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Regulation of the RNA polymerase II holoenzyme-associated cyclin-dependent kinase SRB10 Goto, Susan Lisa 2002

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REGULATION OF THE RNA POLYMERASE II HOLOENZYME-ASSOCIATED CYCLIN-DEPENDENT KINASE SRB10  by Susan Lisa Goto B.Sc, Simon Fraser University, 1997  A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Biochemistry and Molecular Biology)  I accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A March, 2002 © Susan Goto, 2002  In  presenting  degree  this  thesis  in  partial  fulfilment  of  at the University  of  British  Columbia,  I agree  freely available for reference copying  of  department publication  this or  thesis by  of this  and study.  for scholarly  his thesis  or  her  I further  purposes  of  gl&tUftZ/Y) I  The University of British Vancouver, Canada  Date  DE-6 (2/88)  CTr^^'S'  Columbia  2^2-  that that  It  is  for  the Library  an  %T^J^  advanced  shall  permission  understood  that  make  it  for extensive  by the head  gain shall n o t be allowed without  permission.  Department  agree  requirements  may be granted  representatives.  for financial  the  of my  copying  or  my written  ABSTRACT Saccharomyces cerevisiae SRB10 is a cyclin-dependent kinase associated with the R N A polymerase II holoenzyme. Although originally identified based on its role in CTD function and subsequently demonstrated to be a CTD kinase, more recent evidence has revealed another aspect of SRB10 function with respect to transcriptional regulation. SRB10 has been shown to phosphorylate several transcriptional activators involved in cellular metabolism and environmental stress responses, leading to speculation that SRB10 function is modulated in response to signals regarding the overall environmental and physiological status of the cell in order to effect appropriate levels of transcription. However, the signals and mechanisms that regulate SRB10 function are currently unknown. The goal of this research was to determine i f and how SRB10 kinase activity is regulated in response to specific nutritional conditions. Data presented in this thesis demonstrate that SRB10 is negatively regulated by nitrogen limitation. In vitro kinase assays performed with SRB10 immunoprecipitated from yeast grown in nitrogen-limiting conditions revealed a transient reduction in kinase activity. In contrast, no significant effect on the in vitro kinase activity of immunoprecipitated SRB10 was observed for yeast grown in non-fermentable versus fermentable carbon sources. Down-regulation of SRB10 in response to nitrogen limitation is achieved through depletion of SRB10 protein levels, which occurs through a decrease in the stability of the SRB10 protein, representing a novel mechanism of cyclindependent kinase regulation.  ii  TABLE OF CONTENTS ABSTRACT  II  T A B L E OF CONTENTS  HI  LIST OF TABLES  V  LIST OF FIGURES  Vi  LIST OF ABBREVIATIONS  VII  ACKNOWLEDGEMENTS  IX  1. INTRODUCTION  1  1.1 OBJECTIVE 1.2 C O N T R O L OF E U K A R Y O T I C TRANSCRIPTION 1.2.1 Relief of chromatin-mediated repression Histone acetyltransferases Chromatin-remodeling complexes 1.2.2 RNA polymerase II. Core components Carboxy-terminal repeat domain R N A polymerase II holoenzymes 1.2.3 General transcription factors TFIID TFIIB TFIIF TFIIE .' TFIIH 1.2.4 Transcriptional coactivators TBP-associated factors TFIIA Mediator 1.3 F I L A M E N T O U S D E V E L O P M E N T IN SACCHAROMYCES 1.3.1 The phenotype offilamentous growth Diploid pseudohyphal growth Haploid invasive growth 1.3.2 The MAP kinase pathway 1.3.3 The cAMP-dependent pathway 1.3.4 Interaction between MAP kinase and cAMP pathways 1.3.5 Upstream signals 1.3.6 Other proteins involved in filamentous growth iii  CEREVISIAE  1 1 2 2 2 3 3 3 5 5 5 6 6 6 6 7 7 8 8 11 11 11 11 12 14 14 15 17  1.4 THE SRB10 C Y C L I N - D E P E N D E N T K I N A S E 1.4.1 SRBIO is an RNA polymerase II holoenzyme-associated cyclin-dependent kinase 1.4.2 SRBIO is a CTD kinase 1.4.3 SRBIO regulates gene-specific transcription factors 1.5 C Y C L I N - D E P E N D E N T KINASES 1.5.1 General principles 1.5.2 CDK regulatory mechanisms Activation by cyclin binding Activation by phosphorylation Inhibition by CKIs Inhibition by phosphorylation Other regulators 1.5.3 CDKs of Saccharomyces cerevisiae 2. METHODS AND MATERIALS  18 18 19 20 23 23 24 24 25 26 26 27 27 30  2.1 PLASMIDS A N D D N A MANIPULATIONS 30 2.2 Y E A S T STRAINS A N D M E D I A 30 2.3 R N A ISOLATION A N D N O R T H E R N BLOTTING 33 2.4 ANTIBODIES A N D R E C O M B I N A N T PROTEINS 33 2.5 IMMUNOPRECIPITATIONS, IN VITRO K I N A S E A S S A Y S , A N D W E S T E R N BLOTTING : 34 2.6 TRYPTIC PHOSPHOPEPTIDE A N A L Y S I S 35 2.7 M E T A B O L I C L A B E L L I N G WITH [ S]-METHIONINE 35 35  3. RESULTS  37  3.1 EFFECT OF C A R B O N SOURCE O N SRBIO K I N A S E A C T I V I T Y 37 3.1.1 Immunoprecipitated SRBIOphosphorylates GAL4 and the CTD 37 3.1.4 Mutation of SRBIO abrogates in vitro phosphorylation of GAL4 42 3.1.5 Kinase activity of endogenously-expressed SRBIO is not decreased in glucose 43 3.1.6 Growth in non-fermentable carbon may inhibit SRBIO kinase activity 44 3.1.7 Endogenously expressed SRB11 is not degraded in non-fermentable carbon 45 3.2 EFFECT OF NITROGEN LIMITATION O N SRBIO K I N A S E A C T I V I T Y 52 3.2.1 Nitrogen limitation causes a transient decrease in SRBIO activity 52 3.2.2 SRBIO is regulated by apost-transcriptional mechanism 54 4. DISCUSSION  62  4.1 DIFFERENT M E C H A N I S M S R E G U L A T E SRBIO IN C A R B O N V S . NITROGEN-LIMITING CONDITIONS 4.2 SIGNALING SPECIFICITY IN HAPLOIDS A N D DIPLOIDS 4.3 DEPLETION OF SRBIO IS A TRANSIENT P H E N O M E N O N 4.4 A N O V E L M E C H A N I S M OF C D K R E G U L A T I O N 4.5 F U T U R E DIRECTIONS 4.6 C O N C L U S I O N 5. REFERENCES  62 64 65 66 67 68 69  iv  LIST OF TABLES I. II.  Plasmids Yeast strains  31 32  v  LIST OF FIGURES 1. Model for initiation of transcription by R N A polymerase II  10  2. Life cycle of Saccharomyces cerevisiae  13  3. Signal transduction pathways regulating filamentous development  16  4. Mechanisms of C D K regulation  29  5. Immunopurified complexes containing HA-SRB10 phosphorylate G A L 4 and CTD in vitro  38  6. HA-SRB10 kinase activity is not affected by growth in glucose  40  7. Disruption of REG1 does not affect the in vitro kinase activity of immunopurified HA-SRB10  41  8. Tryptic phosphopeptide analysis of in vitro phosphorylated G A L 4  46  9. Mutation of SRB10 abrogates activity of immunopurified kinase complexes  47  10. Endogenously-expressed HA-SRB10 is not affected by growth in glucose  48  11. HA-SRB10 kinase activity does not diminish during prolonged growth in nonfermentable carbon  49  12. Disruption of SRB11 abrogates HA-SRB10 kinase activity but growth in nonfermentable carbon does not  50  13 .Endogenously-expressed SRB11 is not degraded during growth in nonfermentable carbon  51  14. Growth in nitrogen-limiting medium causes a transient decrease in HA-SRB10 kinase activity  53  15 .KIN28 protein levels remain constant during growth in nitrogen-limiting medium  55  16. Northern blotting of SRB10 in nitrogen-limiting conditions  56  17. Metabolic labelling of SRB10 protein  59  18. Overexpression of SRB10 does not inhibit filamentous growth  60  19. SRB10 stability is decreased in nitrogen-limiting conditions  61  vi  LIST OF ABBREVIATIONS Z Ab ATP bp CAK cAMP CDK Ci cpm CTD DNA DTT E. coli EDTA ERK Gal Glu Gly GST GTF GTP HA HAT His IgG Inr IPTG KAB kDa KLB Leu LSM MAb MAPK MEK MEKK mM MOPS MW ng Ni nm  micro sigma antibody adenosine triphosphate base pair C D K activating kinase 3'-cyclic-5'- adenosine monophosphate cyclin dependent kinase Curie counts per minute carboxy-terminal repeat.domain deoxyribonucleic acid dithiothreitol Escherichia coli ethylenediamine tetraacetic acid extracellular signal-related kinase galactose glucose glycerol Glutathione-S-transferase general transcription factor guanosine triphosphate hemagglutinin histone acetyltransferase histidine immunoglobulin G initiator isopropyl-|3-D-thiogalactopyranoside kinase assay buffer kilodalton kinase lysis buffer leucine low sulfate medium monoclonal antibody mitogen-activated protein kinase M A P / E R K kinase M A P / E R K kinase kinase millimolar 3-(N-morpholino) propane-sulfonic acid molecular weight nanogram nickel nanometer vii  NP-40 NSM nt OD o PAb PAGE PAK PCR PIC pmol PMSF Pro RAP RIPA R N A pol II RNA RPB S. cerevisiae S. pombe SAGA SC SCF SD SDS Ser S£9 SLAD TAF TBP TEA/ATTS TFII Thr Trp ts Tyr UAS URA WT YLB 60  Nonidet P40 no sulfate medium nucleotide optical density at 600nm polyclonal antibody polyacrylamide gel electrophoresis p21-activated kinase polymerase chain reaction preinitiation complex picomole phenylmethylsulfonyl fluoride proline R N A polymerase II-associated protein radio-labelled immunoprecipitation assay R N A polymerase II ribonucleic acid R N A polymerase B Saccharomyces cerevisiae Schizosaccharomyces pombe Spt-Ada-Gcn5-acetyltransferase synthetic complete Skpl/Cdc53/F-box, synthetic dropout sodium dodecyl sulfate serine Spodoptera frugiperda synthetic low ammonium dextrose TBP-associated factor TATA-binding protein TEF-1, TEC1, AbaA/AbaA, TEF-1, TEC1, Scalloped transcription factor of R N A polymerase II threonine tryptophan temperature-sensitive tyrosine upstream activating sequence uracil wild type yeast lysis buffer  viii  ACKNOWLEDGEMENTS I thank my supervisor, Ivan Sadowski, for his support, encouragement, and the inspiration of his boundless enthusiasm for the scientific process. I thank the members of my supervisory committee, Drs. Vincent Duronio, Michel Roberge, and George Mackie, for timely reading of my thesis and helpful suggestions along the way. I would also like to thank those members of the Sadowski lab, past and present, who have enriched my experience here in various ways.  I especially thank my father for his unconditional support and quiet encouragement, and my grandparents for the example of integrity, hard work, and generosity their lives have provided. Much gratitude also to the rest of my family and my friends for their camaraderie, counsel, and at times much-needed comic relief.  IX  Introduction  1. INTRODUCTION 1.1 OBJECTIVE The traditional view of transcriptional activation is one in which transcriptional activators bind to specific D N A sequences and more or less passively recruit a series of general transcription factors and R N A polymerase II to the site of initiation. With the discovery of multiprotein holoenzyme complexes and identification of their components, the concept of transcriptional regulation has changed. The presence of holoenzymeassociated protein kinases suggests the possibility that components of the transcription machinery may themselves serve as targets of signal transduction pathways. The R N A polymerase II holoenzyme-associated cyclin-dependent kinase SRB10 has been shown to phosphorylate multiple transcriptional activators involved in response to various nutritional and stress conditions. Such modifications are believed to communicate signals concerning the physiological status of the cell to these gene-specific activators in order to modulate an appropriate transcriptional response. However, little is known of the signals or mechanisms upstream of SRB10 that regulate this activity. The objective of this thesis research was to determine whether SRB10 kinase activity is regulated in response to specific nutritional conditions and if so, to elucidate the mechanisms responsible.  1.2 CONTROL OF EUKARYOTIC TRANSCRIPTION Transcriptional activation is regulated,according to the physiological needs of the cell. Because the amount of R N A polymerase II in a cell is limiting, gene-specific activators are required to increase the amount of transcription from specific promoters. Activators appear to control transcriptional initiation in two major phases: relief of repressive chromatin structure, and formation of a preinitiation complex (PIC) by recruitment of R N A polymerase II (RNA pol II) and accessory factors to the promoter (Kornberg, 1999; Gustafsson and Samuelsson, 2001) (Figure 1).  1  Introduction 1.2.1 Relief of chromatin-mediated repression Histone acetyltransferases Acetylation of specific lysine residues in the N-terminal tails of core histone proteins has been associated with transcriptionally active genes. This, along with the observation that many histone acetyltransferases (HATs) interact with D N A sequencespecific transcription factors, led to the presumption of a role for histone acetylation in transcriptional activation (Brown et al., 2000). Several complexes possessing H A T activity have been isolated. The most extensively characterized is the S A G A (Spt-AdaGcn5-acetyltransferase) complex, which preferentially acetylates histone H3 due to the catalytic activity of Gcn5 (Brownell et al., 1996). In addition to Gcn5, S A G A comprises at least 14 subunits, grouped into the Ada proteins, the TBP-related Spt proteins, a subset of TBP-associated factors (TAFs) (Grant et al., 1998a), and the ATM/PI-3-kinase-related protein Tral (Grant et al., 1998b). Other yeast H A T complexes include the smaller Gcn5-containing A D A complex, and the Gcn5-independent NuA3 (nucleosomal acetyltransferase of histone H3) and NuA4 (nucleosomal acetyltransferase of histone H4) complexes (Brown et al., 2000). Acetylation may weaken the affinity of histones for D N A , increasing the accessibility of D N A to transcription factors (Wade and Wolffe, 1997; Struhl, 1998). Since histone tails lie outside the core nucleosome particle and do not contribute to its organization or stability, acetylation is more likely to affect higherorder chromatin structure (Kornberg, 1999). Thus, relief of inhibition of R N A pol II and transcription factor binding to D N A within core particles requires additional mechanisms that remodel chromatin structure.  Chromatin-remodeling complexes  Chromatin remodeling is generally defined as any event that alters the nuclease sensitivity of a region of chromatin (Aalfs and Kingston, 2000). Two families of A T P dependent chromatin-remodeling complexes, termed SWI/SNF and ISWI, are distinguished based on their ATPase components. Complexes containing members of the yeast SWI (mating type switching) and SNF (sucrose non-fermenting) gene families include the SWI/SNF complexes of yeast, humans, and Drosophila, and the yeast RSC (remodels the structure of chromatin) complex (Kwon et al., 1994; Cote et al., 1994; 2  Introduction Cairns et al., 1996). These complexes contain between 8-16 distinct peptides, including an ATP-hydrolysing subunit homologous to yeast SWI2/SNF2. ISWI (imitation switch)based complexes are much smaller, containing between two and six peptides. Both types of complexes can catalyse the cz's-displacement, or sliding, of nucleosomes (Langst et al., 1999; Whitehouse et al., 1999). At high molar ratios, SWI/SNF can also catalyse the frans-displacement of a nucleosome (Lorch et al., 1999), as well as cause major topological changes in a nucleosomal array (Guyon et al., 1999). SWI/SNF is a relatively rare enzyme in yeast (100-500 copies per cell) (Cote et al., 1994) and is required for transcription of <5% of all yeast genes (Holstege et al., 1998); thus it has been hypothesized that SWI/SNF activity must be targeted to specific loci. Recent data support a model in which gene-specific activators recruit the SWI/SNF complex to target genes via direct interactions (Peterson and Workman, 2000).  