INTRODUCTION

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In response to nitrogen limitation, diploid cells of Saccharomyces cerevisiae undergo a dimorphic transition to a filamentous growth form referred to as pseudohyphal differentiation (14, 19). This filamentous growth form represents a dramatic change in the cellular program in which the cells elongate, adopt a unipolar budding pattern, remain physically connected in chains, and invade the agar (14, 23). This alternative growth form may enable this nonmotile species to forage for nutrients under adverse conditions.

Two signaling pathways that regulate yeast filamentous growth have been defined. The first involves components of the mitogen-activated protein (MAP) kinase pathway that also functions during mating in haploid cells (8, 28, 35). These components include the kinases Ste20, Ste11, Ste7, and Kss1 and the transcription factor Ste12. In addition, the transcription factor Tec1 forms heterodimers with Ste12 that regulate expression of Tec1 itself and additional targets, such as the cell surface flocculin Flo11 required for invasive and filamentous growth (3, 11, 31, 34). Early elements of the pheromone response pathway, including the pheromones, their receptors, and the coupled heterotrimeric G protein, are not expressed in diploid cells and are not required for filamentous differentiation (28). Instead, the MAP kinase pathway is activated by Cdc42, Ras2, and the 14-3-3 proteins Bmh1 and Bmh2 (38, 39, 49), possibly in response to the Sho1 osmosensing receptor (46).

A second signaling pathway functions in parallel with the MAP kinase pathway to regulate pseudohyphal differentiation. This pathway involves a novel G protein-coupled receptor, Gpr1, which is required for both pseudohyphal differentiation (33) and, in conjunction with Ras2, vegetative growth (63). The Gpr1 ligand has not yet been identified. The Gpr1 receptor is coupled to a heterotrimeric G protein  subunit, Gpa2, which is also required for pseudohyphal differentiation and plays a role in nutrient sensing (26, 32). Early studies suggested Gpa2 might stimulate cyclic AMP (cAMP) production by adenylyl cyclase (41). Consistent with this, cAMP stimulates pseudohyphal differentiation and suppresses the filamentation defects of gpr1 and gpa2 mutant strains (26, 32, 33). A recent study has confirmed that Gpa2 regulates cAMP production by adenylyl cyclase in response to nutritional signals (7). Dominant activated Gpa2 mutants or cAMP suppresses the pseudohyphal defect of mutant strains lacking MAP kinase cascade components (32). In summary, a second signaling pathway comprised of the Gpr1 receptor, the Gpa2 G protein, and cAMP regulates pseudohyphal growth in parallel to and independently from the MAP kinase pathway.

The target of cAMP in yeast is the cAMP-dependent protein kinase, protein kinase A (PKA). The yeast PKA kinase is similar to mammalian PKA and consists of a regulatory subunit encoded by a single gene, BCY1, and three catalytic subunits encoded by the TPK1, TPK2, and TPK3 genes (6, 57, 58). In both yeast and mammals, PKA in resting cells is an inactive tetramer composed of two regulatory subunits bound to two active subunits. In response to external signals that increase intracellular cAMP levels, cAMP binds to the regulatory subunit and triggers conformational changes that release the active catalytic subunits. Hydrolysis of cAMP by cAMP phosphodiesterases, the products of the PDE1 and PDE2 genes in yeast, restores PKA to the resting, inactive state (43, 54).

The yeast cAMP-dependent protein kinase is required for vegetative growth (58). Triple mutants lacking the Tpk1, Tpk2, and Tpk3 catalytic subunits are inviable, whereas mutant strains expressing any one of the three Tpk subunits are all viable. These findings led to the model that the three PKA catalytic subunits are largely redundant for function. The PKA catalytic subunits share a conserved C-terminal kinase domain attached to unique N-terminal regions. Tpk1 and Tpk3 share 88% identity in the kinase domain, whereas Tpk2 is more divergent (77 and 75% identity with Tpk1 and -3, respectively). Several candidate PKA targets for vegetative growth have recently been identified. For example, the Msn2 and Msn4 transcription factors are regulated by PKA and repress expression of genes that regulate vegetative growth (4, 18, 56). The Rim15 protein kinase is also phosphorylated and inhibited by PKA and regulates entry into meiosis and stationary phase (48, 60).

