SOK2 May Regulate Cyclic AMP-Dependent Protein Kinase ...

MOLECULAR AND CELLULAR BIOLOGY, Dec. 1995, p. 6854–6863 0270-7306/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 15, No. 12

SOK2 May Regulate Cyclic AMP-Dependent Protein Kinase-Stimulated Growth and Pseudohyphal Development by Repressing Transcription MARY P. WARD,1 CARLOS J. GIMENO,2† GERALD R. FINK,2

AND

STEPHEN GARRETT1*

Departments of Molecular Cancer Biology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710,1 and Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 021422 Received 5 July 1995/Returned for modification 15 August 1995/Accepted 21 September 1995

Yeast cyclic AMP (cAMP)-dependent protein kinase (PKA) activity is essential for growth and cell cycle progression. Dependence on PKA function can be partially relieved by overexpression of a gene, SOK2, whose product has significant homology with several fungal transcription factors (StuA from Aspergillus nidulans and Phd1 from Saccharomyces cerevisiae) that are associated with cellular differentiation and development. Deletion of SOK2 is not lethal but exacerbates the growth defect of strains compromised for PKA activity. Alterations in Sok2 protein production also affect the expression of genes involved in several other PKA-regulated processes, including glycogen accumulation (GAC1) and heat shock resistance (SSA3). These results suggest SOK2 plays a general regulatory role in the PKA signal transduction pathway. Expression of the PKA catalytic subunit genes is unaltered by deletion or overexpression of SOK2. Because homozygous sok2/sok2 diploid strains form pseudohyphae at an accelerated rate, the Sok2 protein may inhibit the switch from unicellular to filamentous growth, a process that is dependent on cAMP. Thus, the product of SOK2 may act downstream of PKA to regulate the expression of genes important in growth and development. The small metabolite cyclic AMP (cAMP) acts as a second messenger to regulate such complex processes as cell cycle progression, development, and transcription (34, 35, 38, 43, 59). In most eukaryotes, including the budding yeast Saccharomyces cerevisiae, the effect of cAMP is mediated by the cAMP-dependent protein kinase (PKA). Like its mammalian counterpart, yeast PKA is a heterotetrameric protein consisting of two catalytic subunits and two regulatory subunits. The yeast catalytic subunits are encoded by three functionally redundant genes, TPK1, TPK2, and TPK3 (63), whereas the regulatory subunit is specified by a single gene, BCY1 (5). Binding of cAMP to Bcy1 results in its dissociation from the catalytic subunits and in stimulation of PKA activity. Yeast cells deficient in PKA activity stop growing and arrest in G1, in a manner similar to that observed for wild-type cells deprived of nutrients (28, 38, 67). Physiological changes associated with a diminution in PKA activity include the accumulation of storage carbohydrates (glycogen and trehalose), increased resistance to stress, and an increase in the expression of genes involved in these (GPH1, GAC1, UBI4, SSA3, and CTT1) as well as other processes (16, 37, 61). Mutations yielding elevated PKA activity give rise to an equally complex array of physiological changes, including accelerated pseudohyphal growth (in diploids), transient arrest in G1, loss of carbohydrate reserves, and sensitivity to various forms of stress, including heat shock and nutrient starvation (1, 5, 21, 65). Together, these phenotypes have been taken as evidence that the yeast PKA pathway plays a central role in regulating the growth and

cell cycle progression of cells in response to a changing environment (2, 3). Although the phenotypes and physiological changes associated with alterations in PKA activity have been described in some detail, there is at present no simple molecular model for the mechanism(s) by which PKA regulates one or more of these processes (3, 62). Sensitivities to stress are thought to be regulated primarily at the transcriptional level, and the expression of several genes (SSA3, UBI4, and CTT1) involved in these stress responses is known to be repressed by active PKA (13, 37, 61). However, the transcription factor(s) that mediates PKA-dependent repression has not been identified. Glycogen accumulation is also partially regulated by a PKA-dependent repression of GAC1 and GPH1 expression (16), but the regulatory proteins involved are also unknown. Only one transcription activator, Adr1, has been shown to be inactivated by PKAdependent phosphorylation. However, this phosphorylation event is not necessary for glucose repression, the only physiological perturbation to which Adr1 is known to respond (7, 8). Recent evidence has also suggested a role for PKA in the activation of transcription. For example, PKA activity seems to mediate the growth-regulated expression of yeast ribosomal protein genes (31, 45), as well as a UV-induced activation of HIS3 expression (12); however, the molecular details and physiological significance of these effects are unknown (60). The mechanism by which PKA regulates progression through G1 is also unclear. Several reports addressing the role of the G1 cyclins (CLN1 and CLN2) in PKA-dependent proliferation have come to opposite conclusions. In one report, CLN expression was shown to be dependent on PKA activity, whereas a second report described no appreciable effect on G1 cyclin mRNA levels in cells arrested by PKA inactivation (15, 26). Moreover, several publications have shown that the addition of glucose led to a transient G1 arrest that could be attributed to the repressive effects of PKA-dependent phosphorylation on G1 cyclin expression (1, 65). Thus, PKA activity may be both

* Corresponding author. Mailing address: Departments of Molecular Cancer Biology and Biochemistry, Box 3686, Duke University Medical Center, Durham, NC 27710. Phone: (919) 613-8608. Fax: (919) 613-8605. Electronic mail address: [email protected]. † Present address: Millennium Pharmaceuticals, Inc., Cambridge, MA 02139. 6854

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TABLE 1. Yeast strains useda Strain

MWY63 SGY398 RS13.58A-1 Y1029 MWY323 MWY362 MWY438 MWY470 MWY319 MWY421 MWY422 MWY423 MWY154 MWY535 MWY525 MWY526 MWY510 MWY512 MWY515 MWY517 CG953 CGX137 CG995 CG934 CGX210 CGX225 CJ56 CJ57 CJ58 CJ59 EY12007-3 SC18 BY606 BY1232 a