1.2.2 R N A polymerase I I Core components Transcription of protein-coding genes in yeast is carried out by a 12-polypeptide complex encoded by the RPB1 to RPB12 genes that is collectively known as R N A pol II (Woychik and Young, 1994). Of these 12, all but RPB4 and RPB9 are essential for cell viability. The two largest subunits, RPB1 and RPB2, are the most conserved and, like their bacterial counterparts p' and P, are involved in binding to D N A and nucleotide substrates, respectively (Hampsey, 1998). The smaller subunits are arranged around the periphery of the complex and aid in positioning of the D N A (Cramer et al., 2000). Carboxy-terminal repeat domain RPB1, the largest R N A pol II subunit, is distinguished by the presence of tandem repeats of the heptapeptide sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser at its carboxyterminus. Although this carboxy-terminal repeat domain (CTD) is highly conserved amongst eukaryotes, its length increases with increasing genome complexity: 26-27 repeats in yeast, 34 in C. elegans, 43 in Drosophila, and 52 in humans (Hampsey, 1998). The CTD is essential for cell viability, with partial truncations causing numerous 3  Introduction phenotypes associated with altered gene regulation (Carlson, 1997). To identify proteins involved in CTD function, Nonet and Young (1989) performed a genetic screen to identify extragenic suppressors of a CTD truncation mutant that restore survival in the cold. Nine novel genes were identified and termed  SRBs  (suppressor of R N A polymerase  B). These genes were subsequently found to encode components of the Mediator complex required for activated transcription (Kelleher et al., 1990). Eukaryotic cells contain two major forms of RPB1, an unphosphorylated IIA form and a CTD-hyperphosphorylated IIO form (Dahmus, 1994; Cadena and Dahmus, 1987). CTD phosphorylation is correlated with conversion of R N A pol II from a promoterrecognition to an elongation-competent complex. Phosphorylation of the C T D may also couple R N A pol II transcription to mRNA processing (Bentley, 1999; Hirose and Manley, 2000), regulate R N A pol II stability (Huibregste et al., 1997; Mitsui and Sharp, 1999), and function in transcription-coupled D N A repair (Mitsui and Sharp, 1999; Ratner et al., 1998). Global changes in CTD phosphorylation have also been observed in various phases of the cell cycle (Akoulitchev and Reinberg, 1998; Parsons and Spencer, 1997), in response to growth factors and mitogens (Garriga et al., 1998; Bonnet et al., 1999), and in response to D N A damage (Ratner et al., 1998). Multiple protein kinases, particularly cyclin-dependent kinases, in yeast and mammals are capable of phosphorylating the CTD: CDK7 and its yeast homolog KIN28 (Feaver et al., 1991; Dubois et al., 1997), CDK8 and yeast SRBIO (Liao et al., 1995; Sun et al., 1998), the catalytic subunit of P-TEFb, CDK9 and its tentatively identified yeast homolog BURlfMurray et al., 2001; Zhou et al., 2000), yeast CTKl(Lee and Greenleaf, 1991), and mammalian CDC2 (Gebara et al., 1997) all specifically phosphorylate serine residues in the heptapeptide repeat. The M A P kinase ERK-1/2 also phosphorylates C T D serine residues to generate a novel form of R N A pol II that can be distinguished electrophoretically from the IIA and IIO forms (Bonnet et al., 1999). A physiological role for this form has not been determined. Phosphorylation of tyrosine residues in the C T D can be carried out by the c-Abl tyrosine kinase, which functions in cell cycle regulation and D N A damage response (Baskaran et al., 1993). A CTD phosphatase (CTDP) has also been identified (Chambers and Kane, 1996). Regulated by TFIIB and TFIIF, it is postulated to play a role in R N A pol II recycling. 4  Introduction RNA polymerase II holoenzymes Discovery and purification of the general transcription factors (GTFs) for R N A pol II (see below) led to a step-wise assembly model for preinitiation complex (PIC) formation. This was based on biochemical studies identifying a defined order of GTF assembly culminating in recruitment of core R N A pol II (Buratowski et al., 1989). The in vivo significance of this model was challenged by the discovery of the SRB proteins (Nonet and Young, 1989) and subsequent purification of SRB protein complexes that contained R N A pol II and a subset of GTFs (TFIIB, TFIIF, TFIIH) along with various regulatory proteins (Koleske and Young, 1994). When supplemented with TBP and TFIIE, this 'holoenzyme' complex was able to both initiate transcription and respond to activators in vitro. A comparable holoenzyme (lacking TFIIB and TFIIH) was identified based on its interaction with the mediator complex (see below) (Kim et al., 1994). A variety of holoenzymes have since been purified from many eukaryotic organisms. These complexes differ with respect to their constituent GTFs and regulatory factors, and in the subsets of genes they transcribe (Hampsey and Reinberg, 1999). Due to the technical challenges involved in purifying intact megadalton-size multiprotein complexes, it is difficult to determine which of these differences in subunit composition represent true in vivo holoenzyme variations and which represent artifacts of the particular method used to prepare them. 1.2.3 General transcription factors The GTFs, including TFIID, TFIIB, TFIIE, TFIIF, and TFIIH, were purified by their ability to facilitate accurate transcription initiation by core R N A pol II, which is incapable of specific promoter recognition on its own (Conaway and Conaway, 1997). TFIID The TFIID complex, composed of TATA-binding protein (TBP) and TBPassociated factors (TAFs), is responsible for promoter recognition. TBP functions in basal level transcription, whereas TAFs are required for response to transcriptional activators (Verrijzer et al., 1996; Green, 2000). TBP is a 27kDa monomer encoded by the yeast SPT15 gene (Eisenmann et al., 1989). Binding of TBP to the T A T A box is the first,  Introduction rate-limiting, step in assembly of the preinitiation complex. Bending of T A T A box D N A around TBP creates a context for interaction with TFIIB (Nikolov et al., 1992). TFIIB Yeast TFIIB is a 38kDa protein encoded by the SUA7 gene that functions in transcription start site selection (Pinto et al., 1992). TFIIB enters the PIC after TBP and recruits R N A pol II in collaboration with TFIIF (Li et al., 1994). Discrete domains of TFIIB interact directly with TBP, R N A pol II, TFIIF and other GTFs (Nikolov et al, 1995; Sun et al., 1996; Ha et al., 1993; Fang and Burton, 1996), as well as gene-specific transcriptional activators (Kim and Roeder, 1994; Roberts et al., 1993). TFIIF TFIIF is composed of two subunits, RAP30 and RAP74, originally identified based on their affinity for R N A pol II (Burton et al., 1988). TFIIF is characterized by tight binding to R N A pol II, suppression of non-specific binding of R N A pol II to D N A , stabilization of the R N A pol II-TFIIB interaction, and stabilization of the PIC by modifying D N A structure in the core promoter (Greenblatt, 1991; Forget et al., 1997; Robert et al., 1998). TFIIF, along with TFIIE and TFIIH, is also involved in the transition of R N A pol II from pre-initiation to elongation (Conaway et al., 2000). TFIIE TFIIE enters the PIC after R N A pol II-TFIIF and prior to TFIIH (Buratowski et al., 1989), interacting directly with the unphosphorylated form of R N A pol II, TFIIF, and TFIIH. Functionally, TFIIE is intimately linked with TFIIH, recruiting it to the PIC and stimulating both its kinase and ATP-hydrolysing activities (Ohkuma and Roeder, 1994). TFIIE and the ATP-dependent helicase within TFIIH participate in unwinding of . promoter D N A , while the transition from initiation to elongation is facilitated by phosphorylation of the CTD by TFIIH kinase activity (Buratowski, 2000). TFIIH As the only GTF with known enzymatic activities, components of TFIIH include a DNA-dependent ATPase, an ATP-dependent helicase, and a CTD kinase (Conaway and Conaway, 1989; Serizawa et al., 1993; Feaver et al., 1991). In yeast, TFIIH was purified as a five-subunit core complex and subsequently, as a holoenzyme containing the core 6  Introduction complex, SSL2, and a two-subunit subcomplex denoted TFIIK (Svejstrup et al., 1994). TFIIH performs crucial functions in both initiation and post-initiation phases of transcription. Its D N A helicase activity is required for formation of an open promoter complex necessary for promoter clearance by R N A pol II (Holstege et al., 1996; Kugel and Goodrich, 1998). Progression from initiation to elongation is facilitated by phosphorylation of the CTD. The TFIIK subcomplex contains a cyclin-dependent kinase encoded by KIN28 and a cyclin H homolog encoded by CCL1 (Feaver et al., 1994; Svejstrup et al., 1996); this CDK-cyclin pair constitutes the principle C T D kinase activity in vivo (Valay et al., 1995). TFIIH also suppresses transcription arrest of early R N A pol II elongation complexes (Spangler et al., 2001). In addition to its roles in transcription, TFIIH is also an essential component in nucleotide excision repair (NER) and may be involved in mammalian cell cycle progression (Hampsey, 1998).  1.2.4 Transcriptional coactivators Interactions between gene-specific transcriptional activators and GTFs have been implicated in gene activation in vivo. However, activators alone fail to stimulate transcription in reconstituted R N A pol II/GTF systems in vitro, indicating the requirement for additional factors, termed coactivators, which mediate the effects of transcriptional activators on the basal transcription machinery. Coactivators are defined as factors that are required for the function of DNA-binding activators, but not for basal transcription, and do not show site-specific D N A binding themselves (Malik and Roeder, 2000). TBP-associated factors Although TFIID is classified as a GTF based on the requirement for TBP in basal transcription, it contains additional subunits, called TBP-associated factors (TAFs), that are dispensable for basal transcription, but necessary for activator-stimulated transcription. The TAFs are highly conserved: in almost all cases, yeast TAFs have recognizable homologs in higher eukaryotes. Except for yeast TAF30 (yTAF30), all are essential for viability, implying at least one obligate, non-redundant function for each (Green, 2000). A subset of TAFs exhibits structural similarities to core histones. These 7  Introduction TAFs may assemble into octamer-like configurations analogous to those of true core histones (Burley and Roeder, 1996). In yeast, genome-wide studies on TAF inactivation have shown that TAFs function as promoter-selectivity factors, with each T A F necessary for transcription of a characteristic subset of genes, ranging from 3% to 67% of the genome (Holstege et al., 1998). The broadest requirement is for the histone H3-like TAF17. Inactivation of specific TAFs has also been shown to impact cell cycle progression in yeast and mammalian cells (Apone et al., 1996; Metzger et al., 1999; Martin et al., 1999). Perhaps surprisingly, TAFs have been found in multisubunit complexes other than TFIID. The yeast S A G A complex and its human counterpart S T A G A , the human P C A F (p300/CBPassociated factor) complex, and TFTC (TBP-free TAF-containing complex) all contain TAFs, along with a single subunit with H A T activity (Martinez et al., 1998; Ogryzko et al., 1998; Brand et al., 1999). Three major mechanisms of action have been proposed. First, TAFs may serve as activator-binding sites, with different TAF-activator combinations defining transcriptional selectivity. Alternatively, TAFs may mediate corepromoter recognition, or provide an essential catalytic activity (Green, 2000). Evidence exists to support each of these hypotheses, and it is likely that different TAFs exert their differential effects through a combination of mechanisms. TFIIA TFIIA was initially identified as a GTF based on in vitro requirements for specific transcription (Matsui et al., 1980). However, it has since been shown to be dispensable for accurate initiation, and instead acts as a coactivator by stabilizing TBP binding to the T A T A box, displacing transcriptional repressors from TFIID, and interacting with specific transcriptional activators and coactivators (Imbalzano et al., 1994; Ozer et al., 1994; Kang et al., 1995; M a et al., 1996; Stargell et al., 2000). Mediator The existence of a 'mediator' of transcriptional activation was initially suggested by 'squelching' experiments in which overexpression of one activator could interfere with the effects of another (Gill and Ptashrie, 1988). This phenomenon was attributed to the sequestration of a factor, present in limiting amounts, required for activated 8  Introduction transcription. When activator interference was reproduced in an in vitro transcription reaction, a distinct crude yeast fraction was able to relieve interference, while excess R N A pol II and GTFs could not (Kelleher et al., 1990). Purification of the activity in this fraction yielded Mediator, a 20-subunit complex believed to function as an adaptor between activators and the basal transcription machinery (Kim et al., 1994; Myers et al., 1998). One prominent group of S. cerevisiae Mediator subunits contains the SRB proteins (Nonet and Young, 1989). SRB2, 4, 5, and 6 were identified genetically as dominant, gain-of-function suppressors of CTD truncation mutants. These four proteins form a stable complex that has been shown to bind the transcriptional activator G A L 4 (Koh et al., 1998). SRB4, SRB6, and SRB7, which was identified as a recessive suppressor, are essential for viability, whereas the other SRB proteins are not. SRB4 and SRB6 appear to play general roles in transcription. Yeast genome-wide expression profiles of srb4 mutants reflect a cessation of mRNA synthesis at virtually all promoters, ts  comparable to the effect of an rpbl mutation, which results in a general loss of R N A pol ts  II transcription (Holstege et al., 1998). The remaining SRB proteins, SRB8-11, form a subcomplex that is not detectable in all forms of Mediator. SRBIO and SRB11 form a CDK-cyclin pair homologous to metazoan CDK8-cyclin C that is capable of phosphorylating the CTD (Liao et al., 1995). SRB8 and SRB9 are required for stable association of SRB10/11 with the holoenzyme (Nonet and Young, 1989). Another group of Mediator components includes GAL11, SIN4, RGR1, ROX3, PGD1/HRS1, NUT1, NUT2, and CSE2. These gene products were previously identified in various screens based on their influence on repression of certain genes, but were not identified as SRBs (for review, see Carlson, 1997). Most of these subunits seem to play a dual regulatory role, activating some sets of genes and repressing others. The third group of Mediator subunits is encoded by the MED genes, which were uncharacterized prior to purification of Mediator. Certain M E D proteins, such as M E D 1 , MED2, and MED11, seem to be specific to S. cerevisiae, while others, such as M E D 6 and M E D 7 , are conserved in all Mediator-like complexes isolated to date (Gustafsson and Samuelsson, 2001).  9  Introduction  IAIA  Figure 1. Simplified model of transcription initiation by R N A polymerase II. (A) Eukaryotic promoters, consisting of the core TATA box (TATA) and initiator (Inr) elements, and regulatory elements such as upstream activating sequences (UAS) are maintained in a quiescent state by the repressive effects of chromatin. (B) Stimulation of transcription by a transcriptional activator bound to the U A S involves the relief of chromatin-mediated repression via histone acetylation and chromatin remodelling (not shown), and recruitment of the R N A pol II holoenzyme through interactions between the activator and its GTF (light grey) and coactivator (dark grey) targets. (See text for details) 10  Introduction 1.3 F I L A M E N T O U S D E V E L O P M E N T I N SACCHAROMYCES CEREVISIAE The yeast Saccharomyces cerevisiae exists in two haploid cell types, a and a, and an a/a diploid cell type, which typically grow by budding to produce ellipsoidal daughter cells that separate from the mother cell at cytokinesis and remain on the surface of an agar growth medium. In response to peptide mating pheromones secreted by the opposite cell type, a and a cells undergo sexual differentiation and cell-cell fusion to form diploids. Diploid cells starved simultaneously for fermentable carbon and fixed nitrogen undergo meiosis and sporulation to form four haploid spores. Under certain conditions, both haploid and diploid yeast cells can undergo a dimorphic transition to a filamentous form (Figure 2).  