In parallel, studies of PKA constitutively activated by cAMP, bcy1 mutation, or activated Ras2 revealed roles in regulating stationary phase, meiosis, and sporulation (5, 59). Activation of PKA prevents glycogen accumulation, heat shock resistance, and survival during nutrient limitation, all hallmarks of entry into stationary phase. Similarly, activation of PKA inhibits sporulation. Thus, activation of PKA promotes vegetative growth in response to nutrients, whereas inactivation of PKA in response to nutrient limitation regulates sporulation and entry into stationary phase.

Several observations suggest PKA might also regulate yeast pseudohyphal differentiation. The dominant active Ras2^val19 mutant protein enhances filamentous growth (14), whereas overexpression of the cAMP phosphodiesterase Pde2 inhibits filamentation (62). In addition, exogenous cAMP enhances filamentous growth (32).

Here we report that the cAMP-dependent protein kinase regulates yeast pseudohyphal differentiation. First, we show that mutation of the PKA regulatory subunit Bcy1 enhances filamentous growth. Second, we demonstrate that the PKA catalytic subunits play distinct roles in regulating filamentous growth: the Tpk2 subunit activates filamentous growth, whereas the Tpk1 and Tpk3 subunits primarily inhibit filamentous growth. The unique activating function of the Tpk2 subunit is linked to structural differences in the catalytic region of the kinase and not to differences in gene regulation or the unique amino-terminal region of the protein. Genetic epistasis experiments support a model in which Tpk2 functions downstream of the Gpr1 receptor and the G protein Gpa2. Importantly, activation of PKA by mutation of the Bcy1 regulatory subunit restores pseudohyphal growth in mutants lacking elements of the MAP kinase pathway, including ste12, tec1, and ste12 tec1 mutant strains. Thus, the MAP kinase and PKA pathways independently regulate filamentous growth. Further analysis reveals that the PKA pathway regulates the switch to unipolar budding and invasion, whereas the MAP kinase pathway is required for cell elongation and invasion. Finally, our studies define a role for the PKA pathway in activating pseudohyphal growth via transcriptional regulation of the cell surface flocculin Flo11 by the Flo8 transcription factor, and both Flo11 and Flo8 were previously shown to be required for pseudohyphal growth (27, 29, 31). Taken together, our studies reveal an intimate role for the cAMP-dependent kinase in the regulation of yeast dimorphism and suggest this role has been evolutionarily conserved in diverse yeast species and fungi, including pathogens of both plants and animals.

	MATERIALS AND METHODS

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Media and growth conditions. Standard yeast media and genetic manipulations were used as described previously (55). Limiting nitrogen media contains 0.17% yeast nitrogen base without amino acids or ammonium sulfate (32), 2% dextrose, 2% Bacto agar, and 50 µM ammonium (SLAD [14]) or 500 µM ammonium (SMAD [1]). SLARG medium, used to induce the dominant active GPA2-2 (Gly132Val) allele, contains 0.5% galactose and 2% raffinose (32).

Yeast strains and plasmids. The yeast strains used in this study are listed in Table 1 and are all derived from the 1278b strain background. The bcy1::G418, tpk1::G418, tpk2::G418, and tpk3::G418 mutations were created by the PCR-mediated gene disruption technique with the G418 resistance cassette from plasmid pFA6-KanMX2 (61). The sok2::HygB, rim15::HygB, flo1::HygB, flo5::HygB, flo8::HygB, and flo11::HygB mutations were generated by PCR-mediated gene disruption with a hygromycin B resistance cassette (17). Independently derived haploid strains (created in strains MLY40 and MLY41a [Table 1]) were mated to produce the homozygous diploid strains (Table 1). To construct multiply mutant strains, haploid strains with single or double gene deletions were crossed, sporulated, and dissected. Tetrads in which G418 resistance segregated 2 resistant:2 sensitive were chosen and confirmed to contain the expected double or triple G418-resistant gene deletions by PCR analysis of genomic DNA. Sterile ste12 haploid strains were complemented with plasmid pSC4 (STE12 URA3 CEN) before mating, while all of the haploid bcy1 mutant strains were complemented with plasmid pXP1 (2µm BCY1) to increase mating efficiency. The pSC4 and pXP1 plasmids were ejected from homozygous diploid strains by selection on 5-fluoroorotic acid medium. When necessary, a control URA3 plasmid was introduced to complement the ura3-52 mutation and allow growth on SLAD medium.

Photomicroscopy. All single-colony photographs were taken directly from petri plates by using a Nikon ECLIPSE E400 microscope with a ×10 primary objective and a ×2.5 trinocular camera adaptor for a final magnification of ×25.