Genotype

Source or reference

MATa tpk1::URA3 tpk2-63(Ts) tpk3::TRP1 bcy1::LEU2 ura3-52 his3 trp1 leu2-3,112 ade8 MWY63 tpk1::ADE8 MATa tpk1w1 tpk2::HIS3 tpk3::TRP1 bcy1::LEU2 ura3-52 his3 trp1 leu2-3,112 ade8 MATa/MATa ura3-52/ura3-52 his3/his3 trp1/TRP1 leu2-3,112/leu2-3,112 lys2/LYS2 ade2/ADE2 Y1029 sok2::URA3/SOK2 SGY398 sok2::URA3 SGY398 phd1::hisG sok2::URA3 SGY398 phd1::hisG MATa/MATa sok1::HIS3/SOK1 sok2::URA3/SOK2 yak1::LEU2/YAK1 ura3-52/ura3-52 his3/his3 trp1/TRP1 leu2-3,112/leu2-3,112 ade8/ade8 RS13.58A-1 (YEplac195) RS13.58A-1 (pMW61) RS13.58A-1 sok2::URA3 MATa TPK1 tpk2::HIS3 tpk3::TRP1 BCY1 ura3-52 his3 trp1 leu2-3,112 ade8 MWY154 sok2::URA3 MWY154 (YEplac195) MWY154 (pMW61) MWY154 (pMW7) MWY154 (pMW8) MWY154 sok2::ADE8 (pMW7) MWY154 sok2::ADE8 (pMW8) MATa/MATa ura3-52/ura3-52 (pRS316) MATa/MATa ura3-52/ura3-52 phd1D1::hisG-URA3-hisG/phd1D1::hisG-URA3-hisG MATa/MATa ura3-52/ura3-52 (pMW61) MATa/MATa ura3-52/ura3-52 (pCG38) MATa/MATa ura3-52/ura3-52 sok2::URA3/sok2::URA3 MATa/MATa ura3-52/ura3-52 sok2::URA3/sok2::URA3 phd1D1::hisG/phd1D1::hisG MATa/MATa ura3-52/ura3-52 leu2::hisG/leu2::hisG (YEp-PDE2-1 and pRS316) MATa/MATa ura3-52/ura3-52 leu2::hisG/leu2::hisG (pRS305-2m and pRS316) MATa/MATa ura3-52/ura3-52 leu2::hisG/leu2::hisG (pRS305-2m and YCpR2V) MATa/MATa ura3-52/ura3-52 leu2::hisG/leu2::hisG (YEpPDE2-1 and YCpR2V) MATa ura3-52 leu2D1 trp1::hisG his1-7 lys2 rad52::LEU2 MATa GAL4 gal80 ura3-52 leu2-3,112 his3 trp1 MATa ura3 his3 leu2 trp1-1 ade2 swi4 MATa ura3 his3 leu2 trp1-1 ade2 swi6::LEU2

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All strains with a CG, CGX, or CJ prefix are congenic to the S1278b genetic background (23).

necessary for, and antagonistic to, G1 cyclin (CLN1 and CLN2) expression. The role of the known cyclin transcriptional complex SBF (Swi4 and Swi6 [44]) in this PKA regulation has not been determined. Recently, we described our efforts to identify growth and cell cycle-specific effectors of the PKA pathway (67). Two genes were identified in a screen for gene dosage suppressors of the conditional growth defect of several tpk2(Ts) mutants. One of the genes, SOK1, encoded a nucleus-localized protein that appeared to suppress the growth defect by a previously described PKA-independent mechanism (18, 25, 67). The present report describes the characterization of the second suppressor, SOK2. Although deletion of SOK2 is not lethal, its loss exacerbates the growth defect of strains with low PKA activity. SOK2 affects several metabolic processes and phenotypes known to be regulated by PKA dependent phosphorylation. The SOK2 coding region predicts a protein that exhibits striking resemblance to a family of fungal transcriptional factors, suggesting that Sok2 may regulate the expression of one or more genes under PKA control. SOK2 may play a more general role in growth control and development, since diploid cells undergo an accelerated program of pseudohyphal development when they are depleted of Sok2 protein. MATERIALS AND METHODS Media and growth conditions. The media used, including yeast rich and minimal media as well as bacterial media, were prepared as described previously (10,

17, 20, 21, 47). Yeast strains were monitored for glycogen accumulation and heat shock sensitivity by methods described previously (17, 25, 67). Strains and plasmids. The yeast strains used in this study are listed in Table 1. Bacterial strains MC1066 [D(lac)X74 galU galK strA hsdR trpC9830 leuB6 pyrF::Tn5] and DH5a [F9/endA hsdR17(r2 m2) supE44 thi-1 recA1 gyrA relA1 D(lacZYA-argF9)U169 (80dlac D(lacZ)M15)] have been described previously (6, 68). Bacterial vectors pUC19 and pBSK1 have been described (Stratagene Product Catalog). The high-copy-number yeast vector YEpADE8 was constructed by T. Toda and has been described previously (18). The high-copy-number URA3 yeast vector YEplac195 has been described previously (19). Other yeast plasmids included were YCp50 (low-copy-number URA3 vector [53]), pGS65 (low-copynumber LEU2 vector [66]), pRS305-2 (LEU2-marked 2-mm vector [20]), and pRS316 (low-copy-number URA3 vector [58]). The high-copy-number LEU2 vector containing PDE2 (YEp-PDE2-1) has been described elsewhere (56). The low-copy-number URA3 vector containing the RAS2Val-19 allele is called YCpR2V and has been described elsewhere (21). The high-copy-number URA3 plasmid containing PHD1 (pCG38), as well as the phd1D1::hisG-URA3-hisG deletion plasmid (pCG36), has been described elsewhere (20). Mapping SOK2. The SOK2 gene was assigned to the right arm of chromosome XIII by probing an ordered set of lambda clones (51) containing the yeast genome with a SOK2 fragment labeled with an enhanced chemiluminescence direct nucleic acid labeling system (Amersham). The SOK2 gene was genetically mapped by crossing a sok2::URA3 strain with strains containing either a rad52::LEU2 disruption or a mutation in GAL80. The results of these crosses (sok2::URA3 3 gal80 5 27 parental ditypes, 1 nonparental ditype, and 36 tetratypes) and (sok2::URA3 3 rad52::LEU2 5 25 parental ditypes, 0 nonparental ditypes, and 10 tetratypes) were used to determine the map distance on the basis of the formula described by Perkins (49). DNA manipulations. Plasmid DNA was prepared from Escherichia coli by the alkali lysis method (36). All enzymes were used according to the specifications of their suppliers (New England Biolabs or Bethesda Research Laboratories), and cloning techniques were as described elsewhere (36). Yeast transformation was by the lithium acetate method (27), and other yeast manipulations were performed as described elsewhere (54).

