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Molecular analysis of the domains in CD45 that affect expression and function Maiti, Arpita 1994

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MOLECULAR ANALYSIS OF THE DOMAINS IN CD45 THATAFFECT EXPRESSION AND FUNCTIONbyARPITA MAITIB. Sc.(Hon.), The University of Toronto, Trinity College, 1990A THESIS SUBMiTTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDepartment of Microbiology and ImmunologyWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIASeptember, 1994©Arpita Maiti, 1994Signature(s) removed to protect privacyIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of LI1The University of British ColumbiaVancouver, CanadaDate__________DE-6 (2188)Signature(s) removed to protect privacy to protect privacySignature(s) removed to protect privacyABSTRACTThe aim of this thesis was to further understand the molecular function ofCD45, a transmembrane glycoprotein that has intrinsic tyrosine phosphataseactivity. Expression of two isoforms of CD45, CD45RABC and CD45RO, and twomutated forms of CD45, one lacking protein tyrosine phosphatase (FTP) activity, andone lacking the cytoplasmic domain, were analyzed in a fibroblast cell line, L tk-. Itwas observed that only a proportion (10-30%) of CD45 expressed in all transfectedcells was expressed on the surface. Deletion of the cytoplasmic tail of CD45 resultedin the expression of two CD45 proteins of 125 kDa and 160 kDa. From pulse-chaseexperiments, it was determined that the higher molecular weight form was derivedfrom the lower molecular weight form.Mutational analysis of the of the cytoplasmic domain of CD45 indicated that aconserved glutamine in the second PTP domain of CD45, expressed in E. coli,resulted in the loss of CD45 PTP activity. This demonstrates that a mutation indomain II can affect FTP activity thought to reside in domain I. In order todetermine the effect of mutations in CD45 on T cell signalling, it was first necessaryto characterize a CD45-deficient T cell line. The BW5147 CD45-negative cell line wascharacterized with respect to p59Y’, a potential in vivo substrate for CD45. It wasdetermined that the expression of CD45 resulted in the reduced tyrosinephosphorylation of p59fYfl, yet CD45 had no appreciable effect on the in vitro kinaseactivity of p59fYfl. However, a 120/130 kDa phosphoprotein was identified only inp59fyn immunoprecipitates from CD45-positive cells after an in vitro kinase assayand this occurred independent of T cell receptor mediated stimulation. Theseresults implicate CD45 in regulating the associations of p59fYI in addition toregulating its tyrosine phosphorylation state.11TABLE OF CONTENTSPageABSTRACT iiTABLE OF CONTENTS iiiLIST OF TABLES vUST OF FIGURES viLIST OF ABBREVIATIONS viiiACKNOWLEDGEMENT xDEDICATION xiINTRODUCTION ICD45 Structure 1The Extracellular Domain 1The Cytoplasmic Domain 5CD45 Function in the Immune System 10Summary of Intentions 11MATERIALS AND METHODS 14RESULTS AND DISCUSSION 281.0 Expression and Characterization of CD45 in L tk Cells 281.0.1 Characterization of Expression of Two Isoforms of CD45 andTwo CD45 Cytoplasmic Domain Mutants Transfected intoLCells 281.0.2 Transport of a CD45 Protein Lacking the Cytoplasmic Domainin L Cells 36Discussion 372.0 Mutational Analysis of the Cytoplasmic Domain of CD45 432.0.1 Generation of Three Mutations in the Cytoplasmic Domainof CD45 431112.0.2 Bacterial Expression and Partial Purification of CD45Mutants 452.0.3 Determination of Phosphatase Activity of CD45 Mutants 46Discussion 503.0 Characterization of a Recipient CD45-Negative T Lymphoma CellLine 533.0.1 Determination of the Levels of Expression of the Src-FamilyKinasep59fyn in CD45-Negative and CD45-Positive Variantsof a BW5147 T Lymphoma Cell Line 533.0.2 Evaluation of the Effect of CD45 Expression on the Phosphotyrosine Levels of p59fYll 533.0.3 Determination of the Effect of CD45 Expression on the In VitroKinase Activity of p59fYfl 55Discussion 58CONCLUSION 61PUBLICATIONS 63REFERENCES 64ivLIST OF TABLESPageTable I. Results of Transfection of L tic Cells with CD45 cDNAs 30Table II. Quantitation of Band Density of Bacterially Expressed Proteinsby Scanning Densitometry 48VLIST OF FIGURESPageFigure 1. Schematic Diagram of the Structure of CD45 2Figure 2. Schematic Representation of the Structure of the CytoplasmicDomain of CD45 6Figure 3. Alignment of CD45 PTPase Domains with a Consensus PTPaseSequence 8Figure 4. Schematic Representation of the Shuttle Vector 23Figure 5. Schematic Diagram of the i-actin Mammalian ExpressionVector and the CD45 Constructs Transfected into L cells 29Figure 6. Western Blot Analysis of CD45 Immunoprecipitates from Lysatesof L Cell Transfectants 32Figure 7. Cell Surface Expression of CD45 as Determined by FlowCytometry 34Figure 8. Surface Expression of CD45 as Determined by Western BlotAnalysis 35Figure 9. Pulse-Chase and Endoglycosidase H Sensitivity of theCD45RABC &yt Cytoplasmic Domain Mutant Transfectedinto L tk- Cells 38Figure 10. Pulse-Chase and Endoglycosidase H Sensitivity of CD45Immunoprecipitates from Untransfected L tk- Cells 39Figure 11. Schematic Diagram of the CD45 Cytoplasmic DomainMutants 44Figure 12. Coomassie Blue Stained Gel of Recombinant CD45 CytoplasmicDomain Proteins Generated in Bacteria 47Figure 13. PTPase Assay of Recombinant CD45 Cytoplasmic DomainProteins Generated in Bacteria 51viFigure 14. Amounts of p59fYIL Isolated from CD45-Negative and CD45-Positive BW5147 Cells 54Figure 15. Tyrosine Phosphorylation of p59fYfl Isolated from CD45-Negativeand CD45-Positive Cells Upon CD3-Mediated Stimulation 56Figure 16. In Vitro Kinase Activity of p59Y” Isolated from CD45-Negativeand CD45-Positive Cells Upon CD3-Mediated Stimulation 57viiLIST OF ABBREVIATIONSATP Adenosine triphosphateBSA Bovine serum albumincAMP Cydic adenosine monophosphatedATP 2’-deoxyadenosine 5’-triphosphatedCTP 2’-deocytidine 5’-triphosphatedGTP 2’-deoxyguanosine 5-triphosphateDMEM Dulbecco’s Modified Eagle MediumDTT DithiothreitoldTTP 2’-deoxythymidine 5’-triphosphateEDTA Ethylenediamine tetra-acetic acidEGF Epidermal growth factorEndo H Endoglycosidase HER Endoplasmic reticulumFACS Fluorescence activated cell sorterFCS Fetal calf serumFITC Fluorescein isothiocyanateHBS Hepes buffered salineHPTP Human protein tyrosine phosphataseHRP Horseradish peroxidaseIgG Immunoglobulin GIL-2 Interleukin-2IPTG Isopropylthio-13-D-galactosideLAR Leukocyte antigen related proteinLRP Leukocyte common antigen related proteinM MolarMicrogramvu’MCS Multiple cloning siteMHC Major histocompatibility complexMicrolitremL MillilitremM Millimolarpmol Micromoleng Nanogramn m Nanometrenmol NanomoleO.D. Optical densityPBS Phosphate buffered salinePKC Protein kinase Cpmol PicomolarPMSF Phenylmethylsulfonyl fluoridePTK Protein tyrosine kinasePTP Protein tyrosine phosphatasePTPase Protein tyrosine phosphatasePVDF Polyvinylidene difluorideSDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresisSH2 Src homology 2SH3 Src homology 3TCR T cell receptorTris Tris (hydroxymethyl) amino methaneUT Untranslated regionixACKNOWLEDGEMENTI would like to acknowledge the technical support of Peter Borodchak andLizabeth Kalt and I would like to thank Dr. Pauline Johnson, Dr. Julie Deans, andDavid Ng for stimulating scientific discussions. I especially want to thank Dr.Pauline Johnson for offering me this opportunity to do my Masters’ degree in herlaboratory, for her guidance, and for setting an example of academic rigour which Ihave tried to apply to my work. Finally, I must acknowledge the work of mysupervisory committee of Dr. Wilf Jefferies, Dr. Robert McMaster, and Dr. LindaMatsuuchi. I would like to thank them for their contributions of time, new ideasand directions, good advice, and moral support.xDEDICATIONI would like to dedicate this body of work to my parents, Mr. S. K. Maiti andMrs. J. K. Maiti, and my sister, Anita. Without their unstinting support andsacrifices too numerous to mention, I would not have been able to achieve half ofwhat I have so far. And to John, who has put up with a lot since he met me butalways listened and helped me to see things from a different perspective.xiINTRODUCTIONCD45 StructureCD45 (leukocyte common antigen, T200, B220, Ly-5) is a family of glycoproteinsexpressed exdusively on nucleated cells of the haematopoietic lineage, reviewed in [1,2,3]. This family of molecules ranges in molecular weight from 180 to 220 kDa andaccounts for as much as 10% of the membrane proteins expressed on the cell surface oflymphocytes. The structural heterogeneity expressed by CD45 relates to its pattern ofexpression in different haematopoietic cells. B cells express predominantly the higherMr 220,000 isoform; thymocytes predominantly the lower Mr 180,000 isoform; and Tcells express different isoforms that correlate with developmental stage and exposure toantigen.The Extracellular DomainCD45 is a transmembrane protein consisting of a heavily glycosylated variableamino terminal extracellular domain ranging from 404-543 amino acids, a singletransmembrane domain, and a large, highly conserved cytoplasmic domain of 705amino acids (Fig.1). The external domain of CD45 includes a variable region at the N-terminus and a cysteine rich region which may contain fibronectin type Ill repeats [4].Alternative splicing of three exons (exons 4,5, and 6) near the amino terminus of themolecule results in the expression of different isoforms of CD45 [5]. The elucidation ofthe primary structure of rat, mouse, and human CD45 from the nudeotide sequence [5,6, 7,8,9] demonstrated that each of the three alternatively spliced exons is encodes forabout 50 amino acids each [10, 11, 12, 13]. Segments of the external domain encoded byexons 4,5, and 6 are referred to as A, B, and C respectively. The CD45 isoform thatIFigure. 1. Schematic Diagram of the Structure of CD45. Regions A, B, and C (residues8-50, 51-99, 100-146, respectively) refer to the alternatively spliced exons 4,5, and 6, thatare present in the CD45RABC isoform. The amino acid numbering is based on themurine B cell isoform of CD45 [9]. The external domain (residues 1-541), thetransmembrane domain (residues 542-563), and the cytoplasmic domain (residues 564-1268) are shown. The acidic region in the second phosphatase domain (residues 958-978) is also shown.2CD451542TTTTTTTTTTTTTTTTTTTTTTI I r r PlasmaL I Membrane564Acidic Region1268ABC 147Aternatively SplicedExonsCysteine-richRegionMembrane ProximalRegionPTPase Domain ISpacer RegionPTPase Domain IIC-terminal Tail3expresses all three alternatively spliced exons, for example, is named CD45RABC, whileCD45RO refers to the isoform that expresses none of the three variable exons.Biochemical studies in the rat show that the regions encoded by the alternatively splicedexons are extensively modified by 0-linked carbohydrates [14]. Variable usage of theexons affects both the length of the molecule as well as the amount of 0-linked sugars.Many of the 0-linked sugars are highly charged by the addition of sialic acid residues[15]. The visualization of purified CD45 by low-angle shadowing [16] demonstratesthat the extracellular domain of the molecule is an extended rod.The cysteine rich region of approximately 360 amino acids contains 15 potentialsites for N-linked glycosylation in murine CD45. Inhibition of N-glycosylation,specifically by interfering with the transfer of dolichol phosphate-linked carbohydratemoieties onto asparagine residues by tunicamycin treatment inhibited the cell surfaceexpression of the 180 kDa and 190 kDa isoforms of CD45 in K562 cells [17]. Thissuggests that the addition of N-linked sugars in the Golgi apparatus is required for cellsurface expression and stability of CD45 molecules.As a transmembrane glycoprotein and putative receptor, many groups are tryingto identify CD45 ligands. It has been reported that the CD45RO isoform interacts withCD22 [18]. Recent evidence has demonstrated that a CD22-immunoglobulin fusionprotein binds to multiple isoforms of CD45 on T cells [19]. Subsequent investigation hasshown that CD22 is a sialic acid binding lectin with a specificity for N-linkedcarbohydrates containing cz2-6 sialic acid moieties on all cell surface glycoproteins [20,21], and therefore is not a CD45-specific ligand. Whether individual isoforms of CD45interact with other ligands in a developmentally regulated or cell-type specific mannerremains to be established.The identity between species in the extracellular domain is approximately 35%,yet there are key conserved residues, cysteines, tyrosines, prolines, and tryptophansthat may result in a conserved three-dimensional structure. Taken together, the proteinand carbohydrate structures in the alternatively spliced region give rise