Construction of TPK chimeric genes. Hybrids between the TPK1 and TPK2 genes were constructed by PCR overlap (20) and cloned into the multicopy plasmid YEplac195 (12). To make the pTPK1-TPK2 hybrid gene, primers 5'-CGGGATCCCGAAGCTGTGCTGCTATTC and 5'-GCCCTTTCTGCAACGAATTCCATACCCAAAAAAAAGATTCTTTCAC were used to generate the promoter portion of the TPK1, and primers 5'-GTGAAAGAATCTTTTTTTTGGGTATGGAATTCGTTGCAGAAAGGGC and 5'-CTAGTCTAGACTAGGAGGACTTAAAGCATGTCG were used to amplify the structural and 3' untranslated region (UTR) region of the TPK2 gene. The products of the first-round PCRs were gel purified and mixed as template to amplify the hybrid pTPK1-TPK2 gene with primers 5'-CGGGATCCCGAAGCTGTGCTGCTATTC and 5'-CTAGTCTAGACTAGGAGGACTTAAAGCATGTCG. The resulting ~2-kb PCR product was gel purified, digested with BamHI and XbaI, and cloned into the multicopy plasmid YEplac195. For the construction of the pTPK2-TPK1 hybrid gene, primers 5'-CGGGATCCCAAGCATCTGTACCTCCAC and 5'-CTCCATTTTGTTCTTCAGTCGACATACCGACAATTTTCAACAGTATG were used to amplify the promoter portion of the TPK2 gene, and primers 5'-CATACTGTTGAAAATTGTCGGTATGTCGACTGAAGAACAAAATGGAG and 5'-GCTGCAGCCGGTGAAAGCTTCTCATC were used to generate the structural and 3'-UTR region of the TPK1 gene. The PCR products were gel purified and combined as the template for a second round of PCR with primers 5'-CGGGATCCCAAGCATCTGTACCTCCAC and 5'-GCTGCAGCCGGTGAAAGCTTCTCATC. The PCR product was gel purified, digested with BamHI and PstI, and cloned into plasmid YEplac195. For the construction of the TPK1-TPK2 hybrid gene, primers 5'-CGGGATCCCGAAGCTGTGCTGCTATTC and 5'-GTCATGTAGTGTATATTTGCCCACTGTAACTCTCGCTTG were used to generate the promoter portion and the coding sequence for the amino-terminal region of the TPK1 gene, and primers 5'-CAAGCGAGAGTTACAGTGGGCAAATATACACTACATGAC and 5'-CTAGTCTAGACTAGGAGGACTTAAAGCATGTCG were used to amplify the coding sequence for the carboxyl-terminal portion and the 3'-UTR of the TPK2 gene. The PCR products were gel purified and combined as the template for a second-round PCR to amplify the TPK1-TPK2 gene with primers 5'-CGGGATCCCGAAGCTGTGCTGCTATTC and 5'-CTAGTCTAGACTAGGAGGACTTAAAGCATGTCG. The PCR product of this reaction was gel purified and cloned into the TA-cloning PCR2.1 vector. The construct was digested with EcoRI, and the desired TPK1-TPK2 hybrid gene was then subcloned into YEplac195. For the construction of the TPK2-TPK1 hybrid, primers 5'-CGGGATCCCAAGCATCTGTACCTCCAC and 5'-GTTCTTGTAAACTATACTTCCTTTGGATACGAGAGATTTC were used to amplify the promoter and the coding sequence for the amino-terminal portion of the TPK2 gene, and primers 5'-GAAATCTCTCGTATCCAAAGGAAGTATAGTTTACAAGAAC and 5'-GCTGCAGCCGGTGAAAGCTTCTCATC were used to amplify the coding sequence for the carboxyl-terminal portion and 3'-UTR of the TPK1 gene. The PCR products were gel purified and combined as the template for a second-round PCR to amplify the TPK2-TPK1 gene with primers 5'-CGGGATCCCAAGCATCTGTACCTCCAC and 5'-GCTGCAGCCGGTGAAAGCTTCTCATC. The product of this PCR was gel purified, digested with BamHI and PstI, and cloned into the multicopy plasmid YEplac195. The junctions of the resulting chimeric genes were confirmed by DNA sequencing.