1.3.1  The phenotype of filamentous growth Diploid pseudohyphal growth When starved for nitrogen in the presence of an abundant fermentable carbon source, diploid yeast cells switch to a filamentous, or pseudohyphal, mode of growth characterized by changes in at least three cellular processes (Gimeno et al., 1992). Cell morphology becomes elongated, daughter cells remain attached to mothers following cytokinesis, and bud site selection changes from a bipolar to a unipolar pattern. This triumvirate of growth characteristics results in the formation of multicellular filaments that project out from the main body of the colony and are capable of invading an agar substrate, perhaps providing a mechanism for otherwise sessile cells to expand their periphery and forage for nutrients or escape from environmental stresses. Haploid invasive growth Haploid cells do not form pseudohyphae in response to nitrogen limitation (Gimeno et al., 1992). However, filament formation in haploids has been observed in colonies growing on rich media (Roberts and Fink, 1994; Lo et al., 1997). Termed haploid invasive growth, this phenomenon depends on a switch from an axial to a bipolar budding pattern similar to that of non-pseudohyphal diploids. Although haploid filamentous cells are elongated and remain attached, haploid filaments do not spread out 11  Introduction across the surface of the growth medium in the manner of diploid pseudohyphae, but are confined to invasion of the agar beneath the colony. Since agar penetration was observed after growth of the colony had slowed, general nutrient deprivation was suggested as the trigger for invasive growth (Roberts and Fink, 1994). More specifically, it has since been demonstrated that haploid invasive growth is induced by the depletion of fermentable carbon sources (Cullen and Sprague, 2000). Thus, rather enigmatically, filamentous growth phenotypes in haploids and diploids appear to be regulated by opposite signals: invasive growth is the result of starvation for fermentable carbon and is unaffected by nitrogen status, while pseudohyphal growth is triggered by nitrogen limitation in the presence of abundant fermentable carbon. The picture is further complicated by recent observations that various short-chain alcohols, byproducts of amino acid metabolism that are produced primarily under conditions of nitrogen starvation, can induce filamentous development. Unexpectedly, these alcohols had a much more dramatic effect on haploids than on diploids (Lorenz et al., 2000a). Despite their dissimilar stimuli, haploid and diploid filamentous growth phenomena appear to share the same signal transduction pathways.  1.3.2 The M A P kinase pathway The dimorphic transition in S. cerevisiae is controlled by at least two different signaling pathways: a M A P kinase (MAPK) cascade and a cAMP-dependent pathway (Figure 3). In the M A P K pathway, an as-yet-unidentified signal is received by the small GTP-binding protein RAS2, which signals through two 14-3-3 proteins, BMH1 and B M H 2 , to CDC42 (Mosch et al., 1996; Roberts et al., 1997). Activated CDC42 interacts with the P A K STE20 and displaces the negative regulator HSL7 (Leberer et al., 1997; Fujita et al., 1999). STE20 then activates the M A P K module consisting of the M E K K STE11, the M E K STE7, and the M A P K KSS1 (Gustin et al., 1998). Unphosphorylated KSS1 interacts with the transcription factor STE12 and the negative regulators DIG1/RST1 and DIG2/RST2, potentiating DIG-mediated inhibition of STE12. Upon activation of KSS1, negative regulation of STE12 by KSS1 and the DIGs is alleviated and transcription of filamentous response genes is induced (Cook et al., 1996; Tedford et al., 1997; Bardwell et al., 1998). Target genes of the filamentation-invasion M A P K 12  Introduction  o  H O  o M K O Q W  .fl o 'o  H  o O 2  %  03  i—i  z « 3 i3 « P o 3  13  Introduction pathway are activated by STE12 in combination with a transcription factor of the T E A / A T T S family known as TEC1. The promoters of these genes contain filamentation and invasion response elements (FREs) consisting of a pheromone response element (PRE) and a T E A / A T T S consensus sequence (TCS) that serve as STE12 and TEC1 binding sites, respectively (Madhani and Fink, 1997). Examples of FRE-containing genes are FLOll,  which encodes a cell-surface flocculin necessary for filamentous  growth (Lo and Dranginis, 1998), and the TEC1 gene itself (Madhani and Fink, 1997).  1.3.3 The cAMP-dependent pathway In addition to its function in the filamentation-invasion M A P K cascade, RAS2 can also activate a cAMP-dependent pathway by interaction with CYR1 (adenylate cyclase), stimulating an increase in c A M P levels, which in turn activates the c A M P dependent protein kinase, protein kinase A (PKA) (Minato et al., 1994). Alternatively, c A M P production by adenylate cyclase can be regulated by GPA2 in response to nutritional signals (Nakafuku et al., 1988; Columbo et al., 1998). Yeast P K A consists of a regulatory subunit encoded by a single gene, BCY1, in combination with one of three catalytic subunits encoded by TPK1, TPK2, and TPK3 (Cannon and Tatchell, 1987). TPK1, TPK2, and TPK3 are essentially redundant during vegetative growth, but play distinct roles in filamentous development (Pan and Heitman, 1999; Robertson and Fink, 1998). TPK2 acts as a positive regulator, while TPK1 and TPK3 inhibit filamentation. Epistasis analysis suggests that TPK1 and TPK3 exert their negative effect upstream of P K A itself, possibly through a negative feedback loop that represses c A M P synthesis (Pan and Heitman, 1999). TPK2 appears to enhance filamentous growth by regulating various transcriptional activators and repressors: these include the activator F L 0 8 and the repressor SFL1, both of which regulate expression of F L O 11 (Pan and Heitman, 1999; Robertson and Fink, 1998; Rupp et al., 1999).  1.3.4 Interaction between MAP kinase and cAMP pathways Activated P K A (due to bcyl mutation or exogenous cAMP) can stimulate filamentous growth in the absence of STE12, TEC1, or both STE12 and TEC1 (Pan and 14  Introduction Heitman, 1999), indicating that the cAMP-dependent and M A P K pathways can function independently. However, the filaments produced in such situations are composed of ellipsoidal rather than elongated cells, suggesting that the M A P K pathway, but not the cAMP-dependent P K A pathway, is responsible for changes in cell morphology. On the other hand, the P K A pathway does appear to control other aspects of filamentous development, since TPK2 is required for both invasion and the switch to a unipolar budding pattern. Thus, the two pathways have both common and distinct functions: both are involved in invasion and cell-cell adhesion, while the P K A pathway affects budding pattern and the M A P K pathway affects cell elongation. This notion is consistent with the work of Mosch and Fink (1997) which suggests that the target genes of filamentation signal transduction pathways occur in functional groupings based on separable cellular processes such as cell morphogenesis, bud site selection, and invasiveness. In addition to these complementary roles, there are numerous examples of crosstalk between the two pathways. Both converge on the FLO 11 promoter, accounting for their shared involvement in cell-cell-adhesion; at the other end, both pathways can be activated by RAS2. Negative regulation of the M A P K pathway by P K A is suggested by observations that c A M P inhibits expression of MAPK-regulated reporter genes, and high levels of c A M P or P K A give rise to filaments composed of ellipsoidal, not elongated, cells (Lorenz and Heitman, 1997; Pan and Heitman, 1999). P K A may also positively regulate the M A P K cascade at a level downstream of STE20 (Mosch et al., 1999).  1.3.5 Upstream signals Although it is now widely accepted that nitrogen limitation in diploids and lack of fermentable carbon in haploids are signals for filamentous development, the precise nature of these signals remains enigmatic. Correspondingly, there is currently no clear picture of how RAS2, which is positioned upstream of both the M A P K and c A M P dependent pathways, becomes activated by conditions leading to pseudohyphal and invasive growth. RAS2-independent signaling through the cAMP-dependent pathway involves a novel G protein-coupled receptor (GPCR) called GPR1, which is associated with the G protein a subunit GPA2; both proteins are required for filamentous growth (Kubler et al., 15  Introduction  glucose  FLOll  t Unipolar budding  v  Cell-cell adhesion Agar invasion  Cell elongation  i  FILAMENTOUS GROWTH  Figure 3. Signal transduction pathways regulating filamentous differentiation in S. cerevisiae. At least two parallel signalling pathways regulate filamentous growth: a cAMP-dependent pathway and a M A P kinase cascade. See text for details. (Adapted from Lengeler et al., 2000). 16  Introduction 1997; Lorenz et al., 2000b) and for glucose-induced c A M P synthesis (Lorenz et al., 2000b; Columbo et al., 1998). Several observations suggest that GPR1 and GPA2 may function as a dual sensor for both fermentable carbon and limiting nitrogen. GPR1 expression is induced by nitrogen starvation (Xue et al., 1998); dominant-active GPA2 mutants or exogenous c A M P signal filamentous growth at normally repressive nitrogen concentrations (Lorenz and Heitman, 1997); and GPA2 association with, and consequent inhibition of, IME2, a kinase essential for promotion of meiosis and sporulation, is enhanced in the presence of abundant nitrogen (Donzeau and Bandlow, 1999). The high affinity ammonium permease MEP2 may also act as a nitrogen sensor (Lorenz and Heitman, 1998). S H O l , a putative membrane-spanning protein, responds to high osmolarity, but may also be capable of sensing a low nitrogen signal and relaying it to the M A P K pathway via STE20 (Maeda et al., 1995; O'Rourke and Herskowitz, 1998).  1.3.6 Other proteins involved in filamentous growth In addition to the ones mentioned above, many other proteins have been implicated in the regulation of filamentous development. The ELM genes, mutations in which cause abnormally elongated cell morphology as well as other characteristics of pseudohyphal cells, may regulate a pathway that inhibits pseudohyphal growth under optimal growth conditions. ELM1 encodes a serine/threonine protein kinase whose absence leads to constitutive pseudohyphal morphology (Blacketer et al., 1993; Koehler and Meyers, 1997). A possible target of E L M 1 is the protein kinase HSL1, which inhibits SWE1, an inhibitor of the penultimate cell cycle regulator CDC28 (Edgington et al., 1999). The role of CDC28 in filamentous development appears to be mediated in part by its G l cyclins. Mutational analysis indicates that CLN3 functions as a negative regulator, while CLN1 and CLN2 play a positive role in a pathway which functions in parallel with the filamentation-invasion M A P K pathway (Loeb et al., 1999). Numerous transcription factors in addition to F L 0 8 , STE12, and TEC1 affect filamentous growth. These include SOK2, ASH1, PHD1, and SWI5 (Ward et al., 1995; Chandarlapaty and Errede, 1998; Gimeno and Fink, 1994; Pan and Heitman, 2000). ASH1, PHD1, and SWI5 all activate FLO 11 expression independently of both the M A P K and c A M P dependent pathways. It has recently been shown that SOK2 negatively regulates 17  Introduction expression of the genes encoding all three of these transcription factors (Pan and Heitman, 2000). SNF1, the AMP-activated kinase responsible for derepression of glucose-repressed genes, is required for both carbon depletion-induced haploid invasive growth and nitrogen starvation-induced diploid pseudohyphal growth, implying a role more complex than its well-known function in relief of glucose repression (Cullen and Sprague, 2000). One target of SNF1 is SIP4, a transcription factor required for activation of genes during the diauxic shift, which was shown to have a negative effect on filamentous growth (Cullen and Sprague, 2000). Another SNF1 target is the transcription factor MSN1 which, along with MS SI 1, is involved in another RAS2-dependent signal transduction pathway regulating FLOll  expression (Gagiano et al., 1999a). MSN1 acts  independently of the M A P K pathway, while MSS11 appears to participate in both pathways, functioning downstream of both MSN1 and STE12. MSS11 may also function downstream of F L 0 8 (Gagiano et al., 1999b).  1.4 T H E SRB10 CYCLIN-DEPENDENT KINASE 1.4.1 SRB 10 is an RNA polymerase II holoenzyme-associated cyclin-dependent kinase The yeast SRB genes were originally identified in a screen for suppressors of the cold-sensitive phenotype conferred by truncations of the RPB1 CTD (Nonet and Young, 1989). Subsequent characterization revealed that the products of the SRB 10 and SRB 11 genes encode a CDK-cyclin pair (Liao et al., 1995). SRB10 encodes a 541-amino acid protein kinase that is -40% identical to the prototypical CDKs, S. cerevisiae CDC28 and S. pombe CDC2. SRB10 is homologous to mammalian CDK8: the two proteins share a high degree of structural similarity as well as several distinct features, including a noncanonical D F G motif in kinase subdomain VII, and the sequence S A C R E in the PSTAIRE domain typical of CDKs (see Section 1.5.1 below) (Tassan et al., 1995). SRB11 is a 323-amino acid protein with 28% identity and 58% similarity, to human and Drosophila cyclin C (Lew et al., 1991; Leopold and O'Farrell, 1991), with 37% identity in the residues corresponding to the highly conserved cyclin box, which is involved in binding to C D K subunits (see Section 1.5.1 below). SRB 10 and SRB 11 are identical to SSN3 and SSN8, which were isolated as suppressors of SNF1 mutations (Kuchin et al., 18  Introduction 1995). SNF1 is a protein kinase required for release from glucose repression, therefore SRB 10 and SRB11 were implicated in negative regulation of transcription. SRBIO and SRB 11 were also identified as UME5 and UME3, respectively, as genes required for the mitotic repression of certain meiotic genes (Strich et al., 1989; Surosky et al., 1994); SRBIO is also identical to ARE1, which is required for a2 repression of mating typespecific genes (Wahi and Johnson, 1995). Association of SRBIO and SRB11 with the R N A pol II holoenzyme was demonstrated by copurification and immunoprecipitation experiments (Liao et a l , 1995). Homologs of SRBIO and SRB11 are components of Mediator complexes isolated from higher eukaryotes; however, they are not detectable in yeast Mediator isolated on the basis of its ability to support activated transcription (Myers et al., 1998; Myers and Romberg, 2000).  1.4.2 SRBIO is a C T D kinase Phosphorylation of the CTD is reduced ~ 10-fold in yeast with a point mutation of SRBIO that eliminates its kinase activity without affecting its incorporation into the holoenzyme, indicating that SRBIO kinase activity is important for CTD phosphorylation (Liao et al., 1995). It was subsequently shown that SRBIO has CTD kinase activity (Hengartner et al., 1998). SRBIO phosphorylates Ser-5 of the CTD heptapeptide repeat, a substrate specificity indistinguishable from that of the other known holoenzymeassociated C D K component, KIN28. Despite this similarity in substrate specificity, KIN28 and SRBIO are not functionally redundant. KIN28, along with its cognate cyclin C C L 1 , is a component of TFIIH and is apparently responsible for CTD phosphorylation following PIC formation (Dahmus, 1996). KIN28 and CCL1 are essential genes; loss-offunction mutations lead to a general defect in R N A pol Il-dependent transcription (Valay et al., 1995; Holstege et al., 1998), indicating a positive role in transcription of almost all genes in the genome. In contrast, SRBIO and SRB 11 are dispensable for growth under most conditions and, in most cases, have a negative regulatory effect on transcription of certain subsets of genes. It has been proposed that the functional distinction between these two kinases is a temporal one (Hengartner et al., 1998). R N A pol II found in holoenzymes lacks phosphate on its CTD (Koleske and Young, 1994), and it is this unphosphorylated form that is preferentially incorporated into the PIC (Maxon et al., 19  Introduction 1994). Subsequent CTD phosphorylation by KIN28 facilitates the transition from initiation to active elongation. SRB 10 would exert its negative effect by phosphorylating the CTD prior to initiation, thereby preventing PIC formation. Support for this hypothesis includes the observation that preincubation of ATP with holoenzymes isolated from a WT SRB 10 strain inhibits an in vitro transcription reaction, whereas preincubation of A T P with holoenzymes from an srblO kinase-dead mutant does not (Hengartner et al., 1998). This model, however, is insufficient to reconcile all the issues concerning SRB 10 function, such as its role in activation as well as repression, and its effect on only specific subsets of genes.  