to the4variability in size and charge of CD45. This diversity in CD45 isoforms may ultimatelyaffect both intra- and intercellular interactions.The Cytoplasmic DomainCD45 has a receptor-like structure and a very large cytoplasmic domain whichincludes tandem PTPase domains [221. The function of the cytoplasmic domain wasunknown until investigators cloning a novel protein tyrosine phosphatase, PTP IB,from human placenta, showed that it exhibited 30-40% amino acid sequence similarityto two regions of CD45 [22]. Evidence that the cytoplasmic domain of CD45 hadintrinsic enzymatic activity was demonstrated by Tonks using purified CD45 [23,241.Subsequently, the generation of soluble recombinant cytoplasmic domain proteins inbacterial and baculovirus expression systems by others confirmed that CD45 was aprotein tyrosine phosphatase [25,261.The cytoplasmic domain of CD45 consists of two tandem PTPase domains of—240 amino acids separated by a 56 amino acid spacer region (Fig. 2). These two PTPasedomains which shall be referred to as domain I and domain II, share --40% sequenceidentity [22]. There is a unique region of 21 acidic residues within the secondphosphatase domain of CD45 (domain II) that contains several potential sites for serinephosphorylation by casein kinase II [221. Additionally, the cytoplasmic domaincontains a 77 amino acid membrane proximal region, and a C-terminal tail of 78 aminoacids.It had been observed that there was a requirement for sulfhydryl compounds forthe phosphatase activity of PTP 1A and PTP lB [27] and that enzymatic activity wassusceptible to inhibition by SH-modifying compounds. Thus, at least one reactivecysteine residue is essential for catalysis. Cysteine 215 of PTP lB and cysteine 1522 ofrat LAR are involved in the formation of a thiol intermediate during the5CD45 CYTOPLASMIC DOMAINMembrane Spacer COOH TailProximal PTPase Domain I Region PTPase Domain IIRegion564 641 876 932 1191 1268______________________958-978IUA\.VH©SAGVGRTG VHCRDGSQQTG\I /GXGXXGFigure. 2. Schematic Representation of the Structure of the Cytoplasmic Domain ofCD45. The representation of murine CD45 cytoplasmic domain shows the tandemPTPase domains, domain I (residues 641-875), domain II (residues 932-1190), includingthe acidic region (residues 958-978), the membrane proximal domain (564-640), thespacer region (residues 876-931), and the C-terminal tail (1191-1268). The position andamino acid sequence of the PTPase consensus sequence is shown for both PTPasedomains. The cysteine essential for activity in domain I is circled.6phosphotransfer reaction [28,29,30]. A point mutation of a highly conserved cysteinein the first tandemly repeated domain (domain I) of CD45 completely abolishedenzymatic activity [25], indicating that domain II may be inactive against substratesused in vitro as it is is unable to compensate for mutations in PTPase domain I.Mutation of the analogous cysteine in domain II resulted in an active enzyme [31] or a2-fold decrease in activity [321. Comparison of the sequences from intracellular andtransmembrane PTPases reveals a short conserved segment of amino acids surroundingthe essential cysteine (Fig. 3). This PTPase consensus sequence includes a GXGXXGmotif which resembles a glycine rich loop that is associated with the nucleotide bindingsite of G-proteins [33] and protein kinases [34].Mutational analysis of CD45 has demonstrated that expression of bacteriallyexpressed and in vitro translated PTP domain I alone does not result in an active PTPase[31,32], suggesting that domain II is required for enzymatic function. In vitro translateddomain II expressed alone, without domain I was also inactive [32]. Additionally, 5non-conserved residues in the PTPase consensus sequence of domain II were altered toconform to the consensus sequence in domain I. This mutation was made in a mutantthat had the essential cysteine 817 of domain I mutated in order to assess the activity ofdomain II only. This mutant did not possess PTPase activity. Taken together, theseexperiments suggest that domain II is not intriniscally active. Yet CD45 isolated fromlymphoid cells and subjected to limited proteolysis to generate a 50 kDa fragmentcomprising part of PTPase domain I, domain II, and the spacer region wasenzymatically active [351. The same group demonstrated that CD45 isolated fromfibroblasts transfected with a CD45 cDNA lacking most of domain I also had PTPaseactivity.Deletion of the membrane proximal region (residues 564-640) abolished PTPaseactivity, but deletion of the C-terminal tail did not [32]. Interestingly, further deletion of13 amino acids at the C-terminus of domain II was sufficient to abrogate phosphataseactivity [321, again suggesting that domain II may be required for the function of7Figure. 3. Alignment of CD45 PTPase Domains with a Consensus PTPase Sequence.Amino acid alignment of the two phosphatase domains of CD45 [9] is shown. Theconsensus PTPase sequence was derived from the alignment of ten representativephosphatase domains. The two PTPase domains of murine CD45 [9], the two domainsof LRP [37], the two domains of human LAR [38], and four cytosolic PTPases, FTP lB[39], T cell PTPase [40], HePTPase [41], and the SH2-domain containing PTPase Syp [42]are shown. In the consensus sequence, invariant residues are indicated by boldfaceletters and residues that are 90% conserved by capital letters. The most conservedregion and presumed active site is underlined. Phosphatase domain boundaries startbefore the al helix and end after the cc6 helix as defined from the crystal structure ofFTP 1B[36].8MouscD45,DcIMourn.CD45,DcIIMous•LP,DcIMourn•LRP,DcIIIuanLR,DcIKanLR,DcIIin1!o.iiPTPas.R.PTPas.SIPFSKFPIKDARKPHNQNKNRYVDILPYDYNRVELSEINGDAGSTYINASYIDGFKEPRKYIAAQGPRDRSWRTQHIGNQEENKKKNRNSNWPYDFNRVPLKHELEMSKESEPESDESSDDDSDSEETSKYINASFVMSYWKPEMMIAAQGPLKPIQATCEAASKEENKEKNRYVNILPYDHSRVHLTPVEGVPDSDYINASFINGYQEKNI(FIAAQGPKEIQNDKMRTGNLPANMKKNRVLQIIPYEFNRVIIPVKRGEENTDYVNASFIDGYRQKDSYIASQGPLLGQQFTWENSNLEVNKPKNRYANVIAYDHSRVILTSIDGVPGSDYINANYIDGYRKQNAYIATQGPLPAHTSRFISM4LPCNKFKNRLVNIMPYELTRVCLQPIRGVEGSDYINASFLDGYRQQKAYIATQGPLAASDFPCRVAKLPKNKNRNRYRDVSPFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPSHDYPHRVAKFPGNRNRNRYRDVSPYDHSRVKLQNAENDYINASLVDIEEAQRSYILTQGPLPSNFVSPEDLDIPGHASI(<FLKKVHTIEDFWRMI WEWKSCSIVMLTELEERGQEKCAQYWP——SDGLVSYGDITVELKKEEECESYTVRDLLVTNTRENKSRQIRQFHFHGWPEVGIPSDGKGINIIAAVESTEDFWRMLWEHNSTIIVMLTKLREMGREKCHQYWPAERSARYQY--—-FVVDPMAEYNMPYILREFKVTDARDGQSRTIRQFQFTDWPEQGVPKTGEGFIDFIGQVNTCGHFWEMVWEQKSRGWMLNRVMEKGSLI<CAQYWPQKEEKEMIFEDTNLKLTLISEDIKSYYTVRQLELENLTTQETREIL————HFHYTTWPDFGVPESPASFLNFLFKVNTVSDFWEMVWQEEVSLIVMLTQLRE—GKEKCVHYWPTEEETYGPFQIRIQDMKECPEYTVRHVTIQYQEERRSVKHILFSAWPDHQTPESAGPLLRLVAEV-T---FW-MVME----KC--YWPWP----PNAFSNFFSGPIWHCSAGVGRTGTYIGIDAMLEGLEAEGKVDVYGYWKLRRQRCLMV--—QVEAQYILI HQALVEYNQFGEKQI(LPKASPEGMKYHKHASILVHCRDGSQQTGLFCALFNLLESAETEDWDVFQVVKSLRKARPGW—--CSYEQYQFLYDIIASIYPAQNKACNPQYAGAIWHCSAGVGRTGTFVVIDAMLDMMHSERKVDVYGFVSRIRAQRCQ1V---QTDMQYVFI YQALLEHYLYGDQKQQQQSGNHPITVHCSAGAGRTGTFCALSTVLERVKAEGILDVFQTVKSLRLQRPHMV---QTLEQYEFCYKVVQEYIDAFSKACNPLDAGPMVVHCSAGVGRTGCFIVIDAMLERMKHEKTVDIYGHVTCMRSQRNYMV---QTEDQYVFIHEALLEAATCGHHKTKEQFGQDGPITVHCSAGVGRTGVFITLSIVLERMRYEGWDMFQTVKTLRTQRPANV———QTEDQYQLCYRALEYLGSFDRESGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVLLEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMESGSLNPDHGPAVIHCSAGIGRSGTFSLVDTCLVLMEKGDDINIKQVLLNMRKYRMGLI———QTPDQLRFSYMAIIEGAKCIKEESPETAAHPGPIWHCSAGIGRTGCFIATRIGCQQLKARGEVDILGIVCQLRLDRGGMI---QTAEQYQFLHHTLALYAGQLPHHKQESIVDAGPVVVHCSAGIGRTGTFIVIDILIDIIREKGVDCDIDVPKTIQMVRSQRSGMVQTEAQYRFIYMAVQHYIETLQVnCSAG-GR-G-FRQ---Qdomain I. Recently, the x-ray crystal structure of PTP 1B, a cytosolic PTPase [361 wasdetermined. The elucidation of the crystal structure of the phosphatase domain of PTPlB will aid in determining more accurately, which regions of the cytoplasmic domain ofCD45 contribute to phosphatase activity and the regulation of that activity.CD45 Function in the Immune SystemSurface expression of CD45 has been shown to be necessary for effectiveactivation of both B and T lymphocytes through their antigen receptors [43,44,45] aswell as for thymic development [46,47]. CD45 is required for the appearance of theearliest T cell receptor (TCR) mediated signalling event; the induction of proteintyrosine phosphorylation. CD45-negative clones of human Jurkat and HPB-ALL cellswere unable to induce the tyrosine phosphorylation of proteins upon anti-TCR or antiCD3 stimulation [48,49]. Engagement of the TCR in cells lacking CD45 fails to lead tophospho-inositide hydrolysis or increases in intracellular calcium [45]. These cells areunable to proliferate or produce cytokines such as interleukin-2 in response to antigen.A CD45-negative cytotoxic T cell done failed to proliferate and was unable to cytolysetarget cells upon antigen presentation [50]. Interestingly, this cell line expressed LRP,another transmembrane PTPase, yet this PTPase was unable to compensate for adeficiency in CD45 expression [3]. Transfection of CD45-defident cells with chimericCD45 molecules, consisting of the cytoplasmic domain of CD45 and the external domainof either the EGF receptor, the p60SC myristylation site, or the external domain of MHCClass I, demonstrates that the cytoplasmic domain is sufficient to restore TCR/CD3mediated signalling events [51,52,53], although it remains to be determined if theextracellular domain is involved in regulating these events by differential isoformexpression or ligand binding.The induction of protein tyrosine phosphorylation events upon T cell stimulationis thought to occur as a result of the activation of protein tyrosine kinases (PTKs)10associated with the TCR/CD3 complex [54,55,56]. Candidate kinases include the srcfamily kinases such asp561ck and p59fYII as well as a member of a new family of PTK,ZAP-70 [57]. Both pS6lCk and p59fY’ have been identified as potential substrates forCD45 [26,58,59,60,61]. CD45 can dephosphorylate the negative regulatory tyrosine(tyrosine 505 and 531, respectively) of p561Ck and p59fY and this is thought to activatethe kinase [59,62,63]. CD45 may be required either to activate these kinases uponstimulation through the TCR/CD3 complex or to maintain these kinases in an activestate ready for T cell stimulation. In addition, CD45 itself may participate in theTCR/CD3 signalling complex. The exact role of CD45 in T cell receptor mediatedsignalling is not yet clear.One approach to study CD45 function is to transfect CD45 mutated genes intoCD45-deficient T cells. Unfortunately, it has been difficult to reconstitute CD45-negative T cells [3,49] and the reasons why this occurs are not understood.Summary of IntentionsThe general aim of this work was to understand further the molecular function ofCD45. Although CD45 isoforms are expressed in a developmentally regulated and cell-type specific manner, the function of individual isoforms is unknown as is the identityof isoform specific ligands. To further complicate matters, lymphocytes often expressmultiple isoforms at any one time. By expressing individual isoforms in a fibroblast cellline, one can begin to look for potential isoform specific ligands. One aim of this thesiswas to transfect two isoforms of CD45 into the L tk fibroblast cell line and characterizetheir expression. Two additional mutants of CD45 were also expressed and these cellswere characterized to determine if these mutations dramatically affected cell surfaceexpression. L cell transfectants were characterized with respect to expression of CD45and transport of CD45 through the endoplasmic reticulum and medial golgi apparatus.These L cell transfectants may be useful for the identification CD45 ligands and in order11to determine how interactions with the external domain of CD45 influences its PTPaseactivity.The role of the cytoplasmic domain in T cell activation has been shown to becrucial, but what aspects of the cytoplasmic domain are required have yet to beestablished. In addition, it is still unclear whether domain II of CD45 has a regulatoryfunction or some other activity. To further examine this question, the second aim of thisthesis was to determine which amino acids in domain II were important for PTPaseactivity.The effect of targeted mutations on phosphatase activity can be studied in vitro,but the elucidation of amino acids important for regulation of CD45 activity andrestoration of T cell signalling require studying cytoplasmic domain mutants in vivo.CD45-deficient cells provide an experimental system that can be used to study theregions of CD45 that are important for physiological functions by assessing the abilityof CD45 mutants to reconstitute T cell signal transduction. In order to determine theeffects of