Cell morphology and budding pattern assays. Cell shape determination was performed based on a method by Mösch and Fink (38) with minor modifications. Diploid strains were grown on SLAD medium for 16 h at 30°C. Cells were washed off the plates and collected for analysis of the proportion of elongated pseudohyphal cells (PH), ovate yeast form cells (YF), and round yeast form cells (round YF) by microscopic examination. Cells were photographed at a magnification of ×200, and at least 300 cells were counted for each strain.

tpk2/tpk2

ste12/ste12

Northern analysis. Total RNA was isolated with the QIAGEN RNeasy Mini kit or by acid phenol extraction, separated by electrophoresis, and transferred overnight by capillary action to nylon membranes (VWR Scientific Products). DNA fragments to be used as probes (200-bp PCR products lying 5' to the TPK1, TPK2, and TPK3 open reading frames (ORFs), a 300-bp PCR product 5' to the FLO11 open reading frame, and a 500-bp fragment 5' to the ACT1 open reading frame) were gel purified and radiolabeled by random priming (Boehringer Mannheim). Hybridization, washes, stripping, and reprobing were performed as described previously (53). The radioactive bands were visualized by autoradiography and quantitated with a Molecular Dynamics PhosphorImager. The TPK2 gene was induced to similar extents (~4-fold) by nitrogen starvation in two independent experiments.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

PKA regulates pseudohyphal growth. We recently found that exogenous cAMP stimulates pseudohyphal differentiation in S. cerevisiae (32). This finding, and other previous studies (14, 62), suggested that cAMP-dependent protein kinase (PKA) might regulate pseudohyphal growth.

View larger version (78K): [in this window] [in a new window]   FIG. 1.   Deletion of the PKA regulatory subunit BCY1 enhances filamentous growth. Homozygous wild-type (MLY61a/) and bcy1/bcy1 (XPY1a/) mutant diploid strains were incubated on low-ammonium sulfate (SLAD; 50 µM) and medium-ammonium sulfate (SMAD; 500 µM) media for 3 days at 30°C. Colonies were photographed originally at a ×25 magnification in this and the following figures.

PKA catalytic subunits play distinct roles in regulating filamentous growth. To establish the functions of the PKA catalytic subunits in filamentous growth, the genes encoding Tpk1, Tpk2, and Tpk3 were individually deleted in the 1278b strain background. The resulting isogenic wild-type and tpk1/tpk1, tpk2/tpk2, and tpk3/tpk3 mutant strains were grown on medium containing 50 or 200 µM ammonium sulfate to assay filamentous growth. As shown in Fig. 2, a clear and striking finding was that the tpk2/tpk2 mutant strain was severely reduced in pseudohyphal growth. This finding suggests that the Tpk2 catalytic subunit is required for pseudohyphal growth and plays a positive signaling role. The tpk2/tpk2 mutant strain does form some rudimentary filaments, which is also the case with most other mutants with defects in filamentous growth (28, 32).

View larger version (58K): [in this window] [in a new window]   FIG. 2.   The tpk2 mutation reduces pseudohyphal growth, whereas tpk1 and tpk3 mutations enhance filamentous growth. Homozygous wild-type (MLY61a/), tpk1/tpk1 (XYP4a/), tpk2/tpk2 (XPY5a/), and tpk3/tpk3 (XPY6a/) mutant strains were incubated on SLAD medium with 50 or 200 µM ammonium sulfate, as indicated, and incubated for 3 days at 30°C.

Point of Tpk1 and Tpk3 inhibitory action. Analysis of tpk double mutant strains revealed that the tpk2 mutation is epistatic to the tpk1 and tpk3 mutations. Thus, the tpk2/tpk2 tpk3/tpk3 and tpk1/tpk1 tpk2/tpk2 double mutant strains exhibited the filamentation defect conferred by the tpk2 mutation, whereas the tpk1/tpk1 tpk3/tpk3 double mutant strain exhibited the enhanced filamentation phenotype of the tpk1 and tpk3 single mutant strains (Fig. 3A). By Northern blot analysis, the TPK2 gene was expressed to the same extent in the wild-type strain and in tpk1/tpk1, tpk3/tpk3, and tpk1/tpk1 tpk3/tpk3 mutant strains, and thus Tpk1 and Tpk3 are not involved in the regulation of TPK2 expression (data not shown). These observations suggest that the negative function of Tpk1 and Tpk3 is exerted on components upstream or downstream of PKA.