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Plasmid pMW48 was constructed by inserting the 3.5-kb SphI-BglII SOK2 fragment of pMW43 (one of the original SOK2 plasmids; plasmid B in Fig. 1) (67) into the SphI-BamHI sites of pUC19. The sok2::URA3 disruption was constructed by digesting plasmid pMW48 with SpeI and inserting the 1.4-kb NheI-SpeI URA3 fragment from YEp24. The resulting plasmid was designated pMW52. The sok2::ADE8 plasmid (pMW53) was constructed by inserting a 4.0-kb XbaI fragment containing ADE8 from YEpADE8 into the single SpeI site of pMW48. A replicating yeast plasmid bearing the sok2::URA3 disruption (pMW54) was created by inserting the 4.8-kb HindIII-SstI fragment of pMW52 (the HindIII site is from the multiple cloning site of pUC19, and the SstI site is situated 1.6 kb 39 of the SOK2 coding region; see Fig. 1) into YEpADE8 at the HindIII-SstI sites. A high-copy-number yeast plasmid bearing URA3 and SOK2 (pMW61) was constructed by inserting a 3.2-kb HindIII-XhoI fragment of pMW48 (the HindIII site is from the multiple cloning site of pUC19, and the XhoI site is situated 1.0 kb 39 of the SOK2 coding region) into the HindIII-XhoI sites of YEplac195. The RAS2 (pMW7) and RAS2Val-19 (pMW8) plasmids were constructed by inserting the 2.0-kb HindIII-SalI RAS2 fragment of pRJ4-27 (14) and pMF100 (14), respectively, into the HindIII-SalI sites of the low-copynumber LEU2 vector pGS65 (66). The sequence of SOK2 was determined by a modification of the method described by Sanger et al. (55), with double-stranded plasmid DNA containing fragments of SOK2 inserted into pUC19 or pBSK1. Northern (RNA) analyses. RNA was isolated by the hot phenol method (33), separated by electrophoresis, and transferred overnight by capillary action to nylon membranes (ICN). DNA fragments to be used as probes (a 1.0-kb XbaI fragment for TPK1, a 450-bp XhoI-BamHI fragment for DED1, a 1.0-kb Sau3APvuII fragment for GAC1, and a 1.0-kb PCR product that lies within the SSA3 open reading frame [ORF]) were gel purified and then radiolabeled by random priming (Boehringer Mannheim). Hybridization and washes were performed as described previously (GeneScreen Plus manual; Dupont), and radioactivity was quantitated on a Molecular Dynamics PhosphorImager. Qualitative pseudohyphal growth assays. The qualitative pseudohyphal growth assay was performed essentially as described previously (20, 21). Strains to be tested were patched onto plates containing synthetic complete medium lacking uracil, grown overnight at 308C, and then streaked to obtain single cells on fresh synthetic complete medium lacking uracil or SLAD medium agar (21). Four strains were always streaked onto one plate. The streaking technique was such that a gradient of colony density existed in the streak, with the highest density existing in the center of the plate. The cultures were grown at 308C, and after an appropriate period of time, representative colonies from the streak were photographed. In those experiments in which transformants were studied, two independent transformants were assayed. In all of the experiments, both transformants exhibited the same phenotype.

RESULTS The SOK2 gene product exhibits homology to fungal transcription factors. The SOK2 gene was previously identified as one of two gene dosage suppressors of the conditional growth defect of a temperature-sensitive PKA strain (67). Whereas the first high-copy-number suppressor (SOK1) isolated in that screen bypassed the growth requirement for PKA, none of the three plasmids containing SOK2 were able to alleviate the growth defect of a strain (tpk1 tpk2 tpk3) lacking all PKA activity (67). Indeed, suppression by SOK2 was restricted, in some conditional PKA mutants, to a narrow range of temperatures and conditions (67). Fragments from one representative SOK2 plasmid were subcloned into the high-copy-number vector YEpADE8 and screened for their ability to suppress the conditional growth defect of the tpk1 tpk2-63(Ts) tpk3 strain MWY63 (Fig. 1). Sequences necessary for suppression were further defined by inserting the URA3 marker into the unique SpeI site within the SphI-BglII fragment (Fig. 1). Insertions at this site abolished suppression, suggesting the SpeI site fell within the SOK2 coding sequence or promoter. The nucleotide sequence of a region encompassing the SOK2-suppressing fragment revealed a single ORF that included the SphI (bp 47 to 52) site (Fig. 2). The ORF corresponding to SOK2 predicted a protein of 785 amino acids when the first ATG encoded the initiating methionine. The SOK2 sequence is identical to an ORF identified as part of the Yeast Genome Sequencing Project (cosmid 9711; accession no. Z49211). Since the SphI-to-BglII fragment was capable of suppressing the PKA growth defect, sequences 59 of the SphI site

FIG. 1. Cloning and restriction map of SOK2. Patches of strain MWY63 [tpk2-63(Ts) ade8] containing the YEpADE8 vector (A) or the YEpADE8 vector with the yeast DNA inserts indicated at the bottom (B to F) were replicated on minimal medium and incubated for several days at 23 and 34.58C. The yeast DNA inserts of some of the plasmids are shown at the bottom of the figure, along with the results of the test of suppression. The predicted SOK2 coding region is marked by a solid arrow, and the position of the URA3 disruption used in these studies is indicated.