CD45 reconstitution with cytoplasmic domain mutants, it is necessary to firstcharacterize the defects in TCR-mediated signalling in the CD45-deficient cells. TheBW5147 CD45-negative (TCR, CD3) cells [64] were deficient in the induction ofphosphoproteins upon stimulation through the T cell receptor (P. Johnson, unpublishedresults). In addition, hyperphosphorylation of the negative regulatory tyrosine 505 ofp561Ck was observed in the CD45-negative BW5147 parental cell line [26]. While p59fYflhas been identified as a potential substrate of CD45 [59,60,61], it was not knownwhether p59Y” was expressed in these cells or if CD45 affected the phosphorylationstate of fyn. Thus, the third aim of this thesis was to determine what the effect of CD45expression was on the phosphorylation state and kinase activity of p59fYfl in the CD45-negative BW5147 cells. Ultimately, the reconstitution CD45-deficient cells with CD45mutants will help us to better understand the role of individual regions of CD45 inphosphatase activity and T cell receptor-mediated signalling.12Thus the three main aims of this thesis were 1) to characterize CD45 transfectedinto L tk cells; 2) to generate mutations in the cytoplasmic domain of CD45 and testtheir PTPase activity after expression in E. coil.; and 3) to characterize a recipient CD45-negative T cell with respect to the tyrosine phosphorylation state and kinase activity ofthe src-family kinase, 59fyn.13MATERIALS AND METHODSCell CultureL tk fibroblasts (American Type Culture Collection, Rockville, Maryland) weremaintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10%fetal calf serum and 100 units/mL of penicillin, 100 g/mL streptomycin, and 0.25p.g/mL amphotericin B (Gibco BRL Life Technologies, Burlington, Ont.). BW5147CD45-positive and CD45-negative T lymphoma cells (available from the ATCC,Rockville, Maryland) were transfected with CD36 and CD3C which resulted in theexpression of surface T cell receptor! CD3 complexes [64] (a generous gift from Dr. B.Malissen). These cells were maintained in DMEM supplemented with 10% horseserum, 100 units/mL penicillin, 100 .tg/mL streptomycin, 0.25 p.g/mL amphotericin Band contained 3 mM histidinol (Sigma Chemical Company, St. Louis, Missouri) tomaintain the expression of transfected plasmids. All cells were incubated at 37°C in 5%CO2. Cells were checked routinely by flow cytometry to ensure similar levels ofTCR/CD3 were expressed in CD45-positive and CD45-negative BW5147 cell lines.AntibodiesAnti CD45 antibodies used were 13/2, a rat antibody against a pan-specific CD45determinant in mice [651 (gift of Dr. I. Trowbridge), Ly5.2, clone 104-2.1, an allele-specific antibody (gift of Dr. S. Komuro), 131, an anti-peptide blotting antiserum againstamino acids 211-250 of the external domain of CD45 (gift of Dr. J. Marth), and anti95kD, a rabbit antiserum against the cytoplasmic domain of CD45 (gift of Dr. H.Ostergaard). CD45 exon-specific antibodies used for flow cytometry were RA3-2C2[66], a rat anti mouse 1gM versus exon A, MB4B4, a rat anti mouse exon B specificantibody [67] (American Type Culture Collection, Rockville, Maryland), and DNL-1.9,an exon C specific rat anti mouse antibody [68] from Pharmingen (San Diego, Ca.).14Murine anti-fyn antiserum was a generous gift of Dr. R. Perimutter. Anti murine CD4antibody GKI.5, (ATCC TIB 207) was obtained fom Dr. H-S. Teh, and anti-CD44antibody 1M7.8.1 (ATCC TIB 235) was a gift from Dr. R. Hyman. Hamster anti-mouseanti-CD3e antibodies (145-2C11) and anti-TCR chain antibodies (H57-597) wereobtained from the ATCC (Rockvile, Maryland). Goat-anti-rat and goat-anti-hamsterfluorescein isothiocyanate (FITC) labeled antibodies were purchased from Pierce(Rockford, illinois) and Southern Biotech Associates Ltd. (Birmingham, Alabama)respectively. The anti-phosphotyrosine antibody (4G10) was purchased from UpstateBiotechnology Inc. (Lake Placid, N.Y.). Protein A-HRP and goat-anti-mouse-HRP werepurchased from Bio-Rad Laboratories Ltd. (Mississauga, Ont.).Transfected CD45 cDNA ConstructsPlasmids transfected into L cells included CD45RABC (#106), CD45RO (#36),CD45RABC C817S (#64), and CD45RABC zcyt (#12) and were constructed by P.Johnson and L. Melito. The Cia I-Xho I fragment encompassing the complete CD45cDNA was doned into the Sal I site of the pHf3apr-1-neo [69] mammalian expressionvector. CD45 cDNAs were missing most of their 5’ and 3’ untranslated regions with theexception of the CD45RO construct (#36) which still contained its 5’UT and 3’ UT.Transfection of L tk- cells with CD45 cDNAsL cells were maintained in 10 mL DMEM, 10% fetal calf serum (FCS), andantibiotics on Nunc 100 mm2 tissue culture dishes (Gibco BRL Life Technologies,Burlington, Ont.) until they were 20-40% confluent, at which point they weretransfected by the calcium phosphate method [70]. 20 pg of DNA was prepared byethanol precipitation and resuspended in 750 ilL of sterile water and added to 250 p.L of2M CaCl2 in an eppendorf tube. lmL of 2X Hepes buffered saline (HBS; 280mM NaC1,50 mM Hepes pH7.l, 1.5 mM NaH2PO4) was aliquoted into 5 mL Falcon 2058 tubes(Baxter-Canlab, Mississauga, Ont.) and the DNA/CaC12mixture was added dropwise15while simultaneously vortexing the Falcon tube. A cloudy fine precipitated was formedwhich was left at room temperature for 30 minutes. 2 mL of DMEM was removed fromthe cells and the DNA/CaPO4precipitate was dotted onto the plate with a pasteurpipet. The plate was rocked gently and a fine precipitate was observed on the cellsunder a Zeiss phase-contrast microscope. The cells were left at 37°C for five hours, atwhich point the media and precipitate was removed and 10 mL of fresh DMEM plus10% FCS was added to the plates. Cells were selected for stable transfectants by theaddition of 1 mg/mL of G418 (Geneticin, Gibco BRL Life Technologies, Burlington,Ont.) 48 hours post-transfection.Flow Cytometry2 X iO cells were incubated with 100 .iL of tissue culture supernatant containingthe appropriate monoclonal antibody for 20 minutes on ice. After washing the cellswith phosphate buffered saline (PBS; 154 mM NaCl, 2.7 mM KC1, 4.3 mM Na21-1P04, 1.5mM KI-12P04)containing 2% FCS, they were incubated for 20 minutes on ice with 100.tL of a 1/100 dilution of FITC labeled secondary antibody. 2 mM ethylenediaminetetra-acetic acid (EDTA) was added to the L cells to prevent re-adherence. Cells werewashed and analyzed on a FACSCAN (Becton Dickinson, Mississauga, Ont.) analyzer.Positive Selection of CD45 L cell Transfectants with Magnetic BeadsAfter 14 days in G418 selection, L cells were positively selected for CD45expression with magnetic beads. 5 X 106 cells were taken off the plates with 2 X Versene(PBS plus 0.7 mM EDTA) for 10 minutes at room temperature, washed in sterile PBSwith 2% FCS and 2 mM EDTA and resuspended in 200 j.tL of the PBS/2% FCS/ 2 mMEDTA solution. 10 jig of purified 13/2 antibody was added to the cells and incubatedfor 30 minutes on ice, resuspending the cells every 10 minutes. Cells were then washed3 times and resuspended in 300 j.ii of the PBS solution, to which 50 p.L of goat-anti-ratmagnetic beads (Collaborative Research Inc., Bedford, Massachussetts) or 15 jiL of16Dynabeads M-450 sheep-anti-rat IgG (Fc) magnetic beads (Dynal Inc., Great Neck, NewYork) were added. The cells and beads were left on ice for 30 minutes, resuspendingthe cells every 10 minutes. The labeled cells were then poured into a 5 mL uncoatedFalcon dish and selected on a magnetic plate. The wash solution and non-adherent cellswere aspirated and the cells washed and selected on the magnetic plate two more times.This selection process was repeated at least four times on all tranfectants before the cellswere doned out and screened by flow cytometry.CD45 Immunoprecipitation and Western BlottingTo immunoprecipitate CD45, the appropriate number of cells were lysed in icecold lysis buffer (1% Triton-X-100, 150 mM Nacl, 20 mM Tris-HC1 pH7.4, 2 mM EDTA, 5mM sodium orthovanadate, 2 mM sodium molybdate, 2 mM PMSF, 10 .tg/mLleupeptin, 10 p.g/mL aprotinin, and 10 mg/mL pepstatin) and left on ice for 10 minutes.Cell lysates were then centrifuged at 12 000g for 10 minutes to remove the insolublepellet and incubated with 200 jiL of Ly5.2 tissue culture supernatant for 30 minutes at4°C rotating end-over-end, after which point 10 iLL of a 50% slurry of protein Gsepharose was added for an additional hour. Surface CD45 was immunoprecipitatedfrom unlysed cells that had been washed in PBS by incubating unlysed cells for 30minutes with 200 j.iL of Ly5.2 tissue culture supernatant at 4°C rotating end-over-endand then washed once in PBS. Cells were then lysed as mentioned above, and 10 j.tL ofa 50% slurry of protein G sepharose was added to the centrifuged lysate which was thenrotated end-over-end for 1 hour at 4°C. Immunoprecipitates were washed 3 times inlysis buffer and resuspended in 10 iLL of 2 X SDS sample buffer (250 mM Tris-HC1pH6.8, 20% glycerol, 200 mM dithiothreitol, 0.02% bromophenol blue) and boiled for 5minutes before loading on 10% SDS polyacrylamide gels. Gels were run by theLaemmli method with prestained standards (Bio-Rad Laboratories Ltd., Mississauga,Ont.) and tranferred to a polyvinylidene difluoride (PVDF) Immobion-P membrane(Milhipore Canada Ltd., Mississauga, Ont.) in a transblot apparatus (Bio-Rad17Laboratories Ltd., Mississauga, Ont.) according to manufacturers instructions andblotted with 131 antibody at a 1/1000 dilution in 5% BSA-TBST (0.1% Tween 20, 150mM NaC1, 20 mM Tris-HC1 pH7.5, including protease and phosphatase inhibitors) for90 minutes after blocking for 90 minutes in 5% BSA-TBST. Membranes were washed inTBST and incubated with a 1/10 000 dilution of HRP conjugated protein A in 5% BSATBST for 45 minutes, washed thoroughly, and developed using the enhancedchemiluminescence assay according to manufacturers instructions (ECL kit, AmershamCanada Ltd., Oakville, Ont.).3S Pulse Chase Labelling and Endoglycosidase H Digestion of ProteinsTwelve 100 mm2plates of cells per clone at 50% confluency (—2.5 X 106 cells)were washed in 5 mL of PBS and then starved of methionine and cysteine by incubationin 4 mL warm DMEM methionine(-) and cysteine(-) media (ICN Biomedicals, Inc., St.Laurent, Que.) for 20 minutes at 37°C. The cells were then pulsed with radiolabel for 30minutes at 37°C. The label mixture consisted of 2.5 mL of DMEM methionine(-)cysteine(-) media supplemented with 250 tL of dialyzed FCS, 25 jiL of Glutamax I(Gibco BRL life Technologies, Burlington, Ont.) and 100 j.tCi/mL of Promix L-[35S] invitro cell labelling mix (specific activity >1000 Ci/mmol, 70% methionine, 30% cysteine;Amersham Canada Ltd., Mississauga, Ont.). After the pulse period, the labeling mediawas removed and the cells washed in 5 mL of PBS before chasing with DMEM plus 10%FCS for 0, 15,30,60, 120, and 180 minutes. At the end of the chase period, the mediawas removed, the cells washed in 5 mL of PBS and then lysed in the plates with 1 mL oflysis buffer (1% Nonidet P-40, 120 mM NaC1, 4 mM MgC12, 20 mM Tris-HC1 pH7.5).The plates were left on ice for 5 minutes, then the cells were dispersed by pipetting,transferred into an eppendorf tube, vortexed, and left on ice for a further 10 minutes.Lysates were centrifuged at 12 000g for 10 minutes at 4°C and the supernatant decantedfrom the insoluble pellet. Labeled cell lysates were precleared twice with 20 p.L of a 50%slurry of CL-4B Sepharose (Pharmacia, Baie d’Urfe, Qué.) for 1 hour at 4°C rotating end-18over-end. Ly5.2 antibody (200 iiL) precoupled to protein G (Pharmacia, Bale d’Urfe,Que.) for 2 hours was then added to the predeared lysates for 1 hour at 4°C rotatingend-over-end to immunoprecipitate CD45. Immunoprecipitates were washed threetimes in low salt buffer (1% NP-40, 10 mM Tris-HC1 pH7.5, 150 mM NaC1, 2mM EDTA),twice in high salt buffer (1% NP-40, 10 mM Tris-HC1 pH7.5, 500 mM NaC1, 2 mMEDTA), and once in 10mM Tris-HC1 pH7.5. Cells treated with Endoglycosidase H(Boerhinger Mannheim Canada, Laval, Qué.) were digested by the addition of 12 p1 of85 mM sodium citrate pH5.5, 4 p.L of Endo H (4 mU), and 2 ,tL of 200 mM PMSF andincubated at 37°C overnight. All immunoprecipitates were boiled for 5 minutes in 2 XSDS sample buffer and run on 16 mm2, 7.5% SDS-polyacrylamide gels overnight at 15mA. Gels were fixed (40% methanol, 10% acetic acid) for 1 hour, placed inEn3hance(NEN DuPont Canada, Mississauga, Ont.) for 1 hour and then placed in Nanopurewater (Millipore Corporation, Bedford, Massachussefts) for 30 minutes with gentlerocking. Gels were then dried for 90 minutes at 70°C and exposed at -70°C with KodakX-Omat AR film and an intensifying screen for three days.Generation of Single Stranded DNA from PhagemidsPlasmid #592 [26], which encodes for