1.4.3  SRB10  regulates gene-specific transcription factors  Although most of the attention has been focused on SRBlO's role as a CTD kinase, a spate of recent studies has uncovered a new aspect of SRB 10 function involving the regulation of gene-specific transcription factors. G A L 4 regulates expression of the GAL genes in response to galactose. In the absence of galactose, G A L 4 binds to upstream activating sequences for galactose (UASG) but is prevented from activating transcription by the inhibitor GAL80. Galactose induces GAL gene expression by binding to G A L 3 ; GAL3-galactose interacts with GAL80, causing a conformational change in the GAL80-GAL4 complex that allows interaction of G A L 4 activation domains with GTFs (Piatt and Reece, 1998; Yano and Fukasawa, 1997). As a consequence of transcriptional activation, G A L 4 is phosphorylated on multiple serine residues (Hirst et al., 1999; Mylin et al., 1989; Sadowski et al., 1991). Only one of these phosphorylations, at Ser699, is necessary for full GAL gene induction (Sadowski et al., 1996). This phosphorylation is mediated by SRB 10: SRB 10 is required for in vivo Ser699 phosphorylation, and purified SRB10-SRB11 complexes specifically phosphorylate Ser699 in vitro (Hirst et al., 1999). SRBlO-mediated Ser699 phosphorylation was subsequently shown to regulate G A L 4 independently of the G A L 3 galactose pathway (Rohde et al., 2000). There is evidence to suggest that SRB 10 is regulated by the environment: SRB 10 protein levels decrease as cells undergo the diauxic shift (Holstege et al., 1998); SRB11 is degraded in response to heat shock, hypoxic stress, and starvation for certain nutrients (Cooper et al., 1997; Cooper et al., 20  Introduction 1999); and SRB 10 appears to function in repression of genes involved response to stress or nutrient limitation (Holstege et al., 1998). Thus, it was proposed that SRB 10 is active under favorable general growth conditions and that G A L 4 Ser699 phosphorylation allows enhanced induction of the GAL genes. Under growth-limiting conditions, SRB 10 would be inactive, and the absence of Ser699 phosphorylation would prevent GAL gene induction from exceeding the overall physiological needs of the cell (Rohde et al., 2000). There are two corollaries to this model: first, that SRB 10 is the target of signals generated by the physiological environment of the cell, and second, that SRB 10 must regulate multiple transcription factors in addition to GAL4. Another example of positive regulation by SRB 10 involves the transcriptional activator SIP4, which binds to carbon source-responsive elements (CSRE) in the promoters of gluconeogenic genes (Vincent and Carlson, 1998). In response to glucose limitation, SIP4 is phosphorylated and its ability to activate transcription increases. Although direct phosphorylation of SIP4 by SRB 10 was not demonstrated, SRB 10 is required for phosphorylation of SIP4 during growth in non-fermentable carbon, the catalytic activity of SRB 10 stimulates the ability of LexA-SIP4 to activate transcription of a reporter, and the SRB 10 and SIP4 proteins physically interact with each other, suggesting that SRB10 directly regulates SIP4 (Vincent et al., 2001). SRBlO-mediated phosphorylations have also been shown to have a negative effect on transcriptional activators that regulate expression of genes involved in cellular responses to starvation or stress. GCN4, a transcriptional activator that induces expression of amino acid and purine biosynthesis genes under conditions of amino acid starvation (Hinnebusch, 1992), is highly unstable. Its degradation is dependent on the ubiquitin-proteasome system, mediated by the S C F  C D C 4  E3 ubiquitin ligase complex  (Meimoun et a l , 2000). A common feature in the ubiquitination of known S C F  C D C 4  substrates is phosphorylation by CDC28, which appears to promote binding of the F-box receptor CDC4 to substrates (Skowyra et al., 1997). CDC28 does not phosphorylate GCN4, but phosphorylation is necessary to target GCN4 for ubiquitination. Purification of the kinase activity responsible for GCN4 phosphorylation led to the identification of SRB10 (Chi et al., 2001). GCN4 was shown to be phosphorylated in vitro by immunopurified SRB 10, and in vivo in an SRBlO-dependent manner. Correspondingly, 21  Introduction GCN4 was stabilized in srblOA and srW0(kinase-inactive) strains. GCN4 is almost completely stabilized in srblOApho85A cells, or upon mutation of all five consensus C D K sites on GCN4, suggesting that these two kinases conspire to limit GCN4 accumulation. MSN2 induces transcription of a set of genes in response to various environmental stresses via the STRE (stress response element). In unstressed cells, MSN2 is inactive due its localization in the cytoplasm; exposure to stress conditions triggers its translocation to the nucleus (Gorner et al., 1998). SRBIO is implicated in the regulation of MSN2, in that multiple MSN2 target genes are induced in srblO mutant cells (Holstege et al., 1998). MSN2 was shown to be phosphorylated by SRBIO in vitro, and phosphorylation of MSN2 was modulated by SRBIO in vivo (Chi et al., 2001). Mutation of SRBIO also resulted in increased nuclear localization of MSN2, although this effect was not dramatic, probably due to the influences of other factors, such as TOR and P K A which regulate MSN2 trafficking in stressed cells (Gorner et al., 1998; Beck and Hall, 1999). It appears that SRBlO-mediated phosphorylation serves to inactivate MSN2 under normal growth conditions by causing its nuclear exclusion; this notion is consistent with recent observations that MSN2 exists in a phosphorylated state during exponential growth on glucose, and becomes hyperphosphorylated in response to stress (Garreau et al., 2000). SRB10 has also been implicated in the regulation of filamentous growth, which occurs as a result of nitrogen limitation in diploids and fermentable carbon depletion in haploids. Diploid cells lacking SRB10 exhibit enhanced pseudohyphal growth and F L O l 1 expression is induced 15-fold in srblO mutants growing in rich medium (Holstege et al., 1998). Both SRB10 and SRB11, as well as SRB8, which mediates their interaction with the holoenzyme, were recently identified in a genetic screen for repressors of  FLOll  (Palecek et al., 2000). It has been shown in our laboratory that SRB10 phosphorylates the transcription factor STE12 (C. Nelson, unpublished results) which, in conjunction with TEC1, activates transcription of filamentous responsive genes. Phosphorylation of STE12 during growth in rich medium inhibits filamentous growth; these phosphorylations are lost during growth of diploids in nitrogen-limiting medium.  22  Introduction  1.5 CYCLIN-DEPENDENT KINASES 1.5.1 General principles Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases that are dependent on association with a cyclin regulatory subunit for their activity (Pines, 1995). Originally identified as essential regulators of cell cycle progression (Hartwell et al., 1974), CDKs have since been found to function in other cellular pathways with diverse effects. C D K catalytic subunits are categorized as such by a high level of sequence identity with other members of the family, typically sharing 35-65% identity with the prototypical CDKs from S. cerevisiae (CDC28) and S. pombe (CDC2) (Hanks and Hunter, 1995). Particularly well-conserved is a domain near the amino-terminus known as the PSTAIRE motif, which is unique to, and diagnostic of, the C D K family. CDKs are proline-directed kinases that phosphorylate serine or threonine residues in S/T-P motifs (Langan et al., 1989). Given the simplicity of this consensus sequence, additional determinants of C D K substrate specificity were anticipated. It has since been demonstrated that, at least in a subset of mammalian CDK-cyclin combinations, the cyclin subunit participates in substrate selection via the interaction of two short consensus sequence motifs, Z R X L (a cyclin-binding motif) and L X C X E (part of the cyclin structure) (Schulman et al., 1998; Adams et al., 1999). The three-dimensional structure of CDKs has been elucidated primarily through crystallographic studies of human C D K 2 , homologous to yeast CDC28/CDC2 (Debondt et al., 1993). As in other protein kinases, CDK2 is bilobed, with an N-terminus composed predominantly of P-sheet (along with the PSTAIRE helix), a somewhat larger, mainly a-helical, C-terminal lobe, and a deep cleft at the junction between the two lobes which acts as the site of A T P binding and catalysis. Enzymatic activity of the monomeric C D K subunit is severely restrained by two mechanisms. First, the N-terminal lobe is displaced relative to the C-terminal lobe, causing misalignment of key catalytic residues involved in A T P phosphate binding. In addition, substrate binding in the active site cleft is blocked by the large, flexible T-loop of the C-terminal lobe (Debondt et al., 1993).  23  Introduction Cyclins were originally discovered as proteins whose levels oscillated throughout the cell cycle (Evans et al., 1983). The subsequent realization that cyclins associated with, and activated, CDKs in diverse eukaryotes revolutionized the field of cell cycle research. As more cyclins were cloned and studied, it became apparent that cyclins are a remarkably diverse protein family, with only some members displaying cell cycledependent fluctuations. Cyclins range in size from approximately 35 to 90 kDa, with primary sequence homology tending to be concentrated in a 100 residue region known as the 'cyclin box', which is required for C D K binding and activation (Kobayashi et al., 1992; Lees and Harlow, 1993). The cyclin box encodes a recognizable structural motif consisting of a five-helix bundle known as the 'cyclin fold' (Brown et al., 1995). Currently, classification of a protein as a cyclin is defined by cyclin-box homology and demonstrable interaction with a C D K (Andrews and Measday, 1998).  1.5.2 C D K regulatory mechanisms Activation by cyclin binding CDKs are known to be regulated by numerous mechanisms, perhaps reflecting the diversity of signaling pathways which converge on them (Figure 4). Not all CDKs, however, are subject to all of these regulatory mechanisms, reflecting the diversity of the C D K family itself. Nevertheless, one aspect of regulation that, by definition, is shared by all CDKs, is activation by association with a cyclin. Cyclin binding activates the kinase by causing conformational changes (Jeffrey et al., 1995). The principal contacts occur between the PSTAIRE helix and T-loop regions of the C D K and the cyclin box domain. Cyclin binding alters the position of the T-loop so that it no longer occludes the substrate-binding site, and exposes an important threonine residue (Thrl60 in CDK2) to allow phosphorylation by CDK-activating kinase (CAK). The cyclin also moves the PSTAIRE helix into the catalytic cleft and rotates it by 90°. This brings the highly-conserved catalytic site glutamic acid residue inside the catalytic cleft where, in conjunction with a lysine residue, an aspartic acid residue, and a magnesium ion, it coordinates the A T P phosphate group for the phosphotransfer reaction.  24  Introduction Thus, cyclin binding confers an increase in kinase activity of several orders of magnitude (Connell-Crowley et a l , 1993). Activation by phosphorylation In addition to cyclin binding, full activation of many CDKs requires phosphorylation at a conserved threonine residue (T160 of human CDK2, T169 of yeast CDC28) in the T-loop. The phosphate group on T160 is inserted in a cationic pocket beneath the T-loop and apparently acts as an organizing center for a network of hydrogen bonds which stabilizes the CDK-cyclin interaction and induces subtle but critical changes in the substrate-binding site (Russo et al., 1996). The kinase responsible for carrying out the activating threonine phosphorylation has been designated C A K , for CDK-activating kinase (Kaldis, 1999). In higher eukaryotes, C A K is itself a cyclin-dependent kinase, consisting of C D K 7 and a cyclin-like subunit, cyclin H (Fisher and Morgan, 1994). CDK7-cyclin H is also a component of the general transcription factor TFIIH, which phosphorylates the R N A pol II CTD and participates in transcription initiation and nucleotide excision repair (Roy et al., 1994; Serizawa et al., 1995). TFIIH-bound C D K 7 exhibits different substrate specificity from C A K : C A K preferentially phosphorylates CDKs while TFIIH-associated CDK7 shows higher activity toward the CTD (Rossignol et al., 1997). In contrast, the CDK7 ortholog in S. cerevisiae, KIN28, possesses no C A K activity either in vitro or in vivo (Feaver et al., 1994; Cismowski et al., 1995). Instead, the major C A K activity is carried out by a monomeric protein kinase, C A K 1 , that is distantly related to CDKs (Espinoza et al., 1996), but is fully active without a cyclin partner or activating phosphorylation (Kaldis et al., 1996; Thuret et al., 1996). No C A K 1 ortholog has yet been identified in any other organism (Liu and Kipreos, 2000), although the existence of one has been hypothesized since CDK7 does not appear to be sufficient for the activation of all CDKs (Larochelle et al., 1998). The requirement for this activating phosphorylation is not universal among CDKs. In mammalian cells, activation of CDK5-p35 and CDK7-cyclin H complexes does not require phosphorylation (Qi et al, 1995; Nigg, 1996), while CDK8 does not even contain a phosphorylatable residue at the analogous site in its T-loop (Tassan et al., 1995). In S. cerevisiae, C A K 1 is required for the activating phosphorylation of KIN28 25  Introduction and CDC28, but not PH085 or SRBIO, although both these kinases contain phosphorylatable residues in their T-loop regions (Espinoza et al., 1998). Inhibition by CKIs Another important mechanism for regulation of CDK-cyclin activity is interaction with C D K inhibitors (CKIs). These proteins were first described as negative regulators of CDC28 in S. cerevisiae. FAR1 binds to and inhibits CDC28-G1 cyclin complexes (Peter and Herskowitz, 1994) while p40 appears to inhibit mitotic CDC28 complexes (Mendenhall, 1993). Recognizable homologs of the yeast CDC28 inhibitors have not yet been identified in metazoans. In mammalian cells, seven CKIs have been described, and are divided into two families based on common structural and biochemical characteristics. INK4 CKIs are characterized by multiple ankyrin repeats, a structural motif previously identified as a protein-protein interaction domain. INK4 CKIs bind to either monomeric or cyclinassociated Cdk4 or Cdk6, and are postulated to diminish interaction between the cyclin and C D K , thereby inhibiting formation of the complex or favouring its dissociation (Jeffrey et al., 2000). S. cerevisiae PH081, aninhibitor of the PHO80-PHO85 cyclinC D K complex, is structurally similar to the INK4 proteins. The Cip/Kip family of CKIs are general C D K inhibitors characterized by obligate binding to preformed CDK-cyclin complexes (Xiong et al., 1993). Study of