were considered unnecessary for suppression. This fragment was sufficient for suppression when it was cloned into two different high-copy-number vectors; therefore, we presume that cryptic transcription and translation signals are provided by yeast sequences immediately downstream of the SphI site. In agreement with this idea, the pMW61 plasmid directs the overproduction of a protein that is recognized by anti-Sok2 polyclonal antibodies and that migrates slightly faster than wild-type Sok2 protein (66a). In addition, Northern analysis of wild-type mRNA with an internal SphI-PstI fragment (Fig. 1) revealed a single mRNA species of 2.6 kb, in agreement with the size predicted for an mRNA encoding a protein of 785 residues (results not shown). Comparison with previously identified proteins revealed that one region of the predicted SOK2 product exhibited significant homology to a family of transcription factors involved in fungal cell cycle control and differentiation (Fig. 3). Sok2 was most similar to the product of the S. cerevisiae pseudohyphal gene PHD1 (20), exhibiting 82% identity within a 104-amino-acid region (residues 412 to 515 of Sok2). Both proteins share a similar degree of amino acid identity with the product of stuA (71% identity), a gene from Aspergillus nidulans that is involved in spatial orientation and cellular differentiation (41, 42). Within the 104-amino-acid region, only nine positions are variant in all three proteins (five of these are conservative changes between Sok2 and Phd1). In contrast to the marked identity among Sok2, Phd1, and StuA within the stretch of 104 amino acids, a significant portion of Sok2 (almost 90% of the protein) does not exhibit significant similarity with any other protein, including Phd1 and StuA. The Sok2 protein also has significant, but more limited, sequence identity with several transcriptional factors from S. cerevisiae (Swi4 and Mbp1), Kluyveromyces lactis (K.1.Mbp1), and Schizosaccharomyces pombe (Cdc10 and Res1) that are essential for transition from G1 to S phase (32, 50). Once again, the sequence conservation between Sok2 and these proteins (Swi4, 34% identity; Mbp1, 35% identity) is restricted to the region of similarity among Sok2, Phd1, and StuA. This

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FIG. 3. Amino acid (aa) sequence homologies among the products of SOK2, PHD1, and stuA. Sequence similarities among residues 406 to 525 of Sok2 and the corresponding regions of Phd1 (residues 178 to 297) and StuA (residues 121 to 240) are displayed. Amino acid identities are denoted by solid boxes. Representations of the three proteins are shown below, with the regions of similarity indicated by solid boxes. The size of each protein (in residues) is shown to the right.

FIG. 2. Nucleotide sequence of SOK2 and predicted amino acid sequence of Sok2.

region has been shown to function as the DNA-binding domain in both Swi4 and Mbp1 (50) and may also serve a similar function in Sok2, Phd1, and StuA. The Swi4 and Mbp1 proteins also contain ankyrin repeats (50), as well as conserved regions in their carboxyl termini that are thought to tether these similar, but functionally distinct, factors to a protein, Swi6, that is common to both transcription complexes (50). The absence of either of these sequences seems to mark Sok2 (as well as Phd1 and StuA) as a member of a family of transcriptional factors distinct from that of Swi4 and Mbp1. Since the SOK2 gene was isolated as a high-copy-number suppressor of a conditional PKA mutant, the predicted polypeptide was scanned for potential PKA phosphorylation sites (9), as well as other consensus motifs with a possible role in the function of Sok2. A single consensus PKA phosphorylation site (KKCT [30]) was identified near the carboxyl terminus of the protein (residues 595 to 598), in a region required for suppressor activity (results not shown). The SOK2 gene was localized to the right arm of chromosome XIII by probing an ordered set of lambda clones containing the yeast genome (51). Genetic mapping (see Materials and Methods) against a gal80 mutation and a rad52::LEU2 marker placed SOK2 14 and 30 cM, respectively, from RAD52 and GAL80, in the order SOK23CENXIII3RAD523GAL80. Disruption of SOK2 exacerbates the growth defect of the conditional PKA mutant. If Sok2 protein were the sole mediator of PKA-dependent growth, Sok2-deficient strains would be inviable. To determine whether SOK2 was essential, the sok2::URA3 disruption was transplaced into the genome of a diploid SOK2/SOK2 strain (Y1029), and two Ura1 diploids were subjected to tetrad analysis. Both transformants contained a single disrupted copy and a wild-type copy of SOK2 as determined by DNA hybridization (results not shown). All tetrads (24 of 24) gave rise to four colonies of equal size, with the Ura1 marker segregating 2:2. Thus, SOK2 was not essential for growth under laboratory growth conditions. Identical results have been obtained with a sok2::ADE8 disruption, as well as a sok2::TRP1 deletion that removes all of the coding region except for the first codon (results not shown). Since the first gene (SOK1) isolated as a high-copy-number suppressor of the conditional PKA defect was not essential (67), we examined whether SOK2 could interact genetically with SOK1. A heterozygous sok2::URA3/SOK2 sok1::HIS3/

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FIG. 4. Disruption of SOK2 slows growth of a conditional PKA mutant. The temperature-sensitive PKA parent (tpk2-63 SOK2 [SGY398]) and six independent sok2::URA3 transformants (transformant no. 1 is MWY362) were streaked onto rich-medium agar and incubated at 308C for several days. The six sok2::URA3 transformants were confirmed by physical analysis to contain a disruption of SOK2.