the cytoplasmic domain of CD45 clonedinto the Cia I-Xho I site of pBluescript SK (+1-) (Stratagene Cloning Systems, La Jolla,Ca.), was transformed into Escherichia coli strain CJ236 (Bio-Rad Laboratories Ltd.,Mississauga, Ont.) which expresses the dut(-) ung(-) phenotype. Likewise, piasmid #26,which encodes for the complete CD45 protein was cloned into the Cia I-Sal I sites ofpBiuescript SK (+1-) and also transformed into CJ236 E. coli. Single stranded DNA wasgenerated by the addition of 300 .tL of helper phage R408 (Bio-Rad Laboratories Ltd.,Mississauga, Ont.) to a log phase culture of plasmid #592 or piasmid #26 in CJ236bacteria grown in Luria Broth supplemented with 100 g/mL ampicillin and 30 p.g/mLchioramphenicol. The R408 helper phage was titrated at 1012 colony forming units/mL.19Single stranded DNA was isolated by established methods [71] and determined to be ata concentration of 100 ng/.tL.Oligonucleotide PurificationOligonucleotides were ordered from the Nucleic Acid-Protein Synthesis Unit(NAPS Unit, U.B.C.) and purified either on a C18 Sep-Pak column (MilliporeCorporation, Bedford, Massachussetts) or by the n-butanol method [72]. Lyophilizedoligonucleotides were dissolved in 3 mL of 0.5 M ammonium acetate (BDH Inc.,Vancouver, B. C.) and loaded onto a C18 column that had been reconstituted with 10 mLof 20% acetonitrile (BDH Inc., Vancouver, B. C.) and equilibrated with 10 mL of waterand 10 mL of 10 mM ammonium acetate. The column was washed with 10 mL of waterand 10 mL of air pushed through with a 10 mL syringe. The oligonudeotides wereeluted with 3 Xl mL of 20% acetonitrile, dried on a Savant speed-vac and resuspendedon 500 iLL of sterile water. N-butanol purification involved the dissolution oflyophilized oligonucleotide in 100 jiL of 30% ammonium hydroxide (BDH Inc.,Vancouver, B. C.) to which 1 mL of n-butanol (BDH Inc., Vancouver, B.C.) was added.The mixture was vortexed, centrifuged at 12 000g and the single aqueous phasediscarded. The oligonucleotide pellet was resuspended in 100 .tL of water and reextracted with I mL of n-butanol once more. The pellet was dried under vacuum andresuspended in 500 jtL of sterile water. The optical density at 260 nm was used todetermine the concentration of the purified oligonucleotides whereconcentration=O.D. /extinction coefficent in j.tmoles /mL. lox the oligonucleotidelength is the calculation used for determining the extinction coefficient. Alloligonucleotides were heated to 80°C and rapidly frozen to prevent self-hybridization.Oligonucleotide-directed MutagenesisMutagenesis was performed as per the method described by Kunkel [73,741. Theannealing reaction was carried out by heating template and oligonucleotide at 80°C and20letting the reaction cool slowly to —30°C. Single stranded plasmid #592 and #26 wereused at a concentration of 100 ng/pL and 10 ng/p.L, while working amounts ofoligonucleotide used were 0.1 pmol, 2 pmol, and 10 pmol. The annealing reactionswere performed in lox annealing buffer (200 mM Tris-HC1 pH7.4, 20 mM MgC12, 500mM NaC1) in a final volume of 10 p.L. After annealing, the synthesis reactioncommenced by the addition of 2-5 U of T4 ligase (8000U/mL; Pharmacia, Bale d’Urfe,Qué.), 1 U of T4 polymerase (6700U/mL; Pharmacia, Baie d’Urfe, Qué.), and I ji.L of lOXsynthesis buffer (5 mM dATP, 5 mM dCTP, 5 mM dGTP, 5 mM dfl’P, 10 mM ATP, 100mM Tris-HC1 pH7.4, 50mM MgCl2, 20 mlvi DTT). The synthesis reaction was placed onice for 5 minutes, at 25°C for 5 minutes, and then at 37°C for 90 minutes. AU of themutagenesis reactions were transformed into competent E. coli strain XL-1 Blue(Stratagene Cloning Systems, La Jolla, Ca.) and selected on Luria broth/l.2% agar platessupplemented with 100 p.g/mL of ampicillin. Oligonudeotides used to generatemutations in the cytoplasmic domain of CD45 were UBC #9 (5’-pGGA-ACT-GGT-ACC-CCT-CAT-AGC-TGC-3’) which mutated glutamine 1180 in PTPase domain II to aglycine, UBC #10 (5’-pGTT-CTT-CTr-CTr-ATr-TTC-CAC-TAA-AGC-3’) which deletedthe spacer region between PTPase domains I and II, and UBC #11 (5’-pGTT-CAT-CTA-AAT-TGG-CGC-CTC-TrT-TCT-TGC-3’) which simultaneously mutated serine 573 andserine 574 to a glycine and alanine respectively. Both the first and last mutationsincorporated a new restriction endonuclease site in the mutant, with the exception ofthe spacer deletion, which resulted in a loss of 168 nucleotides. Colonies were screenedby restriction digest analysis for the generation of mutants. All potential mutants weresequenced by the Sanger chain termination method using double stranded template, theSequenase Version 2.0 DNA sequencing kit (United States Biochemical Corporation,Cleveland, Ohio) and355cc-dATP (>1000 Ci/mmol; NEN DuPont Canada, Mississauga,Ont.) according to the manufacturer’s instructions. Clones generated were C3.1 andC3.2 for the Q1I8OG mutant, H8.8, H9.1, and H9.11 for the spacer deletion mutant, and4.18 and 18.35 for the S573GS574A mutation in the membrane proximal region.21Generation of the Shuttle VectorThe pBluescript SK (+1-) vector (Stratagene Cloning Systems, La Jolla, Ca.) wasmodified to remove a portion of the multiple cloning site (MCS) by cutting with Xba Iand Hind ifi, filling in the cohesive ends and blunt-end ligating the ends. Unique siteslost from the MCS were Spe I, Barn HI, Srna I, Pst I, Eco RI, Eco RV, and Hind Ill. TheXba I site was regenerated by this procedure. Two independent clones of this vectorwere called pBS #1 and pBS #5. pBS #5 was then cleaved with Cia I-Sal I,dephosphorylated, and ligated with a Cla I-Sal I fragment encoding the full CD45cDNA and named #264 and #268. Vectors #264 and #268 were then digested with AccI-Xho I at the 3’ end of the CD45 insert and ligated to a linker oligo with the samecohesive ends. This linker was generated by hybridizing two non-palidrornicoligonucleotides (UBC #43 and UBC #44) comprising Acc 1-Spe I-Bgl 11-Not I-Xho Isites and the resulting plasmids were numbered #647 and #682. Plasmid #682 wasdigested with Cia I at the 5’ end of the CD45 insert and ligated to a Cia I-Xho I-Cia Ipalindromic linker (UBC #29; 5’-pCGA-TAC-TCG-AGT-AT-3’) to incorporate an Xho Isite at the 5’ end of the CD45 cDNA. This plasmid was numbered #822 and henceforthcalled the shuttle vector (Fig. 4). This vector was constructed to easily subdonefragments of the CD45 cytoplasmic domain encoding site-directed mutations. Theresulting mutant in the shuttle vector could then be digested with Bgl 11 to generate a 2kb fragment representing the cytoplasmic domain of CD45 that could be inserted into abacterial expression vector or cut with Cia I-Sal I or Xho I-Not I to express mutants inmammalian expression vectors. The Q118OG domain 11 mutant on plasmid #592 wascut with Sma I-Xba I and ligated into the Sma I-Spe I sites of the shuttle vector to enablethe eventual subcloning of this particular mutant into a bacterial expression vector.Subcloning of Mutants into a Bacterial Expression Vector22Sma I5’ LinkerIFigure. 4. Schematic Representation of the Shuttle Vector. The shuttle vectorcontaining a CD45 insert comprising the whole molecule was constructed on thepBluescript 5K +1- plasmid (see Materials and Methods). The size of the whole vectoris 6.78 kb, and the CD45 insert is approximately 4 kb. Linkers at the 5’ and 3’ end areshown to demonstrate the extra restriction sites engineered for ease of subsequentsubcloning. Selected restriction sites shown because they are unique or are useful forscreening purposes. A region of the multiple cloning site 5’ of the linker in pBluescriptwas removed. This region includes several restriction sites: (5’-3’) Spe I, Barn Hi, Sma I,Pst I, Eco Ri, Eco RV, and Hind ifi.BarnXbaIEcoRiBarn HiShuttle Vector#822Xba IBglll3,Sal I/Acc ISpe IBgl IINot IXho I23The shuttle vector with the Q118OG mutation in CD45 was digested with Bgl II toproduce a 2 kb insert comprising the cytoplasmic domain. This insert was ligated to thepET-3d-6His-IEGR bacterial expression vector (constructed by Dr. P. Johnson) that hadbeen linearized with Barn HI and dephosphorylated and the ensuing plasmid wasnamed C3.lpx. The pET-3d [75] bacterial expression vector includes a 6 histidine tag toenable purification on a nickel column and a Factor Xa cleavage site; IEGR, to allow forpurification of recombinant proteins away from the 6 histidine tag. To subclone thespacer deletion mutant into the pET-3d-6His-IEGR plasmid, the spacer deletion mutantthat had been generated on the #592 plasmid (clone H8.8) was digested to produce aKpn I-Barn HI fragment. This fragment was exchanged with a Kpn 1-Bam HI fragmentincluding the spacer region in the bacterial expression vector expressing the wild-typeCD45 cytoplasmic domain (pET-3d-6His-IEGR-CD45wt) to produce H8.8px, the spacerdeletion mutant in the pET-3d-6His-IEGR expression plasmid in XL-1 Blue E. coil.Finally, the S573GS574A mutation in the pBS-CD45 #26 vector was cut with Bgl II toisolate a 2 kb cytoplasmic domain fragment that was then ligated into the Barn HI site ofpET-3d-6His-IEGR and the clones named 4.l8px and 18.35px. These plasmids weresubsequently transformed into BL2I (DE3) E. coil that contain a 17 polyrnerasecontaining plasmid that can be induced by the addition of IPTG. The 17 polymerasethen binds the 17 promoter in the pET vector system to induce the synthesis ofrecombinant proteins, in this case the CD45 cytoplasmic domain.Bacterial Expression and Partial Purification of Mutant Proteins10 mL of Luria broth supplemented with 100 .tg/mL of ampidillin wasinoculated with 100 j.i.L of an overnight culture of the appropriate mutants in BL21(DE3) E. coil and grown at 37°C to an O.D.600 of 0.6 (—2 hours) and then induced for 2hours at 30°C with 40 mM IPTG. Bacteria were then lysed by spinning the bacteria in 50mL Falcon tubes at 3000 r.p.m. in a Beckman GPR centrifuge for 15 minutes at 4°C. Thebacteria were then resuspended in 2 mL of lysis buffer with 1 mM EDTA pH8.0 (50 mM24Tris-HC1 pH7.5, 100 mM NaC1, 2 mM PMSF, 10 .tg/mL leupeptin, 10 aprotinin,and 10 p.g/mL pepstatin), re-centrifuged at 12 000g and then resuspended in lysis bufferwithout EDTA. 20 iL of lysozyme (10 mg/mL stock solution) was added to the lysatewhich was then frozen in a dry ice-ethanol bath. After allowing the lysate to thaw atroom temperature, 40 jtL of a 20 mg/mL solution of sodium deoxycholate was addedand the solution was left at 4°C for 15 minutes, rotating end-over-end. 2 p.L of DNase I(10 mg/mL) was then added to remove the nudear material by placing the lysate at37°C for 10 minutes. The nuclear material was removed by centrifugation at 12 000gand the resulting supernatant was immunoprecipitated with 10 p.L ofNTA2-agarosebeads which binds the 6 histidine tag, for 90 minutes at 4°C rotating end-over-end. Theimmunoprecipitated recombinant proteins were eluted in 1 M imidazole pH7.2, 0.1%Triton X-100 and subsequently used in a PTPase assay.PTPase AssaysEqual volumes of recombinant protein were assayed for tyrosine phosphataseactivity against an 13-mer fyn peptide phosphorylated on tyrosine 531(TATEPQpYQPGENL), synthesized by Dr. Ian-Clark Lewis (Biomedical ResearchCentre, U.B.C.) using a non-radioactive, colourimetric assay. This substrate wasconsidered to be biologically relevant with respect to CD45 phosphatase activity as ithad been demonstrated that CD45 was able to dephosphorylate tyrosine 531 of p59Y invitro [59] [59,60,63]. This assay measures phosphate release by the development of agreen colour measured at O.D.6ij [76]. Activity was normalized for equal amounts ofrecombinant CD45 cytoplasmic domain proteins by running equal volumes ofrecombinant proteins on a 7.5% SDS-polyacrylamide gel stained with Coomassie Blue(40% methanol, 10% acetic add, 0.2% Coomassie Blue R-250). Amounts of protein werequantified by scanning densitometry of bands using Quantity One software (PDI Inc.,Huntington Station, N.Y.). The recombinant protein was suspended in 10 iLL of 1XPTPase buffer (50 mM Imidazole pH7.2, 1 mM EDTA, 0.1% f3-mercaptoethanol, 2mM25PMSF) and placed in a 96 well half-well plate (A/2 plates, Costar/Nudepore CanadaInc., Toronto, Ont.) and left to equilibrate at 30°C, rotating at 140 r.p.m. The fyn pY531peptide (original concentration 13.8 M) was diluted to a final concentration of 4 mM inlox PTPase buffer (500 mM imidazole pH7.2, 10 mM EDTA, 1% 3-mercaptoethanol, 20mM PMSF), and 10 I.L of this peptide was added to the 10 L sample and rotated at 30°C for the required time points, usually 0,3, and 6 minutes. The reaction was stoppedby the addition of 80 ji.L of malachite green solution (1 part 0.135% Malachite greenoxalate salt, I part 4.2% ammonium molybdate, 0.01% Tween 20, and 2 parts sterilewater), incubated at room temperature for 15 minutes to allow for the development ofthe green colour and read on a plate reader at 0.D. using Softmax software(Biotechnology Lab, U.B.C.). To determine nmol of P04 hydrolyzed/0.D.650, 1 mMKH2PO4was serially diluted twelve-fold in duplicate and assayed by the addition of themalachite green stop solution at 0.D.650 and a standard curve was constructed.CD3 Stimulation and Analysis of the src-family kinase p59fY9 X 106 BW5147 CD45-positive and CD45-negative cells were resuspended in 90).LL of pre-warmed DMEM and equilibrated at 37°C for 10 minutes. 