View larger version (74K): [in this window] [in a new window]   FIG. 3.   Epistasis analysis of tpk and bcy1 mutations. (A) The tpk2 mutation is epistatic to the tpk1 and tpk3 mutations. Isogenic wild-type (MLY61a/) and tpk1/tpk1 tpk2/tpk2 (XPY12a/), tpk2/tpk2 tpk3/tpk3 (XPY13a/), and tpk1/tpk1 tpk3/tpk3 (XPY14a/) mutant diploid strains were incubated on SLAD medium for 3 days at 30°C. (B) The bcy1 mutation suppresses the filamentation defect conferred by the tpk2 mutation. Isogenic wild-type (MLY61a/) and bcy1/bcy1 (XPY12a/), tpk2/tpk2 (XPY5a/), and bcy1/bcy1 tpk2/tpk2 (XPY59a/) mutant diploid strains were grown on SLAD medium for 3 days at 30°C.

TPK2 expression is induced by nitrogen starvation. We next focused on the role of the Tpk2 catalytic subunit in stimulating filamentous growth to address why Tpk2 is unique compared to Tpk1 and Tpk3. We first examined the expression pattern of the TPK2 gene. Wild-type diploid cells of the 1278b strain were grown in liquid media containing a range of ammonium sulfate concentrations from 5 to 5,000 µM. RNA was isolated and analyzed by Northern blotting for the expression of TPK2 and with actin as a control. Expression of the TPK2 gene was induced ~4-fold in response to nitrogen starvation in two independent experiments (data not shown). By comparison, expression of the TPK1 gene was induced by only ~1.5-fold, and the TPK3 gene was not induced, under similar conditions (data not shown). These findings are consistent with Tpk2 playing a unique role in nitrogen-starved diploid cells.

Tpk2 catalytic domain stimulates filamentous growth. That expression of the TPK2 gene is induced by nitrogen starvation suggested this might be the unique feature of Tpk2. To test this, we constructed chimeric genes in which the Tpk2 protein was expressed from the TPK1 promoter and the Tpk1 protein was expressed from the TPK2 promoter (Fig. 4A). These chimeric genes were introduced into the wild-type 1278b diploid strain and analyzed for the phenotype conferred by overexpression of Tpk2 (enhanced filamentation) or Tpk1 (reduced filamentation). Expression of Tpk2 by either its own promoter or the promoter of the TPK1 gene stimulated filamentous growth to similar extents (Fig. 4B). Expression of Tpk1 from either promoter inhibited filamentous growth (Fig. 4B). Thus, differences in the TPK1 and TPK2 gene promoters and transcriptional regulation do not underlie the unique regulatory functions of Tpk1 and Tpk2.

View larger version (44K): [in this window] [in a new window]   FIG. 4.   The unique activating function of Tpk2 maps to the C-terminal kinase domain. (A) The structures of the wild-type TPK1 (open bars) and TPK2 (solid bars) genes and four chimeric TPK genes are illustrated. The pTPK1-TPK2 hybrid gene consists of the promoter region of TPK1 (dashed line) and the ORF and 3'-UTR of the TPK2 gene. The pTPK2-TPK1 hybrid gene consists of the promoter region of TPK2 (solid line) and the ORF and 3'-UTR of the TPK1 gene. The TPK1-TPK2 hybrid gene consists of the promoter plus the coding sequence for the unique amino-terminal portion of the TPK1 ORF (aa 1 to 79) and the carboxyl-terminal portion of the TPK2 ORF (aa 63 to 281) and 3'-UTR. The TPK2-TPK1 hybrid gene consists of the promoter and coding sequence of the amino-terminal portion of the TPK2 ORF (aa 1 to 62) and the carboxyl-terminal portion of the TPK1 ORF (aa 80 to 378) and 3'-UTR. (B) The wild-type TPK1 and TPK2 genes and the hybrid TPK genes were expressed from the high-copy plasmid YEplac195 in wild-type yeast strain MLY61a/ and assayed for filamentous growth following incubation at 30°C for 3 days on SLAD medium.

Tpk2 regulates the switch to unipolar budding and invasive growth. Yeast pseudohyphal growth consists of several physiological events, including cell elongation, a switch from bipolar to unipolar budding, mother-daughter cell adhesion, and invasive growth. We addressed which of these facets are effected by the Tpk2 subunit of PKA by analyzing the isogenic wild-type strain and the tpk2/tpk2 and ste12/ste12 mutant strains (see Materials and Methods). This analysis revealed that the tpk2 mutation impairs the switch to unipolar budding and also agar invasion, whereas cell elongation occurred normally in response to nitrogen starvation (Fig. 5 and Table 3). On the other hand, the ste12 mutation inhibited cell elongation and agar invasion, but had little or no effect on the switch to a unipolar budding pattern (Fig. 5, Table 3, and data not shown), in accord with previous reports (28, 39, 50). We note that robust activation of PKA, either by exogenous cAMP or the bcy1 mutation, results in filaments largely composed of round cells (Fig. 1) (32), further indicating that the PKA pathway does not promote cell elongation. In the microcolony budding assays, bipolar budding in wild-type cells produced quite linear chains of four cells, whereas the ste12/ste12 and tpk2/tpk2 mutant strains produced chains that were often perturbed at the central mother-daughter cell junction (Fig. 5), suggesting that the MAP kinase and PKA pathways may also regulate cell adhesion required for the integrity of pseudohyphal filaments.