SOK1 diploid strain (MWY319) was sporulated, and the progeny were monitored for growth on media lacking uracil or histidine. All of the asci (25 total) gave rise to four viable colonies, including 18 Ura1 His1 colonies. These results suggest that SOK2 and SOK1 do not constitute an essential gene pair. Although the SOK2 gene was not essential for viability, it seemed conceivable that strains partially compromised for PKA activity and bearing a disruption in SOK2 might be either impaired for growth or inviable. Similar combinatorial effects have been described previously (39, 52). Thus, a strain bearing the conditional tpk2(Ts) allele was transformed with the sok2::URA3 mutation and several Ura1 disruptants (containing the sok2::URA3 mutation as shown by Southern analysis; results not shown) were tested for growth at several temperatures. Although the sok2::URA3 tpk1::ADE8 tpk2(Ts) tpk3:: TRP1 mutants were viable at 238C, they grew more slowly than the isogenic SOK2 tpk1::ADE8 tpk2(Ts) tpk3::TRP1 parent at the permissive temperature of 308C (Fig. 4). Neither strain was affected by the subsequent disruption of SOK1 (results not shown). No evidence for genetic interaction between SOK2 and SWI4 or SWI6. In S. cerevisiae, progression from G1 to S requires the function of two partially redundant transcription factors, Swi4 and Mbp1 (32). Swi4 function is necessary for the proper expression of two G1 cyclins, CLN1 and CLN2, and Mbp1 activates expression of genes involved in DNA synthesis. Progression through G1 is also dependent on PKA activity, possibly as a result of its effect on G1 cyclin gene expression (26). Although the sequence similarity between the DNA-binding domains of Swi4 and Sok2 was limited, physiological and molecular studies were consistent with the two proteins sharing overlapping functions. Since SWI4 and SOK2 were dispensable, we examined whether the double mutant (swi4 sok2) exhibited a more-severe growth defect than strains containing either mutation (swi4 strains exhibit a temperature-sensitive growth defect [48]). Diploids constructed by mating a sok2:: URA3 haploid strain with a swi4 strain were sporulated, and the resulting colonies were monitored for growth and uracil prototrophy. In contrast with the synthetic defect observed

between the tpk2(Ts) and sok2::URA3 mutations, no synthetic defect was observed in strains bearing mutations in SOK2 and SWI4 (results not shown). Similar results were observed in a sok2::URA3 swi6 double mutant, suggesting that Sok2 also does not interact genetically with Swi6. SOK2 affects several PKA-regulated phenotypes. Yeast strains containing mutations that diminish or activate PKA are resistant or sensitive, respectively, to acute exposure to high temperature. To examine the effect of SOK2 on thermotolerance, strains with various PKA activities were transformed with a sok2::URA3 disruption or a high-copy-number URA3 plasmid carrying the suppressing SOK2 fragment (pMW61) and were used in standard tests of thermotolerance. Mid-log-phase cultures of a strain with low PKA activity (tpk1w [4]) were diluted onto minimal medium agar, placed at 558C for 10 min, and then incubated at 308C for 3 days. In that strain, deletion of SOK2 significantly enhanced viability after heat shock, increasing survival 50- to 100-fold (Fig. 5A). By contrast, SOK2 overexpression resulted in a 10-fold loss in viability (Fig. 5A). The RAS2Val-19-activating allele confers exquisite sensitivity to heat shock as a result of the increase in PKA activity (29). The effect of Sok2 on this sensitivity was determined by replicating RAS2Val-19 strains to minimal medium agar and placing them directly at 308C or exposing them to 508C for 10 min and then incubating them at 308C for 2 days. Disruption of SOK2 blocked the thermosensitivity conferred by the RAS2Val-19 allele (Fig. 5B). Thus, cellular thermotolerance is inversely proportional to the levels of Sok2 protein and PKA activity of the strain. PKA activity has also been shown to have a significant effect on glycogen accumulation; strains with high PKA activity fail to accumulate glycogen, whereas strains compromised for PKA activity hyperaccumulate this storage carbohydrate. To examine the effect of SOK2 on glycogen accumulation, the glycogen levels of several PKA mutants bearing a high-copy-number SOK2 plasmid or the sok2::URA3 disruption were assessed by inverting patches of the strains over iodine crystals. Overexpression of SOK2 slightly decreased glycogen accumulation in strains compromised for PKA activity (tpk1w), whereas strains bearing the RAS2Val-19 plasmid accumulated higher glycogen

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FIG. 5. SOK2 affects the heat shock sensitivities of strains with diminished or elevated PKA activities. (A) SOK2 affects the heat shock sensitivity of a strain containing low PKA activity. Serial dilutions of the indicated cultures were transferred to agar, exposed to 558C for 10 min, and then incubated at 308C for several days. The strains were MWY421 (tpk1w), MWY422 (tpk1w/YEpSOK2), and MWY423 (tpk1w sok2::URA3). (B) Disruption of SOK2 alleviates the heat shock sensitivity of a RAS2Val-19 strain. Patches of the indicated strains bearing either YCpRAS2 (pMW7) or YCpRAS2Val-19 (pMW8) were replicated to minimal medium agar lacking leucine and incubated at 308C or exposed to 508C for 10 min prior to incubation at 308C. The strains were MWY510 (TPK1 BCY1 SOK2/YCpRAS2), MWY512 (TPK1 BCY1 SOK2/YCpRAS2Val-19), MWY515 (TPK1 BCY1 sok2::ADE8/YCpRAS2), and MWY517 (TPK1 BCY1 sok2::ADE8/ YCpRAS2Val-19).

levels if they contained the sok2::URA3 disruption than if they carried the wild-type SOK2 allele (Table 2). Identical results have been seen with the sok2::TRP1 deletion (66a). Therefore, Sok2 and PKA appear to regulate glycogen accumulation in a similar manner. SOK2 affects GAC1 and SSA3 expression. The pleiotropic effect of Sok2 on such PKA-regulated processes as growth, thermotolerance, and glycogen accumulation was consistent with a model in which TPK expression and, ultimately, PKA activity were positively regulated by the Sok2 transcription factor. In this way, SOK2 overexpression might suppress the growth defect of the conditional PKA mutant by increasing the synthesis of the temperature-sensitive tpk2-63 product. To determine whether this was correct, we examined the effect of SOK2 overexpression and disruption on TPK1 mRNA accumulation. mRNAs isolated from strains with moderate (MWY154 [TPK1 tpk2 tpk3 BCY1]) or low (RS13.58A-1 [tpk1w tpk2 tpk3 bcy1]) PKA activity and bearing either a high-copynumber URA3 plasmid containing the SOK2-suppressing fragment or the genomic sok2::URA3 disruption were used to examine the effect of Sok2 on TPK1 expression. Neither overexpression nor disruption of SOK2 significantly altered TPK1 transcription in strains with moderate (Fig. 6A) or low (Fig. 6B) PKA activity (quantitation on a PhosphorImager showed that the TPK1 message varied by less than 35% when normalized with the DED1 control). Identical results were observed when a radiolabeled TPK2 fragment was used to probe the expression of the truncated TPK2 mRNA (the HIS3 gene is inserted at a BalI site in the middle of TPK2) in the tpk2::HIS3 strains used in this study (results not shown). Thus, Sok2 does not appear to regulate TPK expression. To determine whether the effect of Sok2 on glycogen accu-