this class of CKIs has been complicated by findings that some members (p21, p27) function as assembly factors for CDK-cyclin complexes at low concentrations and act as inhibitors only at higher stoichiometrics, and may also bind to and inhibit proliferating-cell nuclear antigen (Labaer et al., 1997; Zhang et a l , 1993). At present, there are no known members of this class in S. cerevisiae. Inhibition by phosphorylation Phosphorylation of CDKs can also negatively regulate their activity. Inhibitory phosphorylations occur at two sites near the N-terminus, corresponding to T18 and Y19 of S. cerevisiae CDC28, or T14 and Y15 in human CDC2 and C D K 2 (Berry and Gould, 1996). In vertebrate systems, maximal C D K inhibition requires phosphorylation at both sites, while tyrosine phosphorylation appears to suffice for regulation in fungal systems 26  Introduction (Norbury et al., 1991). T18 and Y19 are located within the roof of the ATP-binding pocket, but the exact mechanism through which inhibition occurs is unknown. Phosphorylation may reduce affinity for ATP or interfere with substrate binding (Endicott et al., 1999). The extent of inhibitory phosphorylation is controlled by the antagonistic activities of the dual-specificity Weel and M y t l kinases and the Cdc25 family of phosphatases (Nilsson and Hoffmann, 2000; Parker et al., 1992; Mueller et al., 1995). Other regulators Although most CDK-cyclin complexes interact with high affinity in the absence of additional factors, a few appear to require assembly factors. The stable association between C D K 7 and cyclin H requires a third protein, MAT1 (Fisher et al., 1995). Similarly, CDC37, a protein distantly related to S. cerevisiae CDC37, which is required for CDC28 stability and cyclin binding (Gerber et al., 1995), is postulated to be an assembly factor for the CDK4-cyclin D complex (Dai et al., 1996). The major cell cycle control CDKs also associate with small SUC1 or C K S proteins (Endicott and Nurse, 1995), which appear to be necessary for in vivo C D K function, possibly by acting as adaptors between CDKs and other regulatory components. Additional levels of regulation for both CDK-cyclin complexes and their myriad regulatory proteins include degradation, primarily via the ubiquitin-mediated proteasome system, and subcellular localization. As a general rule, CDKs are constitutively expressed, while cyclins undergo periodic patterns of expression and regulated degradation (Udvardy, 1996). Functional C D K activity is primarily nuclear; this entails the transport of CDKs, which may require additional factors in the absence of nuclear localization signals on the CDKs or cyclins themselves. Proper and timely localization of C D K regulators, including C A K , is also required (Obaya and Sedivy, 2002).  1.5.3 CDKs of Saccharomyces cerevisiae There are six known cyclin-dependent kinases in S. cerevisiae. CDC28, the prototypical C D K , is the central coordinator of cell cycle progression and associates with nine distinct cyclin subunits (for review, see Mendenhall and Hodge, 1998). PH085 is a 27  Introduction  non-essential C D K with 51% identity to CDC28 that was first discovered on the basis of its role in repression of acid phosphatase transcription, but has since been shown to exert pleiotropic effects facilitated by its association with at least ten cyclin partners (for review, see Carroll and O'Shea, 2002). The remaining four CDKs each associate with a single dedicated cyclin and are capable of phosphorylating the carboxy-terminal domain of R N A polymerase II. The CDK-cyclin pairs constituted by KIN28-CCL1 and SRB 10SRB11 are associated with the yeast R N A pol II holoenzyme; KIN28-CCL1 is a component of TFIIH, while SRB10-SRB11 is found in the Mediator subcomplex (Feaver et al., 1994; Liao et al., 1995). CTK1-CTK2 is not detectable in purified yeast holoenzymes (Sterner et al., 1995), while the association of BUR1-BUR2 with the holoenzyme has not yet been determined. Although the past two decades have witnessed the extensive characterization of a variety of CDKs, our understanding of CDKs—their activity and regulation—is by no means complete. In the case of SRBIO, the identification of several transcription factors as targets has provided valuable insight into the cellular function of this C D K . However, the signals, whether extracellular or intracellular, responsible for regulation of SRBIO, as well as the molecular nature of this regulation, remain obscure. The objective of the research presented in this thesis was to determine whether exposure to specific nutritional stresses affects SRBIO activity and, i f so, by what mechanisms.  28  Introduction  29  Methods  2. METHODS AND MATERIALS 2.1 PLASMIDS AND DNA MANIPULATIONS Plasmids used in these experiments are listed in Table I. A l l D N A manipulations were done in E. coli strain DH5oc, except construction of p S G l , for which the dam" strain RJB404 was used in order to facilitate digestion with Clal.  In p S G l , the TRP1 marker  was excised from pIS028 by digestion with Pmll/Clal and replaced with the BamHI/EcoRI fragment containing the URA3 marker from pBS82. In pSG5, the MscI fragment encompassing the D290A srblO-3 mutation from pRY7096 was used to replace the corresponding MscI fragment from WT SRBIO in pIS028. The LEU2 marker was excised from p F H E l O l by digestion with Narl/Aatll and replaced with Narl/Aatlldigested URA3 marker from YIplac211 to form pSG4.  2.2 YEAST STRAINS AND MEDIA A l l experiments used the yeast Saccharomyces cerevisiae. Strains used in this study are listed in Table II. A l l strains are congenic with either W303-1A or E1278b as indicated in Table II. One-step gene disruptions of REG1 and SRB 11 were performed by transforming yeast with Pstl-digested pBM1966 and BamHI/Notl-digested pVZ329, respectively. To add carboxy-terminal H A epitope tags to chromosomal SRBIO, cells were transformed with an integrating plasmid (either pFHElOl or pSG4) containing a 1097bp fragment of the SRBIO coding region fused to an H A tag followed by the ACT1 terminator. For HA-tagging of KIN28 and myc-tagging of SRB 11, a one-step PCRmediated technique was used (Longtine et al., 1998). Briefly, this method utilizes primers with 5' ends (~40 nt) corresponding to the desired target gene sequences and 3' ends (~20nt) that anneal to and allow amplification of sequences encoding an epitope tag, terminator, and selectable marker gene. This amplified D N A is transformed directly into yeast and homologous recombinants carrying the tagged gene are identified. The KANMX6 selectable marker module used here is based on the E. coli Kan gene and R  confers resistance to G418/geneticin. Gene disruptions and integration of epitope tags were confirmed by PCR. Yeast transformations were done according to the lithium acetate method of Gietz et al. (1992). 30  Methods  Table I: Plasmids Plasmid  Description  Source  pIS027  Episomal plasmid, TRP1 marker, ADH1 promoter, HA epitope tag Episomal plasmid, TRP1 marker, ADH1 promoter, SRB 10 with HA epitope tag Episomal plasmid, URA3 marker, ADH1 promoter, SRB 10 with HA epitope tag Episomal plasmid, TRP1 marker, ADH1 promoter, srblO-3 (D290A) mutation with HA epitope tag Episomal plasmid, URA3 marker Integrating plasmid, LEU2 marker, adds HA epitope and ACT1 terminator to C-terminus of  I. Sadowski  pIS028 pSGl pSG5  PRS426 pFHElOl  I. Sadowski This study This study  (Christianson et al., 1992) (Espinoza et al., 1998)  SRB 10 pSG4 pDF33 pDC130 pVZ329 pFA6a-13myckanMX6 pFA6a-3HAkanMX6 pRY7096 pBM1966 pBS82  Integrating plasmid, URA3 marker, otherwise identical to pFHElOl 850 nucleotide fragment of ACT1 in pUC18 Expresses 6His-GST-12CA5-CTD fusion protein in E. coli Single step disruption plasmid for SRB 11, TRP1 marker • • PCR template for 13myc-kanMX6 cassette used for C-terminal myc-tagging of chromosomal genes PCR template for 3HA-kanMX6 cassette used for C-terminal HA-tagging of chromosomal genes CEN, URA3, contains srblO-3 (D290A point mutation in subdomain VII of SRB 10 kinase) Single step disruption plasmid for REG1, LEU2 marker pBR322 backbone with lox URA3 cassette 2  31  This study D. McMaster (Thompson et al., 1993) (Cooper et al., 1997) (Longtine et al, 1998)  (Longtine et al, 1998)  (Liao et al., 1995) M. Johnston (Sauer, 1994)  Methods  Table II: Yeast strains Genotype  Strain W303-1A  3  H617  a  HLY333" HLY334" L5366" YSGl YSG3 YSG5 YSG6  a  a  b  b  YSG8" YSG9  a  YSG10  a  YSG12 " YSGll a  b  a  Source  MATa, ade2, canl, leu2, his3, trpl, ura3  (Thomas and Rothstein, 1989) MATa, ade2, canl, leu2, his3, trpl, ura3, srblO::HIS3 (Balciunas and Ronne, 1995) (Liu et al., MATa, urai-52 1993) MATa, ura3-52 (Liu et al., 1993) (Liu et al., MATa/a, ura3-52/ura3-52 1993) This study MATa, ade2, canl, leu2, his3, trpl, ura3, regl::LEU2 MATa, ade2, canl, leu2, his3, trpl, ura3, HA-SRB10::LEU2 This study This study MATa, ura3-52, HA-SRB10::URA3 This study MATa, ura3-52, HA-SRB10::URA3  MATa/a, ura3-52/ura3-52, HA-SRB10::URA3/HASRB10::URA3 MATa, ade2, canl, leu2, his3, trpl, ura3, SRB11myc::KANMX6 MATa, ade2, canl, leu2, his3, trpl, ura3, HA-SRB10::LEU2, srbllr.TRPl MATa, ura3-52, HA-KIN28::KANMX6 MATa, ade2, canl, leu2, his3, trpl, ura3, HAKIN28::KANMX6  Congenic with W303-1A Congenic with 11278b  32  This study This study This study This study This study  Methods Standard yeast media was made as described (Guthrie and Fink, 1991). Synthetic low ammonia dextrose (SLAD) contains 0.67% (w/v) Yeast Nitrogen Base without amino acids and ammonium sulfate (Difco), 2% (w/v) anhydrous D-glucose, and 0.05mM ammonium sulfate as sole nitrogen source, and has been previously described (Gimeno et al., 1992). Low sulfate medium (LSM) is SD with sulfate salts replaced by corresponding chloride salts; no sulfate medium (NSM) is L S M with no ammonium sulfate added (Guthrie and Fink, 1991).  2.3 R N A I S O L A T I O N A N D N O R T H E R N B L O T T I N G Total R N A was extracted from yeast using a phenol-freeze method (Schmitt et al, 1990). For Northern blotting, 15Lig of total R N A was resolved by electrophoresis on 1% agarose/formaldehyde gels in MOPS buffer (20mM MOPS, 5mM sodium acetate, I m M EDTA) and transferred to nylon membranes as described (Sambrook et al, 1989). Probes were random-prime labelled using Ready-to-Go D N A labelling beads (Amersham Pharmacia) according to manufacturer's instructions. The ACT1 probe was a 850nt BamHI fragment from pDF33; the  SRBIO  probe was a 770nt MscI fragment of  SRBIO  from pIS028 (see Table I).  2.4 A N T I B O D I E S A N D R E C O M B I N A N T P R O T E I N S G A L 4 and STE12 proteins were produced by expression in Sf9 cells from a recombinant A c M N P V virus as previously described (Kang et al., 1993; Olson et al., 2000). Recombinant RPB1 CTD construct (comprising a 6-His-GST-12CA5 fusion with the RPB1 C T D and 98 N-terminal amino acids) was expressed from plasmid pDC130 in E. coli BL21 cells induced with ImM IPTG. Following induction, bacteria were lysed by sonication, and recombinant CTD protein was batch-purified using N i - N T A agarose (Qiagen) as per manufacturer's instructions. Monoclonal antibody against H A (12CA5 epitope) was ascites fluid from Balb/C mice injected with anti-HA-producing hybridoma. Rabbit polyclonal antibody against SRB10 was a kind gift of S. Hahn (Liu et al., 2001). Polyclonal anti-KIN28, monoclonal anti-myc (9E10), and monoclonal anti-p-tubulin antibodies were obtained commercially 33  Methods (from Berkeley Antibody Company, Santa Cruz Biotechnology, and Cedarlane Laboratories, respectively).  2.5 IMMUNOPRECIPITATIONS, IN VITRO KINASE ASSAYS, AND WESTERN BLOTTING For initial experiments involving immunoprecipitation of HA-SRB10 expressed from a 2|i plasmid (see Results), yeast transformed with pIS028 or p S G l were grown in SD-Trp or SD-Ura containing non-fermentable carbon sources (glycerol, lactate, ethanol) to OD6oo=0.8 (i.e. mid-log phase), then induced with 2% glucose or 2% galactose for 2hr. In subsequent experiments, yeast from an overnight culture were inoculated directly into SD-Trp or SD-Ura containing either non-fermentable carbon, galactose, or glucose and grown to OD6oo~0.8-1.0 prior to harvesting. Strains with integrated epitope tags were grown similarly. For time course experiments, yeast were grown to mid-log phase in SD containing 2% glucose, recovered by centrifugation, washed with sterile dt^O, then resuspended in the same volume of S L A D (for nitrogen limitation studies) or SD containing non-fermentable carbon sources. Samples were aseptically removed from the cultures at indicated time points and fresh media added to prevent depletion of nutrients. Cells were harvested by centrifugation, washed once with sterile dH20, flash-frozen in liquid nitrogen, and stored at -70°C until use. For immunoprecipitations, cell pellets were thawed on ice and resuspended in 500ul of kinase lysis buffer (KLB) (50mM Tris pH7.5, 5mM E D T A , 250mM NaCl, 0.1% NP-40, 50mM NaF , I m M PMSF and protease inhibitors). A n equal volume of acidwashed glass beads (425-600fim) was added and cells were lysed by vigorous vortexing or in a Beadbeater (Biospec). Lysates were cleared of insoluble materials by centrifugation at 16,000xg for 20 minutes. Protein concentration of the resulting supernatants was assessed using the BioRad protein assay and comparison with a bovine serum albumin standard curve. For in vitro kinase assays, 2mg of soluble protein were used for immunoprecipitations. Extracts were pre-cleared by incubation with 20li1 Protein G-Sepharose at 4°C for at least 30 minutes. Supernatants were then incubated on ice for one hour with 3 pi anti-HA M A b , followed by agitation for one hour at 4°C with lOp.1 Protein G-Sepharose. Complexes were recovered by centrifugation at 3000g for 3 34  Methods minutes and washed two times in K L B , then two times in kinase assay buffer ( K A B ) (50mM Tris pH7.5, lOmM M g C h , I m M DTT). Kinase reactions were carried out in lOul K A B with ~200ng substrate and lOuCi [y- P] ATP at 30°C for 30 minutes. 32  Reactions were terminated by addition of SDS sample loading buffer and heating to 95 °C for 2 minutes. Reactions were resolved on 7.5% SDS-PAGE and visualized by exposure to Kodak Biomax film. For western blotting of HA-SRB10, 4-8mg of soluble protein were used for immunoprecipitation reactions. Samples were washed four times in K L B , then subjected to SDS-PAGE. Because HA-SRB10 tends to co-migrate with the heavy chain of the antibody used in the immunoprecipitation, gels were run under non-reducing conditions in order to discourage dissociation of the light and heavy antibody chains. Proteins were transferred to nitrocellulose membranes and detected by western blot with E C L reagents used as described by the manufacturer (Amersham Pharmacia). The primary antibody was polyclonal anti-SRBlO (1:3000 dilution); the secondary antibody was horseradish peroxidase-coupled goat-anti-rabbit IgG serum (Gibco-BRL) (1:10000 dilution).  2.6 TRYPTIC PHOSPHOPEPTIDE ANALYSIS G A L 4 protein phosphorylated by immunopurified HA-SRB10 kinase complexes was located on dried SDS-PAGE gels by exposure to X-ray film (Kodak Biomax). Dried gel slices were excised and rehydrated in dE^O. Tryptic phosphopeptide analysis was then performed as previously described (Hung et al., 1997).  