9 I.Lg of purified 145-2C11 was added at time zero to stimulate the cells. The cells were incubated at 37°C forthe appropriate amount of time and then lysed by adding ice cold iox lysis buffer (10%Triton X-100, 1.5 M NaC1, 200 mM Tris-HC1 pH7.4, 20mM EDTA, 5 mM sodiumorthovanadate, 2 mM sodium molybdate, 2 mM PMSF, 10 p.g/mL leupeptin, 10 ).Lg/mLaprotinin, and 10 p.g/mL pepstatin). Cell lysates were placed on ice for 10 minutes andthen centrifuged at 12 000g for 10 minutes to remove the insoluble pellet. Lysates werethen added to 30 p.L of protein A (Pharmacia, Bale d’Urfe, Qué.) that had beenprecoupled to 0.4 iL of murine fyn antiserum and 1 mL of 1X lysis buffer. Theseamounts had been previously determined to be sufficient to precipitate p59Y’ from I0BW5147 cells (unpublished observations). Immunoprecipitates were washed threetimes with 1% Triton X-100, 150 mM NaC1, 20 mM Tris-HC1 pH7.5,2mM EDTA, 5 mM26sodium orthovanadate, 2 mM sodium molybdate and protease inhibitors, before beingdivided into three equal aliquots. 3 X 106 cell equivalents were run on a 7.5% SDSpolyacrylamide gels, transferred to a PVDF membrane, and blotted either with the 4G10anti-phosphotyrosine antibody at a dilution of 1/1200 in 5% BSA-TBST or with thep59Y” antiserum in 5% BSA-TBST at a dilution of 1/1000. 3 X 106 cell equivalents werewashed twice in kinase buffer (10 mM MnCl2, 40 mM Pipes pH7.2) and then used in anin vitro kinase assay.In Vitro Kinase AssaysImmunoprecipitated p59fYll was resuspended in a final volume of 10 j.tL kinasebuffer containing 5 pCi32P-y-ATP (specific activity —3000 Ci/mmol, AmershamCanada, Ltd., Mississauga, Ont.) and incubated at room temperature for 10 minutes.The reaction was stopped with 25 mM EDTA in kinase buffer and theimmunoprecipitate washed three times with kinase buffer prior to running on a 10%SDS polyacrylamide gel. The gel was dried and then exposed to Kodak X-Omat ARfilm with an intensifying screen.27RESULTS1.0 Expression and Characterization of CD45 in L tk cellsAs expression of CD45 in lymphoid cells proved difficult, characteristics ofindividual isoforms were analyzed in L tk- cells. In addition, two mutant forms ofCD45 were also expressed in these cells.1.0.1 Characterization of Expression of Two CD45 Isoforms and Two CD45Cytoplasmic Domain Mutants Transfected into L CellsTwo isoforms of CD45, CD45RABC and CD45RO, encoded by plasmid #106 andplasmid #36, were transfected into L cells (Fig. 5). Additionally, two cytoplasmicdomain variants of CD45 were transfected into L cells in order to evaluate the structuraland functional effects of the cytoplasmic domain on CD45 expression (Fig. 5). Themutant represented by CD45RABC C817S (plasmid #64) consists of an intactcytoplasmic domain with the tyrosine phosphatase activity rendered inactive by themutation of cysteine 817 to a serine. This essential cysteine is required for catalysis ofthe phosphate hydrolysis reaction [25,28]. The CD45RABC Acyt construct (plasmid#12) represents a mutation in which the cytoplasmic domain has been deleted, with theexception of the first six amino acids of the membrane proximal region. Thetranscription of the CD45 cDNA was driven by the f-actin gene promoter in thepHapr-1-neo mammalian expression vector [69]. For each of the isoforms transfectedinto L cells, approximately 25 clones were tested by flow cytometry for CD45 expressionand 2 clones were found to be positive for CD45 expression (Table I). One clone foreach isoform transfected was taken for further study. These clones are L106A6(CD45RABC) and L36B3 (CD45RO). Only one CD45RABC C817S clone (L64C1) out of64 clones tested by flow cytometry was positive for CD45 expression (Table I). In28CD45ROCD45RABC 817SCD45RABC Acyt-Actin MammalianExpression Vector.-“-— III II1I::N I C817SI1:::: I IFigure 5. Schematic Diagram of the -actin Mammalian Expression Vector and theCD45 Constructs Transfected into L cells. The 13-actin mammalian expression vectorand a representation of the CD45 constructs are shown. The CD45RABC, CD45RABCC817S, and CD45RABC Acyt constructs had their 5’ and 3’ untranslated regionshortened by the deletion of nudeotide sequences to promote mRNA stability. TheCD45RABC Acyt construct induded 6 amino acids of the cytoplasmic domain to preventsecretion of the molecule. A, B, and C refer to the three alternatively spliced exons andTM refers to the transmembrane domain.-actinSV2neoamp rInterveningSequencesMultipleCloning SiteCONSTRUCT A B C TMCD45RABC I l:29TABLE I.Number of Number ofCD45 Constructs Neor Colonies CD45-positiveScreened ColoniesCD45RABC 24 2CD45RO 25 2CD45RABC C817S 64 1CD45RABC Acyt 26 7Table. I. Results of Transfection of L tk Cells with CD45 cDNAs. The number ofnoemycin resistant colonies screened and the number of CD45-positive clones obtainedare shown. Colonies were screened by flow cytometry after four rounds of positiveselection with anti-CD45 antibody and secondary antibody conjugated to magneticbeads (see Materials and Methods).30contrast, of the 26 CD45RABC Acyt colonies tested, 7 were positive for CD45 expression(Table I). Clone L64C1 (CD45RABC C817S) and clones L12A5, L12B5, and L12C2(CD45RABC Acyt) were also taken for further study.The total amounts of CD45 protein was determined by Western blot analysis ofCD45 immunoprecipitated from transfected L cells (Fig. 6). It was determined thatCD45RO transfected L cells synthesized similar amounts of CD45 protein as theCD45RABC transfected cells as determined by scanning densitometry. The apparentmolecular weight of CD45 immunoprecipitated from the CD45 RABC C817S clone wasdetermined to be 220 kDa (Fig. 6, lane 5), the same molecular weight as the wild-typeCD45RABC molecule (Fig. 6, lane 3). A lower band detected at 200 kDa may beattributed to degradation of the protein during immunoprecipitation (Fig. 6, lane 5).CD45 immunopredpitated from the CD45RABC Acyt clones migrated as two bands of160 kDa and 125 kDa (Fig. 6, lanes 6-8). The predicted molecular weight of this mutantwas calculated to be 145 kDa, thus the presence of two CD45-specific bands, neither ofwhich corresponded to the predicted molecular weight, was unexpected. It is possiblethat these two forms represent unprocessed and processed forms of the CD45 protein.No CD45 protein was detected in untransfected L tk- cells by Western blot analysis (Fig.6, lane 2).Some donal variation was observed with respect to total amounts of CD45expressed in the three clones of the CD45RABC Acyt mutant. The L12A5 doneexpressed 3-fold to 5-fold less CD45 than the L12B5 and L12C2 clones, as determined bydensitometry (Fig. 6, lanes 6-8). Because of this donal variation, only clones L12B5 andL12C2 of the CD45RABC Acyt mutant were used for further studies, the L12A5 donewas not characterized further. The L12C2 clone of the CD45RABC Acyt transfectantexpressed similar amounts of CD45 (-4.5-fold more) as the L12B5 clone (Fig. 6, lanes 7and 8). The intensities of the 160 kDa band and the 125 kDa band were equivalent inindividual clones of the CD45RABC Acyt transfectant as determined by densitometry(Fig. 6, lanes 7 and 8), indicating that equivalent amounts of these species are expressed31123456.2051168050Figure. 6. Western Blot Analysis of CD45 Immimoprecipitates from Lysates of L cellTransfectants. CD45 was immunoprecipitated with the Ly5.2 antibody conjugated toProtein G from lysates of 2 X 106 CD45RABC (lane 3), CD45RO (lane 4), CD45RABCC817S (lane 5), and CD45RABC Acyt (lanes 6-8) transfected L cells and were blotted withan anti-CD45 antiserum (131) raised against a common peptide epitope in the CD45extracellular domain. Three clones of the CD45RABC Acyt mutant are shown, L12A5(lane 6), L12B5 (lane 7), and L12C2 (lane 8). Protein G plus the precipitating antibody(lane 1) and CD45 inimunoprecipitates from untransfected L cells (lane 2) are shown forcomparison. Molecular weight markers in kDa are shown on the left.7832I2 -______________________________I. ______________________________CD45RABC CD45ROI1c:IiO LFLI-‘9CD45RABC CYTi i tFL IFigure. 7. Cell Surface Expression of CD45 as Determined by Flow Cytometry. L celltransfectants, CD45RABC, CD45RO, CD45RABC C817S, and CD45RABC Acyt werescreened for expression of CD45. Untransfected L cells are shown in the top panel forcomparison. Cells were labeled with secondary antibody alone, FITC-labeled goat-anti-rat immunoglobulin (negative control —), anti-CD4, GK1.5 (negative antibody control), anti CD45, 13/2 ( ), and anti-CD44, 1M7.8.1 (positive antibody control --—--).- Lcdls.IJ 1I9jI •CCS-Wi 10210a Io34in the cells. If the intensities of the two bands are added, the L12B5 and L12C2 dones ofthe CD45RABC Acyt mutant express approximately the same amount of total CD45protein as the CD45RABC C817S mutant (L64C1), which was significantly more thanthe CD45RABC (L106A6) and CD45RO (L36B3) clones. However, this experiment wasonly done once. Due to the lack of sufficient dones for each construct, it cannot bedetermined whether these differences are consistently seen. Indeed, subsequent dataindicates that variation in the levels of expression can occur for individual clones.CD45 cell surface expression detected by flow cytometry indicated that levels ofCD45RABC (clone L106A6) were 3-fold over background (Fig. 7), that the levels of theCD45RO isoform (clone L36B3) were 4-fold over background (Fig. 7), the levels of CD45expresion of the CD45RABC C187S transfectant (clone L64C1) were 3-fold overbackground (Fig. 7), and the levels of CD45RABC &yt (clone L12B5) were 4-fold overbackground (Fig. 7). Untransfected L cells were tested for CD45 expression, as a control(Fig. 7). Examination of surface expression of CD45 by flow cytometry indicated nosignificant differences in the levels of expression.Surface expression of CD45 in the L cell transfectants was also characterized byimmunoprecipitation of CD45 from the cell surface and subsequent Western blotting(Fig. 8). In addition, CD45 remaining in the cell lysate was also immunoprecipitatedafter immunoprecipitation of CD45 from the surface (Fig. 8). Immunoprecipitation ofCD45 from the surface of L cell transfectants indicates that the CD45RABC &yt mutantis expressed at the highest level on the surface, followed by the CD45RABC C817Smutant, and then by the CD45RABC and CD45RO isoforms. Once again thisexperiment was only done once and without examination of multiple clones, it cannotbe determined whether these differences are significant or whether they arise as a resultof experimental variation or donal variation. Immunoprecipitation of all four proteinsindicate that the majority of CD45 is not present on the surface, but is found inside thecell (70-90%). Only 10% of the CD45RABC protein and 20% of the CD45RO protein isexpressed on the surface compared to what is present inside the cell (Fig. 8, lanes 2S, 2C,33and 3S, 3C), as determined by densitometry. Similarly, only 30% of the CD45RABCC817S protein is expressed on the cell surface, with the rest of the protein remaininginside the cell (Fig. 8, lanes 4S and 4C). No CD45 was detected in the parental L cells(Fig. 8, lanes iS and 1C). Bands appearing at about 200 kDa in CD45RABCimmunoprecipitates (Fig.8, lane 2C) may be attributed to degradation of the proteinduring immunoprecipitation or alternatively, the presence of immature, unprocessedforms of CD45 inside the cell.The observation of two distinct bands upon immunopredpitation of total CD45from the CD45RABC Acyt mutant led to the characterization of these forms of CD45 onthe surface. From the immunoprecipitation and Western blotting analysis of CD45 fromthe surface of the CD45RABC Acyt clone LI2C2, it was shown that the major speciesexpressed on the surface of these cells was the 160 kDa form (Fig. 8, lane 5S). A lowerband at about 130-140 kDa may be either a product of degradation or the lowermolecular weight form (Fig. 8, lane 5S). The 160 kDa band that comprised CD45expressed on the surface of the CD45RABC z\cyt mutant cells represented only i5% ofthe total amount of CD45 protein (Fig. 8, lanes 5S and 5C) expressed in these L cells, asdetermined by densitometry. When considering the i60 kDa band alone, 30% of the160 kDa CD45 protein was expressed on the cell surface, and