View larger version (32K): [in this window] [in a new window]   FIG. 5.   PKA pathway regulates unipolar budding, while the MAP kinase pathway is required for cell elongation. (A) Isogenic wild-type (MLY61a/), tpk2/tpk2 (XPY5a/), and ste12/ste12 (MLY216a/) strains were incubated on SLAD medium for 16 h at 30°C. Cells were collected and studied for cell morphology (upper panels) and budding pattern (lower panels). Cell elongation in response to nitrogen starvation occurs in the wild-type and tpk2 mutant strain, whereas the number of elongated cells is severely reduced in the ste12 mutant strain. For the microcolony budding pattern assay, daughter cells were micromanipulated on SLAD medium and incubated at 30°C for 5 to 6 h, at which time, three- and four-cell microcolonies were photographed and scored for budding pattern. Cells were also stained with Calcofluor white and photographed to score the pattern of chitin bud scars (not shown [see Materials and Methods]). (B) The patterns of cell division that give rise to four-celled microcolonies by either the bipolar or the unipolar budding patterns are depicted.

Tpk2 functions downstream of Gpr1, Gpa2, and Ras2. The point of action of Tpk2 with respect to other known regulators of pseudohyphal growth was addressed by epistasis analysis. First, we used the bcy1 mutation to activate PKA and test if this suppresses mutations in the Gpr1 receptor or the linked G protein Gpa2. In isogenic bcy1/bcy1 gpr1/gpr1 and bcy1/bcy1 gpa2/gpa2 double mutant strains, the bcy1 mutation suppressed the filamentation defect conferred by either the gpr1 or the gpa2 mutation (data not shown), suggesting that Tpk2 functions downstream of Gpr1 and Gpa2. We also tested whether the tpk2 mutation would block activation of filamentation in response to dominant activated alleles of GPA2 or RAS2. As shown in Fig. 6, the tpk2 mutation completely suppressed filamentation in response to the Gpa2-gly132val dominant active mutant. On the other hand, the tpk2 mutation only partially suppressed filamentation in response to the dominant active Ras2-gly19val mutant protein (Fig. 6). These findings suggest that Gpr1 and Gpa2 signal upstream of Tpk2, in accord with a role in stimulating cAMP production by adenylyl cyclase. These findings also support a model in which Ras2 signals to activate both the MAP kinase and the PKA pathways (32, 40, 49).

View larger version (49K): [in this window] [in a new window]   FIG. 6.   Tpk2 acts downstream of Gpa2 and in part downstream of Ras2. (A) The tpk2 mutation is epistatic to the activated GPA2Val132 allele. Wild-type (MLY61a/) and tpk2/tpk2 (XPY5a/) mutant diploid strains containing a control vector (pSEYC68) or expressing the dominant active GPA2-2 allele (pML160), which is under the control of a galactose-inducible promoter, were incubated on SLARG medium for 5 days at 30°C. (B) Wild-type (MLY61a/) and tpk2/tpk2 (XPY5a/) mutant diploid strains containing a control plasmid or expressing the dominant active RAS2Val19 allele (pMW2) were grown on SLAD medium at 30°C for 3 days.

PKA regulates filamentous growth independently of Ste12 and Tec1. We next addressed the point of action of PKA with respect to the MAP kinase pathway regulating pseudohyphal growth. To address this, we performed epistasis analysis with the bcy1 mutation (to activate PKA) in mutants lacking Ste12, Tec1, or both Ste12 and Tec1.

View larger version (69K): [in this window] [in a new window]   FIG. 7.   Activated PKA restores filamentous growth in MAP kinase cascade mutants. Isogenic wild-type (MLY61a/) and ste12/ste12 (MLY216a/), tec1/tec1 (MLY183a/), ste12/ste12 tec1/tec1 (XPY77a/), bcy1/bcy1 (XPY1a/), bcy1/bcy1 ste12/ste12 (XPY69a/), bcy1/bcy1 tec1/tec1 (XPY75a/), and bcy1/bcy1 ste12/ste12 tec1/tec1 (XPY76a/) mutant diploid strains were incubated on SLAD medium at 30°C for 3 days.