For each sample, 500cpm  of trypsinized protein was analyzed. Phosphopeptides were resolved by electrophoresis at p H 2.1 in the horizontal dimension, and by chromatography (butanol: acetic acid: dH20: pyridine, 75:50:37.5:15.5) in the vertical dimension. Peptides were visualized by exposure to Kodak Biomax film.  2.7 METABOLIC LABELLING WITH [ S]-METHIONINE 35  For yeast strains with integrated HA-SRB10: yeast were grown to ODeoo^O.S in SD medium, pelleted, washed once with methionine-free medium, and resuspended in lOOjxl of the same medium per 10ml original culture in a 2ml screwcap tube. 30uT [ S]35  35  Methods methionine (Expres s Protein Labeling Mix, NEN) was added, and cultures were labeled 35  for 90 minutes at room temperature. Cells were pelleted and washed three times in cold yeast lysis buffer (YLB) (50mM Tris-HCl pH8.0, 5mM M g C l , 150mM NaCl, 5mM 2  NaF, 2mM ZnCk, 10Lig/ml aprotinin, l p M leupeptin, lOmM PMSF). Cell pellets were resuspended in 400(il Y L B plus an equal volume of acid-washed glass beads (425600Lim, Sigma) and stored at -70°C until use. Yeast strains expressing plasmid-borne HA-SRB10 from the ADH1 promoter were grown to OD6oo=0.6 in SD glycerol medium, then induced with 2% glucose (added to cultures from a sterile 40% stock) for two hours prior to harvesting. Subsequent procedures were identical to those described above. Immunoprecipitation of HA-SRB10 was performed as described in Section 2.5, except that cells were lysed in Y L B and RIP A buffer (lOmM Tris, lOOmM NaCl, I m M E D T A , 0.5% sodium deoxycholate, 0.1% SDS) and washed with Wash buffer #1 (lOmM Tris pH8, 1M NaCl, 0.1% NP-40), Wash buffer #2 (lOmM Tris pH8, 0.1M NaCl, 0.1% NP40, 0.1% SDS), Wash buffer #3 (lOmM Tris pH8, 0.1% NP-40), and RIPA buffer. Metabolic labeling with [ S]-sulfate (NEN) was essentially as described for [ S]35  35  methionine, except that cells were grown in low sulfate medium (LSM) without methionine or uracil (for plasmid selection) and labeled in no sulfate medium (NSM). For pulse-chase experiments: following labeling, cells were washed with S L A D containing I m M sodium sulfate, and split into two equal volumes which were added to 50ml of either S L A D or SD-uracil. 2ml samples were removed for each timepoint, cells were pelleted and frozen in dry ice-ethanol. Subsequent lysis and immunoprecipitation were as described above. To determine the amount of radioactivity present in each sample, the relevant protein bands were excised from dried SDS-PAGE gels, rehydrated in water, left in scintillation fluid overnight, and counted in a scintillation counter.  36  Results  3. RESULTS 3.1 E F F E C T OF CARBON SOURCE ON SRB10 KINASE ACTIVITY 3.1.1 Immunoprecipitated SRB10 phosphorylates GAL4 and the CTD SRB 10 phosphorylates G A L 4 on Ser699; this phosphorylation is necessary for full induction of the G A L gene regulon (Hirst et al., 1999). A previous result in the lab (unpublished) suggested that SRB 10 kinase activity differed during growth on different carbon sources; specifically, that kinase activity toward G A L 4 was significantly decreased when cells were grown in glucose, as compared to either galactose or nonfermentable carbon sources. This decrease appeared to be specific for G A L 4 , in that phosphorylation of a co-immunopurifying protein presumed to be RPB1, the C-terminal domain of which is another known substrate of SRB 10, occurred at similar levels in all carbon sources tested. This observation implies a mode of regulation of the SRB 10 kinase complex in which the kinase itself is present at similar levels, but its enzyme activity is modified with respect to different substrates. As a preliminary toward determining what this mechanism of regulation is, I attempted to repeat the previous result myself. In the following series of experiments, SRB 10 tagged at the carboxy terminus with the hemagglutinin (HA) 12CA5 epitope was expressed from a multicopy plasmid under the control of the ADH1 promoter and used for immunoprecipitation and in vitro kinase assays (as described in Section 2.5). Immunoprecipitated HA-SRB10 complexes were found to phosphorylate both G A L 4 and a recombinant CTD construct (hereafter referred to as CTD), as well as a co-immunopurifying protein presumed to be RPB1 (Figure 5, Lanes 1 and 2). Kinase reactions containing only the recombinant protein substrates with no immunoprecipitated complexes demonstrate the absence of endogenous kinase activity in the protein preparations (Lanes 4 and 5). In addition, no kinase activity was apparent in immunoprecipitates from yeast transformed with a vector control (pIS027) (Data not shown).  37  Results  Figure 5. Immunopurified complexes containing HA-SRB10 phosphorylate G A L 4 and C T D in vitro. In vitro kinase reactions were performed with HA-SRB10 immunopurified from yeast strain W303-1A transformed with pIS028 (Lanes 1-3). Reactions contained 200ng G A L 4 protein (Lanes 1 and 4), 200ng CTD (Lanes 2 and 5), or no substrate (Lane 3). Kinase reactions in Lanes 4 and 5 contain 200ng of G A L 4 and C T D , respectively, with no added immunoprecipitate. 38  Results 3.1.2 Growth in glucose does not affect SRBIO kinase activity Since this assay appeared to be working appropriately, I used this technique to determine whether the kinase activity of SRBIO immunoprecipitated from yeast grown in various carbon sources differed; more specifically, whether kinase activity was decreased in glucose. There is good reason for supposing this to be the case. While yeast can utilize many different sources of carbon, glucose is preferred since it can enter glycolysis directly. When glucose is present, expression of genes required for conversion of other sugars to glucose is repressed. This occurs primarily through MIG1, which recruits the general corepressors SSN6 and TUP1 to the promoters of glucose-repressed genes, which include the GAL genes (Treitel and Carlson, 1995). Repression is relieved by the action of the SNF1 kinase, which phosphorylates MIG1 to stimulate its export from the nucleus (DeVit et al., 1997). The action of SNF1 is antagonized by the type I protein phosphatase GLC7 and its regulatory subunit REGl(Carlson, 1998). Since SRBlO-mediated phosphorylation of G A L 4 Ser699 causes full induction of the GAL genes, downregulation of SRBIO activity in glucose might be expected, as GAL gene products would be extraneous under these conditions. SRBIO has been genetically linked to SNF1 via its identification as a suppressor of snfl mutations, and has also been shown to coimmunoprecipitate with SNF1 (Kuchin et al., 2000). HA-SRB10 was immunoprecipitated from yeast grown in SC medium containing either glucose, galactose, or glycerol/lactate/ethanol as carbon sources. In vitro kinase assays using these immunopurified kinase complexes show no apparent difference in SRBIO kinase activity in the carbon sources tested with respect to either G A L 4 or CTD substrates (Figure 6). This experiment was repeated multiple times with slight procedural modifications, including variations in buffer composition, washing conditions, and culture conditions (see Section 2.5), but the previous observation of a decrease in SRBIO kinase activity toward G A L 4 in glucose-grown cells could not be reliably reproduced. Likewise, no difference was observed in a regl yeast strain (Figure 7). A snfl strain was also constructed but was not amenable for use in this experiment since snfl mutants will not grow on any carbon source but glucose.  39  Results  GAL4  CTD  H-RPB1 -GAL4 CTD  Figure 6. HA-SRB10 kinase activity is not affected by growth in glucose. In vitro kinase reactons were performed with HA-SRB10 immunopurified from yeast grown in synthetic complete (SC) medium containing either glucose (Lanes 3 and 6), galactose (Lanes 2 and 5), or glycerol/lactate/ethanol (Lanes 1 and 4). Reactions contained 200ng of G A L 4 (Lanes 1-3) or 200ng of C T D (Lanes 4-6) as substrate.  40  Results  GAL4  CTD  Gly  Gal  Glu  Gly  Gal  Glu  1  2  3  4  5  6  Figure 7. Deletion of REG1 does not affect the ability of immunopurified HA-SRB10 to phosphorylate G A L 4 and CTD in vitro. HA-SRB10 was immunopurified from W T or regl yeast grown in glucose (Lanes 3 and 6), galactose (Lanes 2 and 5), or glycerol/lactate/ ethanol (Lanes 1 and 4). In vitro kinase assays were performed with 200ng G A L 4 (Lanes 1-3) or 200ng CTD (Lanes 4-6) as substrate.  41  Results 3.1.3 GAL4 phosphorylated by immunoprecipitated SRBIO exhibits identical phosphorylation states regardless of carbon source Assuming that the initial result was accurate—that is, that SRBIO activity toward G A L 4 is decreased in glucose—there are several possible explanations as to why this difference was not detected. SRBIO, in addition to association with its cyclin , SRB11, is part of the multiprotein SRB/Mediator complex affiliated with the R N A pol II holoenzyme. Thus, at any given time, SRBIO has the potential for interaction with many different proteins, including several known kinases. Based on the presence of what is presumed to be RPB1 in the reactions, at least a fraction of immunoprecipitated SRBIO is holoenzyme-associated. Thus, it is a distinct possibility that other kinases are copurifying with SRBIO and effectively masking a possible difference in SRBIO activity. The principal candidate is KIN28, the kinase component of TFIIH, which is also known to phosphorylate both the CTD and G A L 4 . Although SRB 10 and KIN28 are indistinguishable in substrate specificity with respect to the CTD (Hengartner et al., 1998), the two kinases phosphorylate distinct sites on G A L 4 (Hirst et al., 1999). To examine the possibility that G A L 4 is being phosphorylated by other, contaminating, kinases (presumably at sites other than Ser699), I carried out tryptic phosphopeptide mapping of the G A L 4 protein phosphorylated by my immunopurified HA-SRB10 complexes. No significant difference in phosphorylation state can be observed in the tryptic phosphopeptide maps (Figure 8). Spot 1, corresponding to the tryptic peptide phosphorylated predominantly by purified recombinant SRB 10/11 in vitro, appears with equal intensity in the maps from all three carbon sources. There are more peptides visible than would be expected i f SRBIO alone were responsible; since the kinase reaction was performed with an immunoprecipitated complex, more background is perhaps to be expected. However, even i f other kinases are present, the relevant conclusion of this experiment is that the phosphorylation state of G A L 4 does not differ in the glucosegrown HA-SRB10 sample. 3.1.4 Mutation of SRBIO abrogates in vitro phosphorylation of GAL4 Because the results of tryptic phosphopeptide mapping indicate the possible presence of other kinases, I performed experiments designed to determine whether the 42  Results predominant kinase activity in my immunoprecipitations was attributable to SRB 10.1 constructed a variation of pIS028 which carries the srbl0-3 allele. This mutation results in a single amino acid substitution (D290A) that renders the SRB 10 protein catalytically inactive but fully capable of being incorporated into the holoenzyme (Liao et al., 1995). Thus, SRB 10 kinase activity should be eliminated while preserving the integrity of the immunopurified complexes. The HA-SRB10 D290A mutation eliminates in vitro phosphorylation of G A L 4 and significantly reduces phosphorylation of the C T D in comparison with W T HA-SRB10, despite the presence of similar amounts of SRB 10 protein (Figure 9). Therefore, the observed kinase activity is most likely attributable to SRB10. 3.1.5 Kinase activity of endogenously-expressed SRB10 is not decreased in glucose Another possible explanation for the observed lack of difference in kinase activity may be related to the mode of expression of HA-SRB10. The experiments described above utilized HA-SRB10 expressed from a multicopy plasmid under the control of a strong constitutive promoter. Perhaps this overexpression of SRB 10 allows it to overcome endogenous regulatory mechanisms. For instance, excess SRB 10 protein may exhibit a low level of kinase activity without cyclin binding, or associate spuriously with cyclin partners other than SRB 11. Some experimental support for these notions exists. BUR1 encodes another S. cerevisiae C D K and is dependent on its cognate cyclin, BUR2, for kinase activity in vitro. However, overexpression of BUR1 can suppress a bur2 null allele (Yao et a l , 2000). The metazoan homolog of SRB11, cyclin C, was originally isolated from human and Drosophila cDNA libraries by virtue of its ability to complement an S. cerevisiae strain lacking the G l cyclins CLN1-3 (Lew et al., 1991; Leopold and O'Farrell, 1991). In addition, excess SRB 10 protein may titrate other inhibitors or components of degradation pathways. To test this possibility, I constructed yeast strains in which the H A epitope tag was integrated at the chromosomal SRB10 locus, thus facilitating the examination of SRB 10 expressed in its natural chromosomal context, under the control of its own promoter. As with overexpressed HA-SRB10, in vitro kinase assays performed with immunoprecipitated, endogenously expressed H A SRB10 reveal no apparent decrease in kinase activity in glucose-grown cells (Figure 10), 43  Results therefore I decided to abandon the pursuit of this aspect of SRB 10 regulation. However, a decrease in HA-SRB10 kinase activity was observed for immunoprecipitates from yeast grown in non-fermentable carbon (Figure 10, Lane 2).  3.1.6 Growth in non-fermentable carbon may inhibit SRBIO kinase activity A decrease in SRBIO kinase activity in non-fermentable carbon was not surprising in light of recent observations indicating that SRBIO may be regulated by the quality of carbon sources in the environment. SRB11, the cyclin subunit of SRBIO, has been shown to become degraded in response to heat shock and hypoxic stress, as well as when yeast are shifted from fermentable to non-fermentable carbon sources (Cooper et al., 1997; Cooper et al, 1999). SRBIO protein levels are depleted as cells enter the diauxic phase of growth which involves a shift from fermentative to non-fermentative growth (Holstege et a l , 1998). The same result was observed in both haploid and diploid strains of the Z1278b background with integrated HA-SRB10 tags (Data not shown). The E1278b background is commonly used in studies of filamentous development which, in diploids, is believed to be caused by nitrogen limitation. Invasive growth, the haploid counterpart of diploid pseudohyphal growth, is now believed to be triggered by starvation for fermentable carbon (Cullen and Sprague, 2000). SRB10 has been implicated in the regulation of these morphological phenomena by phosphorylation of STE12. Z1278b strains undergo filamentous development much more readily than other backgrounds such as S288C and W303. These differences have been attributed in part to a mutation in the transcription factor F L 0 8 in S288C (Liu et al., 1996) and a hyperactive RAS2 pathway in Z1278b strains (Stanhill et al., 1999). Accordingly, it is of interest to determine whether SRB10 regulation differs in different strain backgrounds, as well as in haploids versus diploids. To further confirm and characterize SRB10 kinase activity during growth in nonfermentable carbon, I performed time courses of yeast grown to mid-log phase in glucose, then shifted to glycerol. According to Cooper et al. (1999), maximum reduction in SRB 11 protein levels occurs approximately four hours following the shift to nonfermentable carbon; thus, one would expect SRB10 kinase activity to diminish concomitantly. Contrary to what was expected, no significant reduction in kinase activity 44  Results toward either G A L 4 or the CTD was observed, even after 48 hours of growth in glycerol (Figure 11). Presumably, kinase activity should be diminished in glycerol due to degradation of the cyclin SRB 11. To test whether this is the case, I disrupted the SRB 11 gene in the integrated HA-tagged SRB 10 strain and compared the resultant kinase activity with that of HA-SRB10 recovered from WT SRB 11 yeast shifted from glucose to glycerol in midlog phase. Also included was a sample that was inoculated from a saturated culture and grown up to mid-log phase in glycerol medium (i.e. the exact growth conditions under which the apparent decrease in kinase activity was observed). In the srbll- strain, there is no apparent kinase activity, even though SRB 10 protein is still present (Figure 12, Lane 2), while in the glycerol samples, there is little to no reduction in activity after 24 hours (Lanes 3-6).  There does appear to be a reduction in the amount of SRB 10 protein  present after 24 hours of growth in glycerol (Lane 6, bottom panel); however, this reduction is not sufficient to significantly reduce kinase activity.  