approximately 70%remained in the cell lysate. To determine the nature of these two species of CD45RABCAcyt, pulse-chase and endoglycosidase H sensitivity experiments were performed.1.0.2 Transport of a CD45 Protein Lacking the Cytoplasmic Domain in L cellsIn order to determine the relationship between the higher and lower molecularweight forms of the CD45RABC &yt mutants, the transport of CD45 cytoplasmicdeletion mutant through the endoplasmic reticulum (ER) and golgi apparatus wascharacterized by pulse-chase studies of the CD45 protein in L cells.36Deletion of the cytoplasmic domain resulted in the expression of two CD45species of 125 kDa and 160 kDa. Pulse-chase analysis of this mutant expressed in L cellsdemonstrated that the first species present was the 125 kDa band (Fig. 9). Digestion ofthe CD45RABC Acyt immunoprecipitates with endoglycosidase H resulted in a shift inmolecular weight from 125 kDa to 80 kDa (Fig. 9), indicating that Endo H digestionreduced the molecular weight of the protein by —40 kDa. The 125 kDa species remainedEndo H sensitive (as represented by a band at —80 kDa in the Endo H lanes) to the endof the chase period of 3 hours (Fig.9). The second species, the 160 kDa form, which wasthe major form of CD45 expressed on the surface (Fig. 8, lanes 5S and SC), was notobserved until the 30 minute time point (Fig. 9). At 120 minutes, it was apparent thatthe 160 kDa species was becoming Endo H resistant, as demonstrated by the presence ofa faint band at 150 kDa in the Endo H lane. Additionally, the 125 kDa and 160 kDabands were of equal intensity at 120 minutes in the Endo H- lane but by 180 minutes, itwas observed that as the intensity of the 160 kDa protein was increasing, the 125 kDaband in the Endo H- lane was becoming less intense (Fig. 9), suggesting that the 160 kDaspecies was derived from the 125 kDa species. At the final time point of three hours, the125 kDa protein was still present but the majority of the CD45 protein was representedby the upper band of 160 kDa (Fig. 9). Pulse-chase data from untransfected L cells wasused as a control (Fig. 10) to determine the amount of background material that wasimmunoprecipitated. Background bands were detected at 55 kDa, 60 kDa, 70 kDa, and80 kDa (Fig. 10).DISCUSSIONThe selection of CD45 expressing clones of L tk- cells required several rounds ofpositive selection and the screening of 25 clones on average per contruct transfected(Table I). CD45 expression levels in L cell transfectants was low, ranging from 3-fold to4-fold over background staining levels (Fig. 5). In comparison, others have shown that37L cells transfected with the CD45RO isoform expressed 9-fold more CD45 overbackground, normal lymphoid cells expressed approximately 50-bOX more CD45 overbackground levels [77] as determined by flow cytometry.Some inconsistencies were observed when comparing the results from Westernblots of total CD45 and surface CD45. From the immunoprecipitation of total CD45from the L cell transfectants, it appeared that clone L64C1 (CD45RABC C817S)expressed the highest levels of CD45, that the CD45RABC tcyt mutant expressedintermediate levels, and that the full length CD45RABC and CD45RO isoformsexpressed low levels of CD45 (Fig. 6). In contrast, from the examination of CD45immunoprecipitated from the cell surface, the cytoplasmic deletion mutant (CD45RBACAcyt) expressed the most amounts of CD45 on the cell surface as well as inside the cell(Fig. 8). This discrepancy in expression levels may reflect the fact that theseexperiments were carried out at different times and that there was a certainamount of clonal variation. The CD45RABC C817S transfectant tended to be stable forCD45 expression for only a month in culture. Although these cells were periodicallychecked for CD45 expression by FACS analysis, the L64C1 clone may have experiencedsome loss of CD45 expression at the time of the surface immunoprecipitation of CD45from the L cells. Unfortunately, this was the only clone of the CD45RABC C817Smutant that was obtained after five rounds of positive selection and the screening of 64colonies (Table I). It is thus difficult to interpret the effect of inactivation of phosphataseactivity on CD45 expression in L cells. More CD45RABC C817S clones will have to betested. When considering the effects of the cytoplasmic deletion mutant on CD45expression (Fig. 6 and Fig. 8), some clonal variation was observed. Clone L12A5 seemedto lose expression of CD45 on the surface over a period of six months after it had beencloned out and screened (unpublished observations). More clones of the isoforms andmutant constructs will have to be characterized with respect to CD45 expression inorder to clear up the discrepancies in this data.40L cells transfected with CD45 expressed only a proportion of total CD45 proteinon the surface. From the immunoprecipitation of CD45 from the surface of L celltransfectants, it was determined that between 10% and 30% of total CD45 in the cell wason the surface (Fig. 8). Whether this is also the same in lymphoid cells will beinteresting to determine. Proteins destined for the plasma membrane should eventuallylocalize to the cell surface, unless they are being degraded prior to or after localizing tothe cell membrane, or if they are kept in the ER by chaperone proteins until they formdimers or multimeric complexes. It is unlikely that all the CD45 remaining in thecytosol is degraded because we observe that between 70% and 90% of total CD45remains inside the cell (Fig. 8) and that this material is recognized by a CD45-specificblotting antibody upon immunoprecipitation. It is possible that CD45 is rapidlyinternalized upon expression on the cell surface and thus only 10-30% of CD45 isactually observed upon surface immunoprecipitation of CD45. Experiments todetermine the rate of internalization of CD45 would address this question.An alternative explanation of the large percentages of CD45 that remain insidethe cell is that there is a requirement for some lymphoid-specific protein with whichCD45 interacts and forms complexes. Since L cells may not express this other proteinthat CD45 interacts with, by virtue of being a fibroblast that does not express lymphoidspecific proteins, a large proportion of CD45 may be localized indefinitely inside thecell. Only a small fraction of CD45 being synthesized, between a tenth and a third, mayescape this requirement for an interacting protein and localize to the cell membrane.Deletion of the cytoplasmic domain resulted in three interesting results. The firstinteresting result was the observation of two bands at 160 kDa and 125 kDa of equalintensity, indicating that there were equivalent amounts of these two CD45 species inthe L cells (Fig. 6). Yet when CD45 was immunoprecipitated from the surface of thesetransfectants, it appeared that predominantly one species, the 160 kDa protein, wasbeing transported to the surface (Fig. 8). Even then, only a certain percentage of theCD45RABC Acyt protein, 15%, was expressed on the cell surface, the remaining 85%41was left in the cell lysate (Fig. 8). Data from pulse-chase experiments explained some ofthese results. From the pulse chase experiment, it was determined that the 160 kDaform of CD45RABC &yt was derived from the 125 kDa form because the 160 kDa banddid not appear until the 30 minute time point (Fig. 9.). In addition, between 120 and 180minutes the higher molecular weight band gained in intensity as the intensity of thelower molecular weight form declined in the Endo H- lanes (Fig. 9). The 125 kDa formremained Endo H sensitive for the whole chase period. In contrast, the 160 kDa formwas Endo H insensitive by 3 hours (Fig. 9). These results suggest that the 125 kDa formis a precursor of the 160 kDa form. Why two forms of CD45RABC Acyt are expressed inL cells and the carbohydrate modifications ocurring to the 125 kDa protein in order togenerate the 160 kDa protein are not known.One possible explanation for the observation of two CD45RABC Acyt species isthat CD45 transport is normally facilitated in some way, perhaps by interacting with atransporter protein. This interaction may occur through the CD45 cytoplasmic domain.Thus, when the cytoplasmic tail of CD45 is deleted, transport of the protein may beadversely affected, causing the sequestration of the protein in the ER and golgiapparatus, resulting in the differential glycoslyation of this mutant. Indeed, the timetaken for 50% of this protein to become Endo H resistant was approximately 135minutes (data not shown). Preliminary data from pulse chase studies of the full lengthform, CD45RABC, showed that it became Endo H resistant by 30 minutes (data notshown), suggesting that the cytoplasmic deletion mutant is transported slower than theCD45RABC isoform and that it does spend more time in the ER.42RESULTS2.0 Mutational Analysis of the Cytoplasmic Domain of CD45In this work, three CD45 cytoplasmic domain mutants were made and expressedin bacteria (Fig. 11). As mentioned previously, deletion of the C-terminal tail of CD45and a further 13 amino acids at the C-terminus of domain II abrogated PTPase activity.Mutation of a conserved tyrosine in this stretch of amino adds (Y1I81F), had no impacton phosphatase activity. One of the goals of the work presented here was to identifyresidues in the deleted area that were crucial for enzymatic activity out of the 13 aminoacids in this region. Glutamine 1180 is invariantly conserved in both PTPase domains ofhuman, mouse, and rat CD45 as well as numerous other protein tyrosine phosphatases(Fig. 3), suggesting that this residue in domain II may be crucial for the structure orfunction of the enzyme. Therefore a point mutation was introduced in the cytoplasmicdomain of CD45 such that glutamine 1180 was changed to a glycine (Q118OG). Asecond mutant was made to delete the spacer region between domain I and domain U,since it had not been established in prevous work, whether or not this region wasrequired for activity [32]. Finally, a third mutant was made to abolish a potential site ofcAMP-dependent kinase or protein kinase C (PKC) phosphorylation. Ultimately, thismutant would be tested in vivo to determine if cAMP-dependent kinasephosphorylation occurs at this site and if so, if it affects CD45 function.2.0.1 Generation of Three Mutations in the Cytoplasmic Domain of CD45Mutations were generated on a single stranded template by the Kunkel methodof site-directed mutagenesis [71,72]. Oligonucleotides were engineered to include arestriction endonudease site for screening mutant colonies. The Q118OG mutation wasengineered to include an extra Kpn I site such that digestion of plasmid DNA would43564 1268573/574 817960978 1180I Domain I Domain II I”’Membrane Spacer COOHProximal Region TailRegionCONSTRUCTC81 7S1 I Domain I H Domain II “‘‘S573G/S574AII______________2 I Domain I H Domain II F”’”Qi 180G3 I Domain I H Domain II fr’’”t876-931‘I : Domain I I H Domain II [0Figure. 