervous tissue and neurosecretory cells (Espindola et al, 1992). In fact, myosin V makes up -0.3% of total proteins in the brain (Cheney et al, 1993). Myosin V is an oligomer that consists of two 190 kDA heavy chains and twelve low molecular weight light chains that bind to six motifs present in the neck region of each heavy chain. Eight of the twelve myosin V light chains are calmodulin molecules and the other four are still unknown. Myosin V functions as a molecular motor in several motility assays The tail region of myosin V has a central coiled-coil region that is possibly involved in dimer formation followed by a unique globular DIL domain. Unlike the muscle myosin, myosin V does not form filaments . Roles for class V myosins in vesicular transport Class V myosins also includes the yeast proteins Myo2p and Myo4p (Mooseker and Cheney, '95). Mutations in yeast Myo2p resulted in growth arrest with the formation of large unbudded cells. Myo2p mutations also resulted in the accumulation of small vesicles indicating a role for Myo2p in vesicle transport (Lillie and Brown, '94; Govindan et al, ;95). In these cells, cortical actin was distributed and was present throughout the cell instead of being localized to the growing bud. Also, Myo2p mutants were defective in transporting the vacuole (the equivalent of lysosomes) into the bud. Thus, these results indicate a role for the yeast Myo2p in regulating the movement of distinct vesicles to the site of polarized growth. Yeast Myo4p plays a role in the asymmetric accumulation of a transcription repressor known as Ashlp (Jensen et al, '96). Ashlp represses the transcription of HO gene, which is 166 required to stop the switching of mating type (Jensen et al, '96). Thus unlike the role for Myo2p in regulation of cell morphology and cytoskeleton, M y o 4 p plays a role in the directed transport of factors required for regulating gene expression. Recent data by Prekeris and Ter r ian , ('97) showed that b ra in myosin V is actually associated wi th synaptic vesicles indicating that myosin V is also involved in the transport of synaptic vesicles. The dilute gene product is a class V myosin that has been identified in mutant mice which have a lightened coat color and die three weeks after birth of nervous seizures (Mercer et al, 91; Mooseker and Cheney, '95). Studies have shown that the dilute gene product, myosin V, is associated with synaptic vesicles (Prekeris and Terrian, '97). These results indicated that myosin V may be involved in the actin dependent movement of vesicles carrying neurotransmitters to the extremities of neurons . Mutations in the myosin V gene product, dilute, are lethal (reviewed in Mooseker and Cheney, '95). Dilute mutant mice suffer from a neurological defect characterized by convulsive limb movement and opisthotonic seizures continuing until the death of animals at 3 weeks after birth. During development of dilute-lethal mice neurons were able to develop dendritic processes and axons. Neither the peripheral nor the central nervous system of dilute-lethal mice show striking histopathological deficiencies. Thus, the abnormalities and the premature death of dilute-lethal mice is probably due to defects in intracellular signaling pathways that are not exhibited morphologically. In fact, electron microscopic analysis of brain sections of dilute-lethal mice showed that smooth endoplasmic reticulum (SER) was absent in the dendritic spines of Purkinje cells. Neither synapse formation nor spine formation was affected in the dilute lethal mouse. Since SER plays a crucial role in regulating intracellular calcium stores, the neurological 167 defects may be a result of the absence of SER in the dendritic spines of Purkinje cells. These studies also indicate an important role for dilute in regulating membrane transport of SER from dendrites to the spine. Possible roles for B E R P in the bra in . The interactions between BERP and myosin V may help us to determine the exact role of BERP in the brain. First, both BERP and class V myosins are mainly expressed in the brain. Second, localization studies of myosin V showed a punctate staining that is most intense in perinuclear regions, but also present in the periphery of cells (Espreafico et al, '92). Myosin V colocalizes with intracellular membranes (Cheney et al, '93). A similar subcellular distribution was observed for BERP in CNS neurons indicating that BERP is also associated with organelles. Our yeast two-hybrid results showed that the BERP C-terminal region associates with the tail region of class V myosins. Transient expression experiments in HEK cells showed that the full-length as well as a truncated version of BERP that contains the C-terminal region also show a punctate labeling indicating that the C-terminal region of BERP is localized to cytoplasmic organelles. In contrast, a truncated version of BERP that lacks the C-terminal sequences had a nuclear localization and was not expressed in a punctate cytoplasmic pattern. Class V myosins have a role in regulating the extension of growth cone filopodia during neuronal development (Wang et al, '96) as well as in the movement of axoplasmic organelles (Kuznetsov et al, '92; Langford et al, '95). Cell transfection experiments showed that a truncated form of BERP inhibited NGF-induced neurite outgrowth, which indicates a role for the full length protein in the normal development and differentiation (neurite outgrowth) of 168 neuronal