Target of PKA for filamentous growth is not Phd1, Msn2, or Msn4. Several targets of yeast PKA have been identified, and we tested which regulate pseudohyphal growth. Previous studies identified two related transcription factors, Sok2 and Phd1, which regulate filamentation and may function downstream of PKA. Phd1 enhances filamentous growth when overexpressed (13), whereas overexpression of Sok2 suppresses the growth defect of strains with reduced PKA activity and sok2 mutations enhance filamentous growth (62). By comparison, phd1 mutations confer only a modest defect in filamentous growth unless combined with an ste12 mutation (30). These observations suggest Phd1 and Sok2 may antagonistically regulate filamentous growth.

View larger version (74K): [in this window] [in a new window]   FIG. 8.   Tpk2 regulates pseudohyphal growth independently of Phd1. (A) Activated PKA pathway induces filamentous growth in the absence of Phd1. Isogenic wild-type (MLY61a/) and phd1/phd1 (MLY182a/), phd1/phd1 tec1/tec1 (XPY89a/), bcy1/bcy1 (XPY1a/), bcy1/bcy1 phd1/phd1 (XPY78a/), and bcy1/bcy1 phd1/phd1 tec1/tec1 (XPY88a/) mutant diploid strains were incubated on SLAD medium at 30°C for 3 days. (B) Overexpression of PHD1 is epistatic to the tpk2 mutation. Wild-type (MLY61a/) and tpk2/tpk2 mutant (XPY5a/) diploid strains containing a control plasmid (vector) or the PHD1 overexpression plasmid (pCG68) were incubated on SLAD medium at 30°C for 3 days.

PKA regulates filamentation via Flo8 and the cell surface flocculin Flo11. Several observations suggested the function of the PKA signaling pathway might be linked to expression and function of cell surface proteins involved in cell-cell adhesion (flocculation). Namely, linear microcolonies produced by tpk2/tpk2 mutant strains were often bent, in contrast to linear four-celled microcolonies produced by wild-type cells (Fig. 5). Second, the Flo8 transcription factor and the Flo11 cell surface flocculin were previously found to be required for pseudohyphal growth (27, 29, 31), and the Flo8 protein has five consensus PKA phosphorylation sites. We therefore tested if these are targets of PKA that regulate filamentation.

View larger version (73K): [in this window] [in a new window]   FIG. 9.   PKA pathway regulates expression of the cell surface flocculin Flo11 via the transcription factor Flo8. (A) flo8 and flo11 mutations block pseudohyphal growth and are epistatic to activated PKA and MAP kinase cascade signaling. Wild-type (MLY61a/) and flo8/flo8 (XPY95a/), flo11/flo11 (XPY107a/), bcy1/bcy1 (XPY1a/), bcy1/bcy1 flo8/flo8 (XPY99a/), and bcy1/bcy1 flo11/flo11 (XPY119a/) mutant strains containing a control plasmid and wild-type and flo8/flo8 and flo11/flo11 mutant strains containing the pTDH1-TEC1 overexpression plasmid were grown on SLAD medium for 3 days at 30°C. (B) The PKA pathway regulates the expression of FLO11 by the transcription factor Flo8. Total RNA was prepared from wild-type (WT) (MLY61) and tpk2 (XPY5), flo8 (XPY95), flo11 (XPY107), tec1 (MLY183), bcy1 (XPY1), bcy1 flo8 (XPY99), and bcy1 tec1 (XPY75) mutant strains and wild-type and flo8 strains containing the pTDH1-TEC1 plasmid grown in synthetic medium lacking uracil. RNA was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a nylon membrane, and probed with portions of the FLO11 and ACT1 genes.