11. Schematic Diagram of the CD45 Cytoplasmic Domain Mutants. Theconstructs used to generate the CD45 cytoplasmic domain proteins are illustrated.Construct 1 represents an inactivated PTPase, construct 2 represents the point mutationof two consecutive serines that may be the site of phosphorylation by cAMP-dependentkinase or PKC, construct 3 is a point mutant in PTPase domain II, and construct 4represents a CD45 protein with the spacer region deleted. The numbering system usedis from the mouse CD45RABC isoform [9].44yield bands of 3.8 kb and 2.2 kb for wild-type colonies and 3.8 kb, 1.2 kb, and 1.0 kb forcolonies carrying mutant plasmids. Deletion of the spacer region between domain I anddomain II resulted in the loss of 168 nudeotides which was detectable after an Eco Ridigest. Mutants with the spacer region deleted yielded bands of 4.8 kb and 1.1 kb upondigestion with Eco Ri, while the wild-type colonies would have bands of 4.8 kb and 1.3kb. The S573GS574A mutation in the membrane proximal region was engineered toindude a Bsa HI site. Thus wild-type colonies would generate bands of 4.8 kb and 2.2kb when digested with Bsa HI while colonies containing mutants would have bands of3.2 kb, 2.2 kb, and 1.6 kb. All mutations were sequenced to verify that all mutationswere accurate and in frame.2.0.2 Bacterial Expression and Partial Purification of CD45 MutantsThe two mutants generated by site-directed mutagenesis in the pBluescript SK(+1-) vector (Stratagene Cloning Systems, La Jolla, Ca.) were then cut with Bgl II andsubdoned into the Barn Hi site of the pET-3d-6His-IEGR bacterial expression vector.Unfortunately, this was not as easily accomplished for the Qii8OG mutant. Therefore ashuttle vector was constructed with convenient restriction sites which allowed easyshutting of mutant constructs out of the original pBluescript vector and into bacterial ormammalian expression vectors. The QII8OG mutant had to be subcloned into theshuttle vector (see Materials and Methods) and once in the shuttle vector, a Bgl IIfragment comprising the cytoplasmic domain mutant was isolated and ligated into theBarn HI site of the pET-3d-6His-IEGR vector. The spacer deletion mutant wassubdoned into pET-3d-6his-IEGR-CD45 by a Kpn 1-Bam Hi fragment. Bacteriatransformed with the mutant CD45 constructs were induced to express CD45 in the logphase of growth.It was noted that levels of recombinant protein expression were equivalent forthree independent clones of the Qii8OG mutant (Fig. 12, lanes 7-9) and for two45independent clones of the S573GS574A mutant (Fig. 12, lanes 10-11) as demonstrated byCoomassie Blue staining of proteins run on an SDS-polyacrylamide gel. Threeindependent dones of the spacer deletion mutant did not express very well and so thismutant was not pursued further (Fig. 12, lanes 4-6). As controls, both wild-type CD45(Fig. 12, lane 2) and a PTPase inactive mutant, C817S (Fig. 12, lane 3), as well as thevector without insert (Fig. 12, lane 1) were induced in BL21 (DE3) E. coil and solubleprotein lysates were isolated and immunopredpitated withNTA+-agarose whichspecifically binds to the 6 histidine tag present at the N-terminus of the recombinantprotein, allowing partial purification of the recombinant protein. From Coomassie bluestained gels, a prominent band at 95 kDa was observed and verified in a previousexperiment to be recombinant CD45 by western blotting with an antiserum against thecytoplasmic domain of CD45 (data not shown). The purification was considered to bepartial because of the presence of contaminating bands at 110 kDa, 106 kDa, 75 kDa, 70kDa, 60 kDa and a major species at 49 kDa which was shown to be due in part, to thedegradation of the 95 kDa recombinant CD45 protein, as demonstrated byimmunoblotting CD45. While the wild-type CD45 construct and the C817S mutantwere expressed at relatively high and equivalent levels, the QII8OG and S573GS574Amutants expressed only a fifth and a third as much recombinant CD45 respectively(Table ifi). No proteins at 95 kDa were observed in the lane containing lysates from thevector control (Fig. 12, lane 1).2.0.3 Determination of Phosphatase Activity of CD45 MutantsRecombinant proteins generated by bacterial expression were assayed using anon-radioactive phosphatase assay [761. The mutation of glutamine 1180 to glydne(Q118OG), contained within the 13 C-terminal residues of CD45 domain II required forPTPase activity, completely abolished the activity of the enzyme (Fig. 13). Thephosphatase assays of the wild-type cytoplasmic domain of CD45 and the C817S46Table II. Quantitation of Band Density of Bacterially Expressed Proteins byScanning Densitometry. The SDS-PAGE gel of bacterially expressed proteins (Fig. 12)was scanned using the Quantity One software (PDI Inc.) and the density of the bandsdetermined. Bands scanned were of the vector alone (pET vector), the wild-type CD45cytoplasmic domain (pET CD45), the mutant in PTPase domain II (Q118OG), the mutantwith the spacer region deleted (bspacer), and the mutant in the potential cAMP-dependent kinase/PKC site, (S573GS574A). Specific clones tested are noted. Lanenumbers refer to lanes in Figure. 12, ND refers to densities that were not determineddue to the faintness of bands.*2/3 of the total volume of these two samples were loaded on the SDS-PAGE gel, theratio of band intensity values were therefore normalized to represent the total volume.48TABLE II.Lane Construct Band Density Ratio of BandO.D. X mm2 Density1 pET Vector alone 0 02 pET CD45 0.979* 1.03 C817S mutant 0.923* 0.944 zspacer mutant ND NDclone H8.85 Aspacer mutant ND NDclone H9.16 zspacer mutant ND NDclone H9.117 Q118OG mutant 0.275 0.20clone C3.1A8 Q118OG mutant 0.216 0.14clone C3.1B9 Q1I8OG mutant 0.399 0.27clone C3.1C10 S573GS5754A 0.481 0.33mutant-clone 4.1811 S573GS574A 0.438 0.30mutantclone 18.3549mutation in the catalytic centre of domain I as positive and negative controlsrespectively, are shown for comparison. It should be noted that the rates ofdephosphorylation were normalized for equivalent amounts ofrecombinant CD45assayed. The normalized activity of the S573GS574A mutant was equivalent to thewild-type recombinant protein (Fig. 13).DISCUSSIONThe fact that mutation of glutamine 1180 to glycine in domain II of CD45abolished PTPase activity suggests that a disruption in domain II can abolish an activitythat is thought to reside in domain I. Thus, a mutational event in domain II affectsdomain I, supporting the two domain enzyme hypothesis, which postulates that bothphosphatase domains contribute to enzymatic activity by interacting with each other.Whether the glutamine at position 1180 of domain II is required for the appropriatefolding of domain II or if it has a functional role remains to be determined. Recentpublication of the x-ray crystal structure of PTP 1B, a single domain tyrosinephosphatase implicates glutamine 262 of PTP lB in the interaction with thephosphotyrosine in the catalytic site [36] and suggests a role for glutamine 1180 indomain II of CD45, in the catalytic site. Since the glutamine homologous to Q1180 isinvariantly conserved in all phosphatases, it may play a role in stabilizing thedephosphorylation reaction.PTP domain II may be an important structural requirement for activity in domainI. This could occur by the involvement of residues in domain II in the catalytic site ofthe enzyme or in the stabilization of an enzymatically active domain I. The amide sidechain of glutamine 262 of PTP lB is involved in forming hydrogen bonds with thephosphate substrate. Mutation of glutamine 1180 in domain II to glycine may destroythis potential structural requirement for activity in domain I by making an amide groupless accessible to the phosphate substrate in order to form an H-bond. The fact that50deletion of CD45 domain II or even specific mutations in domain II result in an inactivePTPase supports the idea that domain II can modulate the activity of domain I and thatit is required for the optimal functioning of domain I as a phosphatase. Howphosphatase domain II can regulate domain I function may be related to its uniqueability to recruit substrates. There is some evidence to support this theory as it has beenshown that bacterially expressed proteins encompassing domain II of HPTPa may havedistinct substrate specificities from domain I [78].A point mutation in domain II has deleterious effects on the phosphatase activitywhich is thought to reside in domain I of CD45. While it may be true that proteinsexpressed in bacteria may not fold as well as in eukaryotic cells, this result suggests thatdomain II has a role to play in maintaining the enzymatic activity of CD45. The Q118OGmutant will be a useful mutant that will be worth testing as a full-length form of CD45in L cells to determine if domain II regulates the activity of domain I in vivo.52RESULTS3.0 Characterization of a Redpient CD45-Negative T Lymphoma Cell LineThe CD45-defident variant of a BW5147 cell line which expressed the TCR/CD3complex was previously characterized with respect to induction of tyrosinephosphorylated proteins upon TCR-mediated stimulation and the phosphorylation andactivity of p56lCk (P. Johnson, personal communication). In this work, the CD45-negative and CD45-positive BW5147 cells were characterized with respect to the levelsof p59Y’ expression, phosphotyrosine levels of p59fYfl, and p59fYIt in vitro kinaseactivity.3.0.1 Determination of the Levels of Expression of the Src-family Kinase p59fY inCD45-Negative and CD45-Positive Variants of a BW5147 T Lymphoma CellLinep59Y was immunoprecipitated from equivalent numbers of unstimulatedCD45-negative and CD45-positive cells and immunoblotted with p59Y antiserum.Equivalent amounts of p59f)m were precipitated from the CD45-negative and CD45-positive T cells and migrated at -59 kDa, the predicted molecular weight of fyn (Fig. 14).The control lane shows a band at 50 kDa that can be attributed to crossreactivity of theprotein A-HRP secondary antibody to the heavy chain of the immunoprecipitatingantibody (Fig. 14).3.0.2 Evaluation of the Effect of CD45 Expression on the Phosphotyrosine Levels ofp59fyn53p59fYlL was immunoprecipitated from equivalent numbers of CD45-negative andCD45-positive cells both prior to and after stimulation through the TCR/CD3complexwith soluble anti-CD3 antibodies and Western blotted with the 4G10 antiphosphotyrosine antibody. Like p56lCk [26, 63], p59Y was more phosphorylated inCD45-negative cells than in CD45-positive cells (Fig.15). This difference wasmaintained upon T cell stimulation. A transient increase in tyrosine phosphorylation offyn in CD45-negative BW5147 cells was observed 90 seconds after stimulation (Fig. 15)although this subtle change in phosphorylation was not always detectable. In contrast,the tyrosine phosphorylation of p59fYIl was observed to increase steadily over time afteranti-CD3 stimulation (Fig. 15) in the CD45-positive cells.3.0.3 Determination of the Effect of CD45 Expression on the In Vitro Kinase Activity ofp59fynp59fYfl immunoprecipitated from 3 X 106 cells was subjected to an in vitro kinaseassay both before and after the cells were stimulated through the TCR/CD3 complex. Aslight increase in autophosphorylation of p591Ywas observed 90 seconds after T cellstimulation in the in vitro kinase assay of jijn immunopredpitates from CD45.-negativecells, but no such increase in activity was observed in the CD45-positive cells (Fig. 16).Instead, more significant differences were observed in other proteins phosphorylated inthe fyn immunoprecipitates.A prominent phosphorylated species of 120/130 kDa band was detected (Fig. 16)in an in vitro kinase assay on p59fYIl immunoprecipitates from CD45-positive cells. Thisphosphorylated 120/130 kDa protein was observed in p59fYfl immunoprecipitates inboth unstimulated and stimulated CD45-positive cells. In the in vitro kinase assay ofp59fYtl isolated from CD45-negative cells, this phosphorylated species was not present.However, a faint phosphorylated band at 120/130 kDa was detected in the CD45-negative cells five minutes after T cell55stimulation. Upon stimulation with soluble anti-CD3 a 30 kDa phosphoprotein wasobserved (Fig.16), which was enhanced in intensity upon CD3 stimulation. This 30 kDaphosphorylated species was not observed in the kinase assay of fyn isolated from CD45-positive cells.DISCUSSIONAnalysis of the phosphorylation state and kinase activity of p59fYtL led to thesurprising finding that although the presence of CD45 in the cell substantially affectedphosphorylation state of the kinase, it did not result in increased activity. In fact, aslight increase in p59Y autophosphorylation was observed in the CD45-negative cells.This is a paradoxical result when one considers that the induction of tyrosinephosphorylated proteins upon T cell stimulation is less efficient in the CD45-negativecells (P. Johnson, personal communication). This situation was recently reported inthree other CD45-negative cell lines [611. The transient nature of the increasedautophosphorylation of p59fY in CD45-negative cells suggests that in the absence ofCD45, other protein tyrosine phosphatases may dephosphorylate this kinase.No correlation was found between dephosphorylation of p59fYfl and an increasedkinase activity in CD45-positive cells, counter to the current model for src-family kinaseregulation which postulates that dephosphorylation of the src-family kinases leads totheir activation. A correlation was observed between the presence of CD45, thephosphorylation state of the kinase, and the proteins associated with the kinase.Differences were observed in the phosphoproteins that associated with p59fYtL in CD45-negative and CD45-positive variants of BW5147 cells. A 120/130 kDa protein wasfound to co-precipitate with fyn and be phosphorylated in an in vitro kinase assay byp59fYfl or another co-precipitating kinase in CD45-positive T cells regardless ofstimulation, suggesting that the association was constitutive and was not occurring as aresult of TCR-mediated stimulation. This 120/130 kDa protein was not observed in in58vitro kinase assays from fyn immunopredpitates from CD45-negative cells. The absenceof the 120/130 kDa band in the in vitro kinase assay of fyn immunoprecipitates fromCD45-negative cells may be due in part, to the saturation of tyrosine phosphorylationsites prior to the addition ofy-32P-ATP. This would suggest that the absence of CD45 inthese cells results in the hyperphosphorylation of the 120/130 kDa