3.1.7 Endogenously expressed SRB11 is not degraded in non-fermentable carbon The persistence of SRB 10 kinase activity suggests that SRB11 protein must still be present in glycerol-grown cells. Hence, the validity of the Cooper et al. finding is called into question. In this study, myc-tagged SRB 11 carried on an A R S - C E N plasmid was expressed from the ADH1 promoter. Since endogenous SRB11 is normally expressed at much lower levels, the observed degradation of SRB 11 protein may simply have been a consequence of its overexpression. Indeed, recent evidence suggests that cyclin C, the metazoan homolog of SRB11, is degraded when expressed exogenously in excess of its cognate Cdk CDK8 (Barette et al., 2001). To investigate the stability of endogenously expressed SRB11,1 constructed a yeast strain in which the C-terminus of SRB11 is tagged with the myc epitope (9E10). SRB11-myc was then immunoprecipitated from yeast grown to mid-log phase in glucose and transferred to glycerol (Figure 13). SRB11 protein is still present even after 24 hours of growth in glycerol. This supports the result presented in Figure 12, in which the SRB 10 kinase is still active after prolonged growth in glycerol.  45  Results  "S5 b § o  o -G  •  f-  <S1  a e3 c o  OH  f  o OH  O  M 13  o  c  03  4 — - * * .a  o  a  13 a» *i  i *  o  o  in u <u q , u o  « o c  i  Gal  j  o  >_ o O fC  u 'S3 +-* o o o  91#  • X  c  lH  ••  a g  o  o  o 1  X  o  46  I • t  •• • •  •3 e  Results  Figure 9. Mutation of SRBIO abrogates activity of immunopurified kinase complexes. In vitro kinase reactions were performed with anti-HA immunoprecipitates from yeast strain W303-1A bearing a vector control (Lane 1), or expressing W T HA-SRB10 (Lane 2) or a kinaseinactive HA-SRB 10 mutant (D290A) (Lane 3). Reactions contained 200ng of either G A L 4 (Panel A) or C T D (Panel B). Duplicate immunoprecipitations were western blotted with anti-SRBlO PAb (Panel C). 47  Results  HA-SRB10 WT  Gly  Gal  Glu RPB1  Kinase assay  GAL4  Western  « 1  2  «  «  3  ^HA-SRB10 4  Figure 10. Endogenously expressed H A - S R B 10 is not affected by growth in glucose. W T yeast strain W303-1A was grown in glucose (Lane 1), and W303-1A with an H A epitope tag integrated at SRBIO (ySG3, Lanes 2-4) was grwon in glycerol/lactate/ethanol (Gly), galactose (Gal), or glucose (Glu). Immunoprecipitations with anti-HA M A b from 2mg of clarified yeast lysate were used for in vitro kinase assays with 200ng G A L 4 as substrate (Top panel). Duplicate immunoprecipitations were western blotted with anti-SRB 10 PAb (Bottom panel). 48  Results  HA-SRB10 WT  t=0  4  12  24 48hrs RPB1 GAL4  CTD  Figure 11. SRBIO kinase activity does not diminish during prolonged growth in nonfermentable carbon. Mid-log phase glucose cultures of ySG3 (integrated H A - S R B 10) were transferred to glycerol medium and incubated for the indicated times. Immunopurified H A - S R B 10 was used in in vitro kinase assays with 200ng G A L 4 (Top panel) or 200ng CTD (Bottom panel) as substrate. Lane 1 (WT) contans a control immunoprecipitate from yeast strain W303-1A which lacks the HA-epitope tag. 49  Results  HA-SRB10 Glu-->Gly  •"I-RPB1 Kinase assay  «w <f#  Western  «**  ^ G A L 4  1 ^ 1  2  3  ^HA-SRB10 4  5  6  Figure 12. Deletion of SRB 11 abrogates SRB 10 kinase activity but growth in nonfermentable carbon does not. Top panel: In vitro kinase assays with HA-SRB10 recovered from an srbll strain grown in glucose (Lane 2), or SRB11 strain grown to mid-log pahse in glycerol (Lane 3) or grown to mid-log phase in glucose and transferred to glycerol for the indicated times (Lanes 4-6). Lane 1 contains a control immunoprecipitate from a strain lacking the HA-epitope tag on SRB 10. Kinase reactions contained 200ng G A L 4 protein as substrate. Bottom panel: SRB 10 protein in the reactions was western blotted with anti-SRBlO PAb.  50  Results  myc-SRB11 Glu-->Gly WT t=0  12  24hrs myc-SRB11 ^Tubulin  Figure 13. Endogenously expressed SRB11 is not degraded during growth in glycerol. Mid-log phase glucose cultures of yeast strain ySG9 (integrated SRBll-myc) were harvested, washed, and resuspended in glycerol medium for the indicated times (Lanes 2-5). Top panel: S R B l l - m y c was immunoprecipitated with anti-myc (9E10) M A b and detected by western blot with the same antibody. Lane 1 contains a control immunoprecipitate from W303-1A which lacks the integrated myc-epitope tag. Bottom panel: cell lysates were western blotted with M A b against beta-tubulin as protein loading control.  51  Results 3.2 E F F E C T OF NITROGEN LIMITATION ON SRBIO KINASE ACTIVITY 3.2.1 Nitrogen limitation causes a transient decrease in SRBIO activity Growth in fermentable versus non-fermentable carbon does not seem to affect the intrinsic kinase activity of the SRBIO protein, although the techniques used here do not rule out regulation by other mechanisms such as association with inhibitors which may not co-precipitate. SRBIO also plays a role in regulating the cellular response to nitrogen starvation. Specifically, SRBlO-mediated phosphorylation of STE12 at two sites inhibits filamentous development when abundant nitrogen is available. These phosphorylations are lost when yeast are grown under conditions of nitrogen limitation. Therefore, I wanted to investigate the effect of nitrogen starvation on SRBIO kinase activity. For the following series of experiments, yeast strains of the E1278b background bearing H A epitope tags integrated at chromosomal loci were used. Diploid yeast strains bearing a chromosomal SRBIO HA-tag were grown to midlog phase in synthetic complete (SC) glucose medium, harvested, washed, and resuspended in synthetic low ammonium dextrose (SLAD) medium. After 4 hours in S L A D , SRBIO kinase activity is almost completely lost (Figure 14A, Lane 3). When immunoprecipitates were western blotted, this loss of activity turned out to be the result of depletion of the SRBIO protein (Figure 14A, western blot). Loss of the protein and its corresponding kinase activity appears to be a transient phenomenon, since protein levels and phosphorylation of substrates are significantly increased by 12 hours. The same pattern of protein loss and subsequent return is observed in haploid strains (Figure 14B). This is notable since, unlike diploids, haploid yeast do not exhibit filamentous development in response to nitrogen-limiting conditions. Rather, haploid invasive growth is triggered by starvation for fermentable carbon. The similarity in SRBIO regulation observed here indicates that this difference is not effected at the level of the SRBIO kinase. Interestingly, an additional protein band appears in the H A - S R B 10 western blots (indicated by the asterisk). Since the appearance of this lower molecular weight band seems to coincide with the disappearance of H A - S R B 10, this band may represent a truncated form of the SRBIO protein. If so, it must be an N-terminal truncation, as the H A epitope tag used for immunoprecipitation is located at the Cterminus of the protein. 52  Results  B  HA-SRB10 WT t=0 2  8 12 24hrs -HA-SRB10  -Tubulin  Figure 14. Growth in nitrogen-limiting medium causes a transient decrease in SRBIO kinase activity. (A) Mid-log phase cultures of diploid yeast strain ySG8 (integrated H A - S R B 10) were harvested, washed, and resuspended in nitrogen-limiting medium (SLAD) for the indicated times. Top panel: Immunopurified HA-SRB 10 was used in in vitro kinase assays containing 200ng G A L 4 or 200ng STE12. Bottom panel: SRBIO protein immunoprecipitated with anti-HA M A b was detected by western blot with anti-SRBlO PAb. Lane 1 contains a control immunoprecipitate from an isogenic strain (L5366) which lacks the H A epitope tag. (B) Haploid yeast strain ySG5 (integrated HA-SRB 10) was grown and western blotted as described in (A).  53  Results In order to determine whether this effect was specific for SRB 10 and not merely a consequence of a general decrease in protein levels in response to nitrogen starvation, the same experiment was performed using a different protein, namely KIN28. Unlike SRB 10, KIN28 protein levels remained constant during growth in nitrogen-limiting medium (Figure 15).  3.2.2 SRB10 is regulated by a post-transcriptional mechanism The next logical question that arises is: at what level is SRB 10 regulated? Northern blotting of SRB10 in yeast grown under nitrogen-limiting conditions reveals that transcript levels remain relatively constant after 24 hours of growth in S L A D (Figure 16). Thus, SRB 10 must be regulated by a post-transcriptional mechanism. In order to determine whether the loss of SRB 10 protein is due to a cessation of translation or an increase in the rate of protein turnover, it was necessary to perform pulse-chase labelling experiments to determine whether the half-life of the SRB 10 protein is decreased when cells are grown in nitrogen-limiting conditions. Unfortunately, I was unable to detect endogenously expressed HA-SRB10 by [ S]-methionine labelling (Figure 17A). This is 35  probably due to low levels of SRB 10 protein expression as well as the compression of protein bands on SDS-PAGE due to co-migration of SRB 10 with the heavy chain of the antibody used in the immunoprecipitation. However, HA-SRB10 expressed off a multicopy plasmid from the ADH1 promoter was readily detected by [ S]-methionine 35  labelling (Figure 17B). The overexpression of SRB 10 conferred by this plasmid does not appear to inhibit filamentous development in response to nitrogen starvation. Colonies of diploid yeast transformed with this plasmid display a comparable degree of filamentation on S L A D plates as yeast transformed with a vector control (Figure 18A, top). In addition, SRB 10 protein immunoprecipitated from SLAD-grown cells transformed with the multicopy HA-SRB10 plasmid exhibits the same degradation pattern as SRB 10 protein expressed at endogenous levels (Figure 18B). I therefore proceeded with experiments using SRB 10 expressed from the multicopy plasmid. A further complication was encountered with respect to pulse-labeling experiments. Ordinarily, cells are pulse-labeled with [ S]-methionine, then chased into medium containing excess 35  •ye  methionine in order to prevent the further incorporation of residual [ S]-methionine. 54  Results  HA-KIN28 WT t=0  2  4  8 12  24 hrs SLAD «-HA-KIN28 *~ Tubulin  5  6  7  Figure 15. KIN28 protein levels remain constant during growth in nitrogen-limiting medium. Mid-log phase cultures of haploid yeast strain ySG12 (integrated HA-KIN28) were harvested, washed, and resuspended in nitrogen-limiting medium (SLAD) for the indicated times. Top panel: HA-KIN28 was immunoprecipitated with anti-HA M A b and detected by western blot with the same Ab. Lane 1 contains a control immunoprecipitate from strain HLY334 which lacks the integrated H A epitope tag. Bottom panel: cell lysates were western blotted with M A b against beta-tubulin as protein loading control.  55  Results  |  t=0  2  4  8  12  24hrs SLAD  SRB10  W&ffi&&& jfilfljltfliii  j^^m&a  *&ktig&&^  WmM^S^ iijiiiTiliiiiiiii  ^^y^^.  WKm 1NVP iHBr H B p  Figure 16. Northern blotting of S/?fl/0 in nitrogen-limiting conditions. WT diploid E1278b strain L5366 was grown to mid-log phase in synthetic complete medium, harvested, washed, and resuspended in nitrogen-limiting medium (SLAD). At the indicated times, culture samples were removed, total R N A prepared, and expression of SRB 10 (Top panel) and A C T i (Bottom panel) was detected by Northern blot.  56  Results Since methionine constitutes a nitrogen source, albeit a poor one, it was necessary to determine whether the relatively high methionine concentration used in the chase (25mM) prevented the filamentous growth response. Unfortunately, this proved to be the case: yeast grown on S L A D supplemented with 25mM methionine formed smooth, round colonies with no evidence of filamentation (Figure 18A, bottom). Pulse-chase experiments were therefore done using [ S] in the form of inorganic sulfate for labeling. 35  Yeast harbouring the 2u. H A - S R B 10 plasmid were grown in low sulfate medium (LSM), labeled in no sulfate medium (NSM), and chased into either synthetic complete (SC) medium or S L A D (supplemented with ImM sodium sulfate). SRBIO stability appears to be decreased in the nitrogen-limited condition (Figure 19), thus supporting the idea that the decrease in SRBIO protein levels in S L A D as compared to SC medium is caused by enhanced protein turnover rather than translational repression. The effect is not overly dramatic; however, this may be due in part to the nature of the experimental technique. In the pulse-chase experiment, a large number of cells are labeled in a very small volume of medium. It is possible that during the labeling period, the limited nutrients in the medium are depleted to the point of starvation. Thus, SRBIO degradation may be initiated in both the nitrogen-rich and nitrogen-limited samples prior to the 'chase'. Enhanced protein turnover is a more likely explanation than translational regulation for another reason. Control of mRNA translation is mediated primarily through sequence elements in the 5'- or 3'-untranslated regions of mRNAs (Proud, 2001). Since the SRBlO-expressing plasmid used in these experiments contains only the SRBIO open reading frame fused to a heterologous promoter and terminator (from ADH1), translational regulation of SRBIO is highly unlikely. It is also worth noting that [ S]35  methionine-labelled SRBIO expressed from the 2p. plasmid resolved as two separate bands on SDS-PAGE (see Figure 17B). This was also observed with [ S]-sulfate35  labelled SRBIO (Figure 19), and western-blotted H A - S R B 10 (Figure 18B) expressed from the multicopy plasmid. Both of these bands were included in the analysis of SRBIO stability but, interestingly, they appear to have slightly different degradation patterns (Figure 19). The higher mobility band is initially the less abundant of the two, but seems to be more resistant to degradation than the lower mobility band, which starts out as the predominant form, but is degraded to a greater degree over time. The existence of these 57  Results two forms of SRB 10 may be an artifact of its overexpression, as only one band is typically observed in western blots of endogenously-expressed SRB 10. Coincidentally, a similar phenomenon was documented in a recent paper (Chi et al., 2001) where a posttranslational modification resulting in a gel mobility shift was observed for overexpressed, but not endogenously-expressed, GCN4. The authors suggest that the overexpressed protein is more susceptible to a protein kinase that does not normally act (or acts poorly) on GCN4 expressed at endogenous levels. Whether this is true for SRB 10, and whether the presumed modification affects the validity of the result presented here, remains to be seen. Ideally, I would prefer to monitor degradation of endogenously-expressed SRB 10; however, at the present time, technical limitations preclude this possibility.  58  Results  CN  Q a  LO  00  00  CO  CM  io  00  CN  8 as  ca a CO  CN  ca o)  •  LO  00  oq 00 CO  LO CN LO  3 T3 .5 u  © TJ-  00  00 ±3  59  C~>  Results  vector  2iiHA-SRB10  SLAD  SLAD+Met  B S kDa  2^HA-SRB10  § t=0  2  4  8 12 24hrs SLAD  mm 6 8  8  Ml  _  •  | | f  ^SRB10  Tubulin 1  2  3  4  5  6  7  Figure 18. Overexpression of SRBIO does not inhibit filamentous growth. (A) Diploid 11278b yeast strain L5366 transformed with pSGl (2p:, H A - S R B 10) or pRS426 (2u, vector) were streaked on S L A D (Top panels) or S L A D plus 25mM methionine (Bottom panels) and incubated at 30°C for 2 days. (B) L5366 transformed with pSGl was grown to mid-log phase in synthetic complete medium, then washed and resuspended in S L A D for the indicated times. Cell lysates were immunoprecipitated with anti-HA M A b and H A - S R B 10 protein was detected by western blotting with anti-SRBlO PAb. Tubulin was detected by straight western blotting of cell lysates with M A b against beta-tubulin as protein loading control. 