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mulation or thermotolerance could be due to a direct effect on gene expression, we examined the levels of GAC1 and SSA3 mRNA in the same TPK1 and tpk1w strains used to probe TPK1 mRNA. GAC1 mRNA levels were significantly elevated (3.1-fold) in strains from which SOK2 was deleted and were diminished (6.1-fold) by overexpression of SOK2 in a strain exhibiting moderate PKA activity (Fig. 6A). GAC1 mRNA levels were similarly affected (2.1-fold increase and 3.7-fold reduction) by SOK2 disruption and overproduction in strains with an attenuated PKA activity (Fig. 6B). SSA3 expression was also affected by disruption and overexpression of SOK2. For example, disruption of SOK2 from the TPK1 strain resulted in a 2.3-fold increase in SSA3 mRNA (by comparison with its isogenic SOK2 parent) after exposure to heat shock (Fig. 6C). Basal expression of SSA3 was higher in the tpk1w strain but was still affected by SOK2 deletion (2.9-fold increase in SSA3) and overexpression (9.7-fold decrease in SSA3) (Fig. 6B). PKA-dependent growth is not affected by PHD1. Deletion of SOK2 had a moderate, though pleiotropic, effect on various PKA-dependent processes, including growth. The significant sequence similarity (82% identity) between the putative DNAbinding domains of Sok2 and Phd1 suggested that the two proteins are functional homologs. To determine whether PHD1 also interacted with the PKA pathway, we examined the effects of overexpressing and deleting PHD1 on various PKA and sok2 mutants. First, the conditional tpk2-63 mutant (SGY398) was transformed with a high-copy-number URA3 plasmid bearing PHD1 (pCG38). In contrast to SOK2, expression of PHD1 from a high-copy-number plasmid did not suppress the temperature sensitivity of the PKA mutant. Second, a deletion of PHD1 was introduced into a temperature-sensitive PKA strain (SGY398) containing either the wild-type SOK2 allele or the sok2::URA3 disruption. Once again, the loss of PHD1 had no effect on the growth of either the tpk2(Ts) SOK2 or the tpk2(Ts) sok2::URA3 strain at any temperature. Finally, altering the expression of PHD1 (by deletion or placement of PHD1 on a high-copy-number plasmid) had no significant effect on glycogen accumulation or resistance to heat

TABLE 2. Effects of overexpression and deletion of SOK2 on PKA-associated phenotypes Strain

Thermotolerancea

Genotype

508C

Glycogen accumulationb

558C

MWY421 MWY422 MWY423

tpk1w (YEp) tpk1w (YEp-SOK2) tpk1w sok2::URA3

r/s s r

1 1/2 1


TPK1 (YEp) TPK1 (YEp-SOK2) TPK1 sok2::URA3

r r r

1 1 1


TPK1 (YCp-RAS2) TPK1 (YCp-RAS2Val-19) TPK1 sok2::ADE8 (YCp-RAS2) TPK1 sok2::ADE8 (YCp-RAS2Val-19)

MWY517

r s r

1 2/1 1

r

1/2

a Thermotolerance assessed as growth at 308C after exposure to the indicated temperatures for 10 min. Resistance to heat shock can be compared only among strains within each class. r, resistant; s, sensitive; r/s, partially resistant. b Glycogen accumulation as assessed by inverting agar plates over iodine crystals. 1, high; 2/1, low; 1/2, intermediate.

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FIG. 7. SOK2 represses pseudohyphal growth. Diploid strains were streaked to obtain single cells on nitrogen starvation (SLAD) medium agar and were grown at 308C. Representative microcolonies were photographed after 2 days. The strains were CG953 (wild type), CGX137 (phd1/phd1), CG995 (YEpSOK2), CG934 (YEpPHD1), CGX210 (sok2/sok2), and CGX225 (phd1/phd1 sok2/sok2), panels A to F, respectively. The scale bar represents 100 mm.

FIG. 6. Effects of SOK2 on TPK1, GAC1, and SSA3 expression. (A) Expression of TPK1 and GAC1 in TPK1 strains with various levels of Sok2 protein. Total RNAs from TPK1 strains containing SOK2 in single copy (SOK2 [MWY525]) or in high copy (YEpSOK2 [MWY526]) or lacking SOK2 (sok2 [MWY535]) were probed sequentially with the indicated DNAs. (B) Quantitation of TPK1, GAC1, and SSA3 in tpk1w strains with various levels of Sok2 protein. Total RNAs from tpk1w strains containing SOK2 in single copy (SOK2 [MWY421]) or in high copy (YEpSOK2 [MWY422]) or lacking SOK2 (sok2 [MWY423]) were probed sequentially with TPK1, GAC1, SSA3, and DED1 DNAs and then quantitated with a Molecular Dynamics PhosphorImager. (C) Expression of SSA3 in TPK1 strains containing various levels of Sok2 protein with (1) and without (2) exposure to heat shock (HS). Total RNAs from TPK1 strains containing SOK2 in single copy (SOK2 [MWY525]) or in high copy (YEpSOK2 [MWY526]) or lacking SOK2 (sok2 [MWY535]) were sequentially probed with the indicated DNAs.

shock (results not shown). Thus, in contrast to SOK2, PHD1 displays no obvious general role in the PKA pathway. SOK2 inhibits pseudohyphal formation. Although PHD1 overexpression induced pseudohyphal growth under noninducing conditions, diploids homozygous for a null allele of PHD1 exhibited normal pseudohyphal development on nitrogen starvation. One explanation for that result was that S. cerevisiae has one or more genes that are functionally redundant with PHD1