protein. Absence ofthe p120/130 from the phosphotyrosine blot of p59fY’ immunoprecipitates suggests thatit is either not phosphorylated in the cell or that the stoichiometry of phosphorylation isbelow the detection level of the phosphotyrosine blot. Currently, the identity of this120/130 kDa protein is unknown. Yet in CD45-negative cells, a faint 120/130 kDaphosphorylated band can be seen in fyn kinase assays 90 seconds after TCR/CD3stimulation, indicating that this association is much weaker and much less efficient inCD45-negative cells and requires T cell stimulation for it to occur. This data indicatesthat CD45 is required for the efficient coupling of this 120/130 kDa in vitro substrate top59fY’\ Currently, it is not known what mediates the association of p59fYfl with the120/130 kDa protein but it has recently been demonstrated that tyrosinephosphorylated 120 kDa proteins do associate with both the SH2 [79,80] and the SH3domains [81]of p59fY’\ These proteins are rapidly phosphorylated upon T cellactivation implying that they are mediators of TCR induced signalling events. Theidentity of these 120 kDa proteins remains to be established.In addition, a 30 kDa phosphoprotein was observed in CD45-negative cells whichwas enhanced upon CD3 stimulation. As p59fYh1 is hyperphosphorylated at its carboxyterminal tyrosine 531 in CD45-negative BW5147 cells [62,63], it is possible that thismust occur for the p30 to bind. It will be interesting to determine if this protein isinvolved in the regulation of TCR-mediated signalling events.Thus, it has been determined that p59fY is hyperphosphorylated in the absenceof CD45 but that this does not influence its kinase activity, assessed in vitro. Rather, theconstitutive association of a 120/130 kDa protein to fyn is adversely affected by theabsence of CD45. These results implicate CD45 in regulating associations of p59fYhl to59other proteins and may be involved in coupling the kinase to downstream TCR/CD3induced signalling events. Both the phosphorylation of fyn and its assodation with the120/130 kDa protein will be useful parameters with which to analyze the function ofCD45 in deficient cells lines in which the expression of CD45 has been restored.60CONCLUSIONThe work presented in this thesis has furthered the understanding of themolecular function of CD45. From the study of two isoforms of CD45 transfected into Ltk cells, CD45RABC and CD45RO, it was determined that expression of the threealternatively spliced exons at the N-terminus of CD45 did not significantly affectexpression of the molecule. In contrast, deletion of the cytoplasmic domain of CD45resulted in the expression of two species of 125 kDa and 160 kDa. It appears frompulse-chase experiments, that the higher molecular weight form is derived from thelower molecular weight form and that the difference in molecular weight is dueadditional glycosylation. The carbohydrate modifications occurring to the lowermolecular weight form in order to generate the higher molecular weight form are notknown.The protein tyrosine phosphatase activity of CD45 was analyzed after targetedmutations in the cytoplasmic domain and it was concluded from this study that PTPasedomain II is required for the activity of PTPase domain I. The point mutation ofglutamine 1180 to a glycine in domain II abrogates phosphatase activity, suggesting thata disruption in domain II can abolish an activity which is thought to reside in domain I.This result supports the idea of a two domain enzyme in which domain II regulates theactivity in domain I, potentially by interacting both with domain I and with putativesubstrates.The effect of CD45 on the src-family kinase p59fYIl was characterized in a T cellline. p59fYI1 is thought to be one of the kinases involved in signalling through the T cellreceptor complex. It was determined that CD45 affects the tyrosine phosphorylation ofp59fYfl, as fyn was hyperphosphorylated in CD45-negative BW5147 cells. Yet CD45 didnot appear to affect the kinase activity of p59fY” as demonstrated by itsautophosphorylation. Rather, CD45 appears to affect the association between p59Yand other phosphorylated proteins, in particular, a 120/130 kDa protein. The61constitutive association of the p120/130 to p59fY11 was not observed in CD45-negativeBW5147 cells. These results implicate CD45 in regulating the associations of p59fY’ toother proteins and CD45 may even be involved in coupling the kinase to downstream Tcell receptor-mediated signalling events.Characterization of the CD45-deficient BW5147 cell line with respect to inductionof tyrosine phosphorylated proteins upon T cell stimulation, p561Ck, and in this work,p59fYfl, has now provided a suitable cell line with which to try and reconstitutedeficiencies in aspects of signalling by transfecting various CD45 constructs back intothese cells. In addition, further mutational analysis of the CD45 cytoplasmic domain inthe work presented supports the two domain model for an active CD45 PTP enzymeand provides us with the tools to test this model further in lymphoid cell lines, such asthe CD45-deficient BW5147 cells.62PUBUCATIONSA list of publications arising from work presented in this thesis is included.Submitted:1. Maiti, A., P. Borodchak, T. Brocker, M. D. Jabali, B. Malissen, and P. Johnson.(1994) Effect of CD45 on phosphorylation and protein associations of p56lCk and p59fYflduring T cell receptor stimulation. submitted to I. Biol. Chem.In preparation:1. Maiti, A., I. Haidi, W. Jefferies, and P. Johnson. (1994) Deletion of thecytoplasmic domain of CD45 retards transport of CD45 significantly in L cells.2. Ng, D., A. Maiti, and P. Johnson. (1994) Point mutation in the secondphosphatase domain of CD45 abrogates phosphatase activity.63REFERENCES1. Trowbridge, I. S., H. Ostergaard and P. Johnson. 1991. 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E. Rudd. 1993. T cell receptor C/CD3p59fY11(T)associated p120/130 binds to the SH2 domain of p59fYfl(T). I. Exp. Med. 178:2107-2113.80. Tsygankov, A. Y., C. Spana, R. B. Rowley, R. C. Penhallow, A. L. Burkhardtand J. B. Bolen. 1994. Activation-dependent tyrosine phosphorylation of fyn-associatedproteins in T lymphocytes. I. Blot. Chem. 269:7792-7800.81. Reedquist, K. A., T. Fukazawa, B. Druker, G. Panchamoorthy, S. E. Shoelsonand H. Band. 1994. Rapid T cell receptor-mediated tyrosine phosphorylation of p120, anfyn/lck src homology 3 domain-binding protein. Proc. Nat!. Acad. Sd. ii. S. A. 91:4135 -4139.75Figure. 8. Surface Expression of CD45 as Determined by Western Blot Analysis.CD45 was immunoprecipitated from the surface (S) of 5 X 106 untransfected L cells(lane 1), CD45RABC (lane 2), CD45RO (lane 3), CD45RABC C817S (lane 4), andCD45RABC Acyt (lanes 5) transfected L cells using the Ly5.2 antibody. This wasfollowed by reprecipitation of CD45 from the lysate remaining after surfaceiminunopredpitation of CD45 from the L cell transfectants (C) using the Ly5.2 antibodyconjugated to Protein G. Immunoprecipitates were subsequently blotted with an antiCD45 antiserum (131) raised against a common peptide epitope in the CD45extracellular domain. Molecular weight markers in kDa are shown on the left.1 2 3 4 5scscscscsc•d..iI.I. —2051168050S35-+-+Figure. 9. Pulse-Chase and Endoglycosidase H Sensitivity of the CD45RABC AcytCytoplasmic Domain Mutant Transfected into L tk cells. The transport of theCD45RABC Acyt isoform (clone L12B5) was characterized in L cells by the pulse-chasemethod and endoglycosidase H treatment. Approximately 2.5 X 106 cells expressing theCD45RABC Acyt isoform of CD45 were pulsed with media containing355-methionineand35S-cysteine and chased for the indicated time with normal media. CD45 wasimmunoprecipitated from the cell lysates using the Ly5.2 antibody conjugated toProtein G and run on a SDS-PAGE gel (-) or digested with Endo H (+) prior toelectrophoresis. Arrows indicate the high (160 kDa) and low (125 kDa) molecularweight forms of CD45RABC Acyt. Molecular weight markers in kDa are shown on theleft. CD45 proteins of 160 kDa (*), 150 kDa (s), 125 kDa (IIi), and 80 kDa (Y) are shown.Time:Endoll:205—0’ 15’ 30’ 60’ 120’ 180’-+-+-+-+—ww116—80———9ØF- — — — — —*§I50—38Time: 0’ 15’ 30’ 60’ 120’ 180’EndoH: -+-+-+-+-+-+_ — — — !: .205—116—8O——I—e-’” s—aI Iso—I I•Figure. 10. Pulse-Chase and Endoglycosidase H Sensitivity of CD45Immunoprecipitates from Untransfected L tic cells. As a control, approximately 2.5 X106 L cells were pulsed with media containing35S-methionine and35S-cysteine andchased for the indicated time with normal media. CD45 was immunoprecipitated fromthe cell lysates using the Ly5.2 antibody conjugated to Protein G and run on a SDSPAGE gel (-) or digested with Endo H (+) prior to electrophoresis. Molecular weightmarkers in kDa are shown on the left.391234567891011106___8050-_Figure. 12. Coomassie Blue Stained Gel of Recombinant CD45 Cytoplasmic DomainProteins Generated in Bacteria. After partial purification, equal volumes ofrecombinant CD45 cytoplasmic domain proteins were visualized by Coomassie Bluestaining of an SDS-PAGE gel. Only a 2/3 volume of the CD45 wild-t9pe and CD45C817S mutant were run on SDS-PAGE gels. Proteins generated by bacterial expressionwere pET-3d-6His-IEGR, the vector control (lane 1); wild-type CD45, the positive control(lane 2); the C817S mutant, the negative control whereby a point mutation inactivatedthe PTPase (lane 3); three clones of the spacer deletion mutant, H8.8 (lane 4), H9.1 (lane5), and H9.11 (lane 6); three dones of the Q118OG mutant, C3.1A (lane 7), C3.1B (lane 8),and C3.1C (lane 9); and two dones of the S573GS574A mutant, 4.18 (lane 10) and 18.35(lane 11). Molecular weight markers in kDa are shown on the left of the gel.I--—471.2-A/0.6 - AZ. :- - -0E 0.4- /z, -4- -0.2 -0- — — —c- —,-0.2- I-1 0 1 2 3 4 5 6 7Time (mm.)Figure. 13. PTPase Assay of Recombinant CD45 Cytoplasmic Domain ProteinsGenerated in Bacteria. After partial purification, equal volumes of recombinant CD45cytoplasmic domain proteins were assayed for tyrosine phosphatase activity against a13-mer tyrosine phosphorylated peptide comprising the negative regulatory site ofp59fYfl. PTPase activity was normalized for equal amounts of protein (Table II).Recombinant proteins tested for PTPase activity were pET-3d-6His-IEGR, the vectorcontrol (--s--) wild-type CD45, the positive control (--A--); the C817S mutant, thenegative control (—o—); the Q118OG mutant, C3.1A (“ ), two clones of theS573GS574A mutant, 4.18 (—A---) and 18.35 (--0--). Only a 2/3 volume of the CD45wild-type and CD45 C817S mutant were run on SDS-PAGE gels. Activity wascalculated as nmoles of phosphate hydrolyzed by the construction of a standard curveusing serial dilutions of 1 mM KH2PO4.51CD4S:205—116—80—50—Figure. 14. Amounts of p59fYfl Isolated from CD45-Negative and CD45-PositiveBW5147 Cells. p59fYn was inimunoprecipitated from lysates from 3 X106unstimulatedCD45-negative and CD45-positive BW5147 cells with afyn antiserum conjugated toProtein A and immunoblotted with the samefyn antiserum. Protein A conjugated to fynantiserum was run as a control (C). Molecular weight markers in kDa are shown on theleft.54TimeCD45:205—116—C0 Oa- +-+90”—5’ 30’+- +- +Figure. 15. Tyrosine Phosphorylation of p59fYfl Isolated from CD45-Negative andCD45-Positive Cells Upon CD3-Mediated Stimulation. At the indicated amounts oftime alter stimulation with 3 p.g of anti-CD3 antibody, 3 X 106 CD45-positive (+) andCD45-negative (-) cells were lysed in 1% Triton X-1 00 and p59fYIl wasimmunoprecipitated from the lysates. At zero time the cells were lysed either with noCD3 antibody, (0), or lysed simultaneously with the addition of CD3 antibody (Oa) andblofted with the anti-phosphotyrosine antibody (4Gb). The control lane (C) containedprotein A conjugated to anti-Jjn antibody. Molecular weight markers in kDa are shownon the left.I80—50—33—.56Time:CD45:205—Figure. 16. In Vitro Kinase Activity of p59fYfl Isolated from CD45-Negative andCD45-Positive Cells Upon CD3-Mediated Stimulation. At the indicated amounts oftime after stimulation with 3 jtg of anti-CD3 antibody, 3 X 106 CD45-positive (+) andCD45-negative (-) cells were lysed in 1% Triton X-100 and p59fY’ wasimmunopredpitated. At zero time the cells were lysed either with no CD3 antibody,(0), or lysed simultaneously with the addition of CD3 antibody (Oa) and subjected to anin vitro kinase assay with32P-ATP prior to separation by SDS-PAGE. The control lane(C) contained protein A conjugated to anti-fyn antibody that was also subjected to an invitro kinase assay. 32P incorporation was measured on X-ray film. Molecular weightmarker in kDa are shown on the left.C - +0 Oa 90” 5’ 30’- + + - + - +116—80—50—33—57


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