60  Results  Time(min)  0  15  30  OH  RICH  45  60  75  90 120 150 180  jWII <wy «—» «r>* 1  •  "*!l!"ii"*  r  •mmm  mmmm  v  .  ^~SRB10  <MN» m  s* .  SRB10  SLAD  Figure 19. Stability of SRB10 protein is decreased in nitrogen-limiting conditions. Yeast strain L5366 transformed with pSGl (2^, HA-SRB10) was grown to mid-log phase in low sulfate medium (LSM), pulse-labelled for 90 min. in no sulfate medium (NSM) containing [ S] sulfate, and chased into either synthetic dropout medium (RICH) or nitrogen-limiting medium (SLAD) for the indicated times. Cell lysates were immunoprecipitated using anti-HA MAb and analyzed by SDS-PAGE and autoradiography (Top panel). Quantitation of results was done by excision of the protein bands from the gel and counting in a scintillation counter (Bottom panel). (RICH=open squares; SLAD=closed circles) 35  61  Discussion  4. DISCUSSION 4.1 DIFFERENT MECHANISMS REGULATE SRB10 IN CARBON VS. NITROGEN-LIMITING CONDITIONS In this study, nitrogen-limiting conditions were observed to cause loss of SRB 10 kinase activity by enhanced degradation of SRB 10 protein, while kinase activity in yeast starved for fermentable carbon remained relatively unchanged. The persistence of SRB 10 kinase activity during growth in non-fermentable carbon was somewhat unexpected in light of previous observations concerning SRB 10 and its cyclin SRB11. SRB10 protein levels decrease during the diauxic shift (Holstege et al., 1998), and SRB 11 was shown to be degraded in yeast shifted from fermentable to non-fermentable carbon (Cooper et al., 1999), although the latter finding was challenged by my own experiments indicating that SRB 11 expressed at endogenous levels (in contrast to the overexpression of SRB 11 in the Cooper et al. study) is not in fact degraded during nonfermentative growth (Figure 13). A known target of SRB 10 phosphorylation, G A L 4 , is hypophosphorylated in cells growing in non-fermentable carbon, but becomes rapidly hyperphosphorylated in response to fermentable sugars (Sadowski et al., 1996). In addition, SRB 10 has been identified as a negative regulator of haploid invasive growth. Since invasive growth is triggered by the absence of a fermentable carbon source (Cullen and Sprague, 2000), the implication is that SRB 10 must be inactivated under these conditions. The results presented in this thesis do not preclude the possibility that SRB 10 is down-regulated during growth in non-fermentable carbon. Indeed, a decrease in SRB 10 protein levels was observed during prolonged growth in glycerol (Figure 12, Lane 6), although this was not sufficient to significantly reduce the kinase activity observed. However, the immunoprecipitation/ in vitro kinase assay technique used here may be too sensitive to differentiate between subtle differences in activity which may be significant in vivo. If SRB 10 protein levels are affected by carbon source, the less dramatic effect (as compared to nitrogen starvation) may reflect the less dire nature of fermentable carbon "starvation". Although fermentable carbon is preferred, yeast cells can grow indefinitely, albeit more slowly, with non-fermentable carbon, whereas the need for nitrogen sources is much less easily bypassed. Alternatively, SRB 10 may be regulated 62  Discussion by an entirely different mechanism in non-fermentable carbon. The immunoprecipitation/kinase assay used in this study essentially measures the intrinsic kinase activity of SRBIO; therefore, other mechanisms of SRBIO regulation, such as association with inhibitors, are likely to be overlooked. Mechanisms other than regulation of SRBIO protein levels might be expected, given that another set of observations indicates that at least some aspects of SRBIO function are required during growth in non-fermentable carbon. SRBIO is required for phosphorylation of SIP4, an activator of gluconeogenic genes, during growth in non-fermentable carbon (Vincent et al., 2001). The authors reconcile the non-fermentative growth requirement for SRBIO with the apparent depletion of SRBIO levels during the diauxic shift (Holstege et al., 1998) by proposing that SIP4 sequesters a certain fraction of SRBIO and protects it from degradation. SRBIO and SRB 11 were also recently identified in a screen for genes required for entry into stationary phase (Chang et al., 2001), a resting state analogous to the Go phase of mammalian cells. Cells growing fermentatively on glucose execute the diauxic shift when glucose becomes limiting. The diauxic shift involves a transition from fermentation to respiration, which utilizes the byproducts of the previous stage of fermentative growth. When these non-fermentable carbon sources are finally exhausted, the cells enter stationary phase. Thus, the requirement of SRBIO and SRB 11 genes for stationary phase entry implies that the kinase complex is present during non-fermentative growth after the diauxic shift. This function of SRBIO may involve its CTD kinase activity, as CTD phosphorylation appears to be regulated during entry into stationary phase (Patturajan et al., 1998). This idea is supported by the fact that SRB9, which assembles into a holoenzyme-associated subcomplex with SRBIO and SRB11, was also isolated in this screen. CTD kinase activity may also function in the SRBlO-mediated effect on SIP4 mentioned above, since the authors were unable to demonstrate direct phosphorylation of SIP4 by SRB 10. These data support the idea that regulation of SRB10 with respect to carbon source may reflect substrate specificity issues rather than the enhanced degradation seen under nitrogen-limiting conditions.  63  Discussion 4.2 SIGNALING SPECIFICITY IN HAPLOIDS AND DIPLOIDS Filamentous growth phenotypes in haploid and diploid yeast are the outcome of seemingly opposite environmental signals. Haploid invasive growth is elicited by starvation for fermentable carbon in the presence of abundant nitrogen, while diploid pseudohyphal growth is the consequence of nitrogen starvation in the presence of abundant fermentable carbon. Both cell types must necessarily be capable of sensing carbon and nitrogen status in order to appropriately modulate their general metabolism yet they respond very differently with respect to filamentation. At least two signal transduction pathways are involved in filamentous development, a M A P K pathway and a cAMP-dependent pathway (see Sections 1.3.2 and 1.3.3.). Although the nature of the nitrogen signal that acts through RAS2 has not been elucidated, GPR1, which functions in the c A M P pathway, has been implicated as a glucose sensor (Lorenz et al., 2000). However, each of these pathways activates both invasive and pseudohyphal growth, so specificity is not achieved through the operation of different signal transduction pathways or receptors. This is in agreement with my results regarding the regulation of SRB 10. Degradation of SRB 10 protein in response to nitrogen limitation was observed in both haploid and diploid yeast strains (Figure 14), indicating that the nitrogen signal is propagated and subsequently received by SRB 10 in both cell types. It has been conjectured that SRB 10 is regulated by general signals regarding the overall fitness of the growth environment. These signals may be transmitted via an entirely different signal transduction pathway, although the pivotal nature of nitrogen and carbon requirements suggests that at least some components of the nitrogen- and carbon-sensing machinery utilized in the filamentous signaling pathways may be shared. Thus, the difference must occur in the output of these pathways downstream of SRB 10. SRB 10 phosphorylates the transcription factor STE12, a target of the M A P K cascade, demonstrating its interaction with this pathway at this level. It is probable that SRB 10 also regulates others in the myriad of transcriptional activators and repressors that function in filamentous development, although this has not yet been determined. Differences in the transcriptional output of common signal transduction pathways in haploids and diploids may be attributable to the al-a2 repressor, which is expressed only in diploid cells (Johnson, 1995). A model has been proposed in which al-oc2 represses transcription of a 64  Discussion glucose-responsive transcription factor for filamentous response genes and a hypothetical repressor of a corresponding nitrogen-responsive transcription factor (Madhani, 2000). Hence, in diploid cells, only the nitrogen-responsive transcription factor would be expressed, while haploid cells would express only the glucose-responsive factor. A common target of these putative cell-type specific transcription factors would be a gene such as FLOll, which is required for both invasive and pseudohyphal growth (Lo and Dranginis, 1998) and possesses an unusually large promoter region containing an unprecedented multitude of positive and negative regulatory elements, including those that respond to nitrogen and carbon sources (Rupp et al., 1999). Other candidates for differential regulation in haploids and diploids might be factors involved in bud site selection, since the failure of haploid cells to form filaments in response to nitrogen starvation has been attributed in large part to their inability to switch from an axial to unipolar budding pattern under these conditions.  4.3 DEPLETION OF SRBIO IS A TRANSIENT PHENOMENON Enhanced degradation leading to depletion of the SRBIO protein is a transient occurrence, with SRBIO protein levels decreasing noticeably after two hours and reaching their lowest levels approximately four hours following a shift to nitrogenlimiting conditions. After four hours, the level of SRBIO protein, and concomitant kinase activity, begin to increase again (Figure 14). This observation is somewhat surprising in that starvation for an essential nutrient would be expected to evoke a sustained response. Transient alterations in gene expression are typically the result of exposure to sudden stressful environmental changes that require an initial adaptation to the new growth conditions, followed by establishment of a new steady-state. The transience of an aspect of the response to nitrogen limitation is a little surprising, since cells cannot necessarily adapt per se to prolonged starvation for an essential nutrient. This result strongly suggests that inactivation of SRBIO is required for the initiation of filamentous differentiation, but not for its maintenance. Temporary inactivation of SRBIO immediately following a shift to nitrogen-limiting conditions may allow a burst of transcription from genes regulated by transcription factors that are normally inhibited by SRBIO under ideal growth conditions. These genes may encode products that are 65  Discussion specifically required for the initiation of filamentous development; alternatively, the initial burst of transcription may be more general, providing a loading dose of filamentous response gene transcripts. Translation of this pool of transcripts could then be regulated to adjust the corresponding proteins to levels that are suitable for adaptation to the new growth conditions.  4.4 A N O V E L M E C H A N I S M O F C D K R E G U L A T I O N Numerous mechanisms are known to regulate C D K activity in response to external signals. These include regulation of cyclin gene expression, regulated proteolysis of cyclins, post-translational modification of CDKs by phosphorylation of inhibitory residues or dephosphorylation of activating residues, interaction of CDK-cyclin complexes with protein inhibitors, and regulation of the subcellular localization of C D K complexes and/or C D K regulatory proteins (Obaya and Sedivy, 2002). I have demonstrated that the CDK-cyclin pair constituted by SRB10-SRB11 in S. cerevisiae is negatively regulated through enhanced degradation of the C D K subunit in response to nitrogen limitation. To my knowledge, this phenomenon has not been documented for any other C D K in yeast or higher eukaryotes and thus represents a novel mechanism of C D K regulation. Proteolysis via the ubiquitin-mediated proteasome pathway has been shown for several cyclins, as well as some C D K inhibitors. C D K levels, however, are generally relatively stable. In yeast, the major cell cycle C D K , CDC28, has been extensively studied and its regulation serves as the prototype for C D K regulation in general. SRBIO differs from CDC28 in a number of characteristics that impact the manner in which it is regulated. CDC28 requires an activating phosphorylation on Thrl69 in its T-loop region. This phosphorylation is catalyzed by the CDK-activating kinase, C A K 1 . In contrast, amino acid sequence alignment indicates that SRBIO does not even contain a phosphorylatable residue in the position corresponding to CDC28 Thrl69 (Liu and Kipreos, 2000). This site is instead occupied by an aspartic acid residue. Since substitution of an acidic residue for a serine or threonine can often mimic the effect of serine or threonine phosphorylation, this aspartic acid residue may confer constitutive activation in the absence of phosphorylation (Johnson et al., 1996). This is consistent 66  Discussion with the finding that, while CDC28 activation requires phosphorylation by C A K 1 , SRBIO does not (Espinoza et al., 1998). CDC28 is also known to associate with nine different cyclins at various times during the cell cycle. Association with multiple cyclins confers a wide range of activities and substrate specificities, as well as providing multiple targets for regulatory mechanisms. Only one cyclin subunit has been identified for SRBIO. Thus, regulatory mechanisms that are vital for CDC28 function appear to be inapplicable to the situation of SRBIO. Divergence of SRBIO from the prototypical C D K may therefore be reflected in novel mechanisms of regulation, such as proteolysis of the C D K catalytic subunit. Another important distinction of the SRBIO kinase complex is its association with the R N A pol II holoenzyme. This multiprotein complex is thought to function as a central coordinator of cellular signals and responses and hence may be the source of regulatory mechanisms governing the activity of its many constituent proteins, including SRBIO.  4.5 F U T U R E D I R E C T I O N S Although I have shown that SRBIO protein is degraded in response to nitrogen limitation, it has not been determined how this degradation is accomplished. Proteolysis of many cyclins is mediated by the ubiquitin-proteasome pathway, therefore it will be of interest to determine whether this is also true for CDKs. The degradation of G l cyclins is dependent, at least in part, on the presence of PEST sequences which are believed to mark proteins for rapid turnover (Rechsteiner and Rogers, 1996), while the mitotic cyclins appear to be targeted for degradation by an element known as the destruction box (Glotzer et al., 1991). Examination of SRBIO for similar sequences could indicate a propensity for ubiquitin-mediated proteolysis. A more extensive examination of SRBIO regulation in various carbon sources is also warranted, since data presented in this thesis combined with observations from other laboratories suggest that SRBIO is regulated by distinct mechanisms in response to starvation for different nutrients. Future studies will also involve elucidation of the signaling pathways that control SRBIO. A genetic screen designed to identify components of the GAL3-independent pathway that regulates SRBlO-mediated phosphorylation of G A L 4 Ser699 has been initiated (Rohde, 2000) and should shed some light on the upstream factors affecting 67  Discussion SRBIO activity. Identification of additional downstream targets of SRBIO will also be informative.  4.6 C O N C L U S I O N  In conclusion, the work presented in this thesis demonstrates that the R N A polymerase Il-associated C D K , SRBIO, is negatively regulated in response to nitrogen limitation. This occurs through enhanced degradation of the SRBIO protein, which represents a novel mechanism of C D K regulation. 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