(20). Given the sequence similarity between the Sok2 and Phd1 proteins, we examined the onset of pseudohyphal development in homozygous sok2/sok2 (CGX210) and sok2/sok2 phd1/phd1 (CGX225) diploid strains, as well as an isogenic diploid strain (CG995) containing the high-copy-number SOK2 plasmid, pMW61. Although SOK2 overproduction had no significant effect on pseudohyphal growth, the homozygous sok2/sok2 diploid strains exhibited significantly enhanced pseudohyphal growth when they were grown on nitrogen starvation (SLAD) medium (Fig. 7). Indeed, the degree of enhancement elicited by loss of Sok2 function was comparable to that conferred by PHD1 overexpression (Fig. 7). Moreover, deletion of PHD1 abrogated most of the effect of the sok2::URA3 disruption (Fig. 7), demonstrating that the enhancement of pseudohyphal growth elicited by the loss of Sok2 function was, for the most part, a Phd1-dependent process. Overproduction of cAMP phosphodiesterase 2 inhibits pseudohyphal growth and is epistatic to RAS2Val-19. A role for cAMP-dependent protein kinase in pseudohyphal development was previously suggested by the observation that the constitutively active RAS2Val-19 mutation strongly enhances pseudohyphal growth (21). The effect of the RAS2Val-19 allele on pseudohyphal development could reflect either its documented stimulation of cAMP synthesis (64), and the corresponding activation of cAMP-dependent protein kinase, or the perturbation of a cAMP-independent pathway (46). To investigate the contribution of cAMP to the stimulation of pseudohyphal growth, a high-copy-number plasmid containing the low-affinity cAMP phosphodiesterase gene, PDE2 (56), was transformed into diploid strains containing the RAS2Val-19 allele. The RAS2Val-19/YEpPDE2-1 (CJ59) strain exhibited more prolific pseudohyphal growth than the RAS2/YEp control strain (CJ57), whereas the enhancing effect of the RAS2Val-19 allele was significantly reduced by the presence of the highcopy-number PDE2 plasmid (Fig. 8). Thus, the RAS2Val-19 mutation probably enhances pseudohyphal growth by causing an increase in cAMP accumulation. Since the only known function of cAMP in S. cerevisiae is to stimulate cAMP-dependent protein kinase, it seems likely that the cAMP-dependent

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FIG. 8. Overexpression of PDE2 blocks pseudohyphal growth. Diploid strains were streaked to obtain single cells on nitrogen starvation (SLAD) medium agar and were grown at 308C. Representative microcolonies were photographed after 2 (A, B, C, and D) and 5 (E and F) days. The strains were CJ57 (wild type [A and E]), CJ56 (YEpPDE2 [B and F]), CJ58 (RAS2Val-19 [C]), and CJ59 (RAS2Val-19 YEpPDE2 [D]). The scale bar represents 100 mm.

enhancement in pseudohyphal growth is mediated by an increase in cAMP-dependent protein kinase. In keeping with this interpretation, overexpression of the PDE2 gene blocked even basal pseudohyphal formation of a wild-type RAS2 strain (compare Fig. 8E and F). DISCUSSION We have identified a new gene, SOK2, that appears to encode a transcription factor involved in PKA-dependent growth control and cellular differentiation. The simplest interpretation of our results supports a model in which the product of SOK2 functions downstream of the PKA catalytic subunit to regulate the expression of a set of genes involved in proliferation (growth and cell cycle progression), metabolism, and pseudohyphal growth. If this is correct, Sok2 would be the first downstream effector of the yeast PKA pathway with a general role in cell growth. PKA-dependent growth may be mediated by Sok2, a new member of a class of fungal transcription factors involved in cell cycle control and differentiation. Several observations support a model in which Sok2 acts downstream of PKA to regulate the expression of genes essential to growth and metabolism. First, deletion of SOK2 exacerbates the growth defect of a PKA-compromised strain (Fig. 4). Thus, lesions in the PKA pathway can be either alleviated (Fig. 1) (67) or exacerbated by changes in SOK2 expression. Second, SOK2 has a pleiotropic effect on several PKA-regulated phenotypes and processes that are not essential to growth (Fig. 5 and 6; Table 2). The simultaneous suppression of several phenotypes conferred by a primary defect is often used as indirect proof that the suppressor defines a function integral to the pathway under study (24). Finally, the model is supported by the observation that TPK1 expression is unaffected by SOK2 overexpression or deletion

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(Fig. 6). The strong sequence similarity between Sok2 and several fungal transcription factors (Phd1, StuA, Swi4, and Mbp1) would argue against Sok2 regulating the posttranscriptional synthesis or activity of PKA. The postulated role for Sok2 in transcriptional control is consistent with the prevailing view that many effects of yeast PKA are mediated by changes in gene expression (13, 26, 31, 43, 67). One could posit that Sok2 is itself a substrate of the PKA and that Sok2 function is regulated directly by PKA phosphorylation. However, the significance of the consensus PKA phosphorylation site found in the carboxyl-terminal tail of Sok2 can, at this time, only be inferred from the observation that the site falls within a region necessary for Sok2 function. The sequence similarities among Sok2 and several yeast transcriptional factors (Swi4 and Mbp1) involved in transition from G1 to S are intriguing, since PKA activity is required for progression through G1 (26, 38, 67). However, the sequence similarities appear to reflect a common DNA-binding motif rather than redundant physiological functions, because deletion of SOK2 failed to affect the growth of swi4 or swi6 strains. This view is supported by the fact that the conditional growth of a PKA mutant is unaffected by overexpression of the Swi4dependent G1 cyclin CLN2 (unpublished results). Thus, the sequence similarity between Sok2 and at least one other member (Swi4) of the class of yeast cell cycle transcription factors may point to a common mechanism of DNA recognition (32) rather than a shared function. By contrast, a reasonable case for identical, or overlapping, DNA-binding specificities can be made for Sok2 and Phd1, a yeast protein implicated in pseudohyphal growth. This possibility is supported by the striking sequence similarity (82% identity) displayed between the putative DNA-binding motifs of these two proteins, as well as the observation that pseudohyphal growth can be stimulated to similar degrees by the overproduction of PHD1 or the loss of SOK2 (Fig. 7). The fact that a functional PHD1 gene is required for the accelerated pseudohyphal growth of the sok2 strain suggests that Phd1 and Sok2 serve to activate and repress, respectively, the expression of an identical set of genes involved in the switch to pseudohyphal growth. That two proteins with such similar DNA-binding domains might have antagonistic functions is easy to envisage, given the modular nature of transcriptional factors (11, 34) and the lack of sequence similarity between Sok2 and Phd1 in regions outside their putative DNA-binding domains. The observations that Sok2 functions as a repressor of genes involved in glycogen accumulation and the response to stress (Fig. 6) and that it may also repress genes involved in pseudohyphal growth (Fig. 7) raise an interesting possibility regarding the mechanism by which it mediates the PKA signal in controlling growth. It is formally possible, for example, that Sok2 negatively regulates the expression of one or more genes antagonistic to cell growth and division. In this scenario, Sok2 repressor activity would be stimulated by PKA phosphorylation, resulting in the maximal repression of genes involved in the stress response as well as cell cycle progression. At odds with this simple interpretation is the apparent contradiction that pseudohyphal growth is inhibited by Sok2 and yet stimulated by PKA activity. However, these observations could be accommodated by proposing that the effect of phosphorylation on Sok2 repressor activity is promoter specific. Whereas Sok2 repression of GAC1, SSA3, and growth-specific genes might be dependent on phosphorylation, the same modification might inhibit Sok2 from repressing the expression genes involved in the pseudohyphal switch. Alternatively, Sok2 might represent but one of several factors that mediate PKA control of pseudohyphal growth. Indeed, the fact that sok2 phd1 mutants