cells. These results are consistent with the possible role for class V myosins in filopodial growth. Several reports indicate that a group of RING finger proteins are involved in vesicular transport including EEA1 (Stenmark et al, 96), VAC1 (Burd et. al., '97), and ARD1 (Mishima et al, '93). In particular, Neurodapl is another brain RING finger protein, which associates with membranes of Golgi and endoplasmic reticulum and localizes to postsynaptic densities of axosomatic synapses (Nakayame et al, '95). Thus, it seems a group of RING finger proteins may be specialized in the regulation of organelle transport. A l l of the current data support a role for B E R P in vesicular transport, most l ikely through its association wi th class V myosins. Other possible roles for B E R P Based on the reported roles for myosins, BERP may be involved in diverse types of organelle transport in neurons, including endoplasmic membrane transport to specific synaptic sites as well as transport of synaptic vesicles. Another possible role for BERP may be targeted transport of specific proteins required for axonal growth. Interestingly, the C-terminal region of class V myosins contains a domain that is present also in two unrelated proteins known as the human AF6 and its Drosophila homologue canoe (Ponting, '95). The exact function of these proteins are not known but recent mutational studies showed that canoe is involved in genetic interactions wi th members of the Notch pathway and therefore indicating a role for canoe in cell-cell interactions that determine cell fate (Lewis, '96; Spana and Doe, '96). Because this domain is present in the well characterized protein dilute (myosin V), this domain was named DIL domain. As described earlier BERP 169 interacts with the tail region of myosin V (the last 641 amino acids of myr 6), which contains the DIL domain ( the last 338 amino acids of myr 6 contain the DIL domain). This indicate a possible association of BERP with the DIL domain of other proteins like AF6 and canoe. In addition, this would indicate that BERP may be involved in cross talk between proteins involved in cell fate determination (Notch pathway) and those involved in organelle transport and molecules targeting (myosin V). Notch protein is a transmembrane protein that translocates to the nucleus and regulates asymmetric cell division of neuroblasts in the CNS (Lewis, '96). The yeast myosin V protein, Myo4, is involved in asymmetric transport of a transcription factor to the yeast bud which is required to stop the switching of mating type (Jansen et al, '96). Thus, such a cross talk between members of the Notch pathway and myosin V is possibly mediated by proteins like BERP. We have shown that a truncated version of BERP containing the RBCC domain is expressed in the nucleus, which may also indicate a possible role for the full length BERP in the regulation of gene expression in early development perhaps through signals of the Notch pathway. Finally, a possible role for BERP in regulating signals to the cell cytoskeleton is indicated by its possible association with alpha actinin (Geizert et al, '96). Also, BERP contains an ABP-like repeat, a domain that is present as several repeats in the cytoskeletal protein ABP-280. These repeats are involved in interactions with focal adhesion proteins such as integrins. The possible pathways that BERP might be involved in are summarized in Figure 49. Evidence for B E R P as a putative tumor supressor gene: The first evidence comes from the chromosomal mapping of BERP gene to 1 lpl 5. The human chromosome 11 pi 5 has been termed the multiple tumor associated chromosomal region 170 1", because of the numerous cancer related genes in this region. The presence in llpl5 of g involved in brain, breast, bladder, and pancreatic cancer, testicular tumors, hepatocellular 171 Receptor ? B E R P Interaction with myos in V : (~) Organelles Ves icular transport? M y o s i n V Outgrowth? Cytoskeletal rearrangement? M H • A F 6 - l i k e D I L domain Figure 49. Summary of some of the proposed functions for BERP. 172 carcinoma, rhabdomyosarcoma, and germ-line neoplasia has been described (Koi, '93). In addition, various familial syndromes, including Beckwith-Wiedemann syndrome (Elliott, '94), map to llpl5. These observations have led to the suggestion that lip 15 harbors numerous tumor suppressor genes. The telomeric end of chromosome 11 (llpl5) shows loss of allelic heterozygosity (LOH) in many types of tumors, implying the presence of tumor suppressor genes. Indeed subchromosomal transfer of DNA fragments from 1 lpl 5 caused in vitro arrest of tumor cells (Koi et al., '93). RING finger proteins have been recently shown to play crucial roles in cellular signal transduction, growth and differentiation. A number of these proteins have been directly implicated in cancer and tumor development. It is therefore expected that BERP may be a novel tumor suppressor gene. The RBCC domain is present in several known tumor supressor proteins such as PML, TIF1 and RFP (de The et al., '90; '91; Goddard et al, '91, Kakizuka et al, '91; Kastner et al, '92 Mu et al, '95a; Liu et al, '95; Miki et al, '91; Le Douarin et al, '95; Durbec et al, 96; Trupp et al, '96; Takahashi et al, '88; Isomura et al, '92). In addition, cell transfection experiments have shown that expressed truncated BERP that contains the RBCC domain is present in the cell nucleus. 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