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are viable and exhibit pseudohyphal growth when starved for nitrogen suggests that genes redundant for both functions may exist. In either case, it seems likely that further molecular and genetic characterization of Sok2 function will contribute to the identification and characterization of PKA-regulated genes and processes that are essential to growth. PKA stimulates yeast pseudohyphal growth. The results presented here also provide further support for a relationship between the cAMP-PKA pathway and filamentous growth. A role for yeast PKA in this process was first suggested by the observation that activating mutations in the RAS2 gene enhanced the pseudohyphal phenotype (21). However, Gimeno et al. were careful to point out that the actual biochemical cause of that enhancement could only be inferred since Ras2 had also been implicated in processes unrelated to cAMP production (40). It appears that the effect of the activated RAS2 allele was, as previously suggested, the result of an increase in cAMP synthesis, since the accelerated pseudohyphal growth of a RAS2Val-19 strain was largely reversed by overexpressing the low-affinity phosphodiesterase gene PDE2 (Fig. 8). PDE2 overexpression has previously been shown to reduce the cAMP concentration of a RAS2Val-19 strain (46, 56). Thus, it seems likely that the inhibitory effect of the high-copy-number PDE2 plasmid on pseudohyphal growth also resulted from a similar reduction in cellular cAMP. Interestingly, the pseudohyphal growth of a wild-type RAS2 diploid was completely abolished by the overexpression of PDE2 (Fig. 8). Presumably, the different effects of PDE2 overexpression on the timing and execution of the pseudohyphal switch of wild-type and activated RAS2 strains reflect the limited capacity of the phosphodiesterase to deplete the cAMP levels of a constitutively activated RAS2Val-19 mutant. Together, these results suggest that fluctuations in yeast PKA activity may provide a primary and essential signal for the switch between budding and filamentous growth, with low PKA activity favoring bud formation and high PKA activity inducing the transition to pseudohyphal growth. A role for cAMP in the dimorphic switch has been suggested for several different fungi (57). Recently, Gold et al. (22) provided compelling evidence to support a key role for cAMP and PKA in regulating the switch from budding to filamentous growth in the fungal pathogen Ustilago maydis. However, the situation for U. maydis appears to be different from that for S. cerevisiae; in U. maydis, high PKA activity favors budding rather than filament formation (22). Thus, S. cerevisiae and U. maydis may represent an interesting example of two organisms that exploit the same signal transduction pathway (cAMPPKA) to regulate, in opposite directions, what appears to be a similar response (budding or filamentous growth). Given the sequence similarity between Sok2 and other proteins implicated in fungal differentiation and morphogenesis (Phd1 and StuA), it would not be surprising to find that U. maydis also regulates the switch between budding and filamentous growth by the phosphorylation-dephosphorylation of a transcription factor(s) bearing structural similarity to Phd1 and Sok2. ACKNOWLEDGMENTS We thank Linda Breeden, Stephen Johnston, Mike Resnick, and Michael Wigler for their generous gifts of plasmids and strains and Margaret Werner-Washburne for the SSA3 DNA fragment. M.P.W. thanks Stef Bogaerts for invaluable assistance with the Northern blots and Anupama Dahanukar for help with the PhosphorImager analyses. We are especially grateful to Kelly Tatchell for suggesting that we test the expression of GAC1 in strains lacking SOK2. M.P.W. was supported by a predoctoral training grant from NIH and a Katherine Stern Fellowship. C.J.G. was supported by a Howard

MOL. CELL. BIOL. Hughes Medical Institute predoctoral fellowship. This work was also supported by NIH grant GM44666 (S.G.). G.R.F. is an American Cancer Society Professor of Genetics, and S.G. was supported by an American Cancer Society Junior Faculty Scholar award (ACS JFRA no. 395). REFERENCES 1. Baroni, M. D., P. Monti, and L. Alberghina. 1994. Repression of growthregulated G1 cyclin expression by cyclic AMP in budding yeast. Nature (London) 371:339–342. 2. Broach, J. R. 1991. RAS genes in Saccharomyces cerevisiae: signal transduction in search of a pathway. Trends Genet. 7:28–33. 3. Broach, J. R., and R. J. Deschenes. 1990. The function of RAS genes in Saccharomyces cerevisiae. Adv. Cancer Res. 54:79–138. 4. Cameron, S., L. Levin, M. Zoller, and M. Wigler. 1988. cAMP-independent control of sporulation, glycogen metabolism, and heat shock resistance in S. cerevisiae. Cell 53:555–566. 5. Cannon, J., and K. Tatchell. 1987. 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