View larger version (20K): [in this window] [in a new window]   FIG. 10.   A model for the regulation of pseudohyphal growth by the PKA and MAP kinase pathways. The three catalytic subunits of PKA play distinct roles in regulating yeast pseudohyphal growth. The Tpk2 catalytic subunit plays a positive role to activate filamentous growth, whereas the Tpk1 and Tpk3 catalytic subunits play negative roles to inhibit filamentous growth. Epistasis analysis indicates that PKA signals downstream of the Gpr1 receptor and G protein Gpa2. PKA and the MAP kinase cascades function independently to regulate budding pattern and cell elongation, respectively, during filamentous growth. In contrast, PKA (via Flo8) and the MAP kinase cascade (via Ste12 and Tec1) coordinately regulate the cell surface flocculin Flo11, agar invasion, and cell adhesion.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Our studies have analyzed the signal transduction pathways that regulate yeast S. cerevisiae pseudohyphal differentiation in response to nitrogen limitation. Our findings support a model in which the cAMP-dependent protein kinase (PKA) plays a central role in regulating filamentation (Fig. 10). First, mutation of the PKA regulatory subunit Bcy1 dramatically enhances filamentation and, in part, bypasses the requirement for nitrogen starvation. Second, we show that the three PKA catalytic subunits play distinct roles in which the Tpk2 catalytic subunit activates filamentous growth, whereas the Tpk1 and Tpk3 subunits inhibit filamentation. This specialization of function is correlated with the sequence of the subunits, in that Tpk1 and Tpk3 are the most closely related (88% identity), whereas Tpk2 is more divergent (75 and 77% identity to Tpk1 and Tpk3). The unique activating function of the Tpk2 subunit was mapped to the catalytic region and not to the unique amino-terminal region or the TPK2 gene promoter. Third, epistasis analysis is in accord with a model in which PKA functions downstream of the novel G protein-coupled receptor Gpr1 and the G proteins Gpa2 and Ras2. Fourth, activated PKA can drive filamentous growth in the absence of the Ste12 and Tec1 transcription factors that are components of the MAP kinase cascade that also regulates filamentous growth. We show that PKA regulates filamentous growth independently of Phd1 and several PKA targets, including the transcription factors Msn2 and Msn4, and the protein kinases Yak1 and Rim15. On the other hand, we define a role for PKA in regulating expression of the cell surface flocculin Flo11 by the transcription factor Flo8, both of which are known to be required for pseudohyphal growth (27, 29, 31).

In conclusion, as outlined in Fig. 10, our studies define a signal transduction cascade regulating the cAMP-dependent protein kinase that positively, (and negatively) regulates yeast pseudohyphal differentiation. While this report was in preparation, similar findings were reported with respect to TPK2 and TPK3 function in filamentous growth, suggesting that another target of PKA may be the transcription factor homolog SFL1, and defining Flo11 as a common target of the PKA and MAP kinase cascades (51, 52).

Relationship between the PKA and MAP kinase signaling cascades. Our studies also provide a perspective on the relationship between the MAP kinase and PKA signal transduction pathways that regulate yeast pseudohyphal growth. First, our studies demonstrate that activated PKA (as a result of a bcy1 mutation) can support filamentous growth in the complete absence of Ste12, Tec1, or both Ste12 and Tec1. These observations are in accord with previous findings that exogenous cAMP restores filamentation in ste20 and ste12 mutant strains and also represses expression of the Fg(TyA)::lacZ reporter gene (32). Taken together, these findings indicate that the function of the MAP kinase pathway can be largely bypassed by activated A kinase, suggesting the two signaling pathways can function independently.

PKA catalytic subunits have unique functions. Our studies reveal that the cAMP-dependent kinase has a novel role in regulating pseudohyphal differentiation in addition to the well-established roles of PKA in vegetative growth, meiosis, and stationary phase. One of the most interesting findings to emerge from our studies is that the three PKA catalytic subunits have unique functions. The more divergent Tpk2 subunit enhances filamentous growth, whereas the two more closely related subunits, Tpk1 and Tpk3, inhibit filamentous growth. In contrast, several previous studies found similar phenotypes in strains expressing any one of the three catalytic subunits (either Tpk1, Tpk2, or Tpk3), leading to the common view that the three PKA catalytic subunits are redundant for function (58). On the other hand, there were some clues that the three Tpk subunits might have distinguishing features. For example, mutant strains expressing only Tpk1 or Tpk3 can utilize acetate as a carbon source, whereas strains expressing only Tpk2 cannot (58). Our findings demonstrate that the three PKA catalytic subunits are specialized with respect to pseudohyphal differentiation to play both positive and negative signaling roles. This dual function may be analogous to the negative and positive signaling roles of the Kss1 MAP kinase in filamentous differentiation (8, 35).

cAMP and PKA signaling in yeasts and pathogenic fungi. cAMP and PKA also regulate mating, meiosis, and sporulation in the fission yeast Schizosaccharomyces pombe (42, 64). Like S. cerevisiae, S. pombe has two mating types that communicate with peptide pheromones. In S. pombe, a second signal, nitrogen starvation, is also required for mating. Two pathways regulate mating in S. pombe. The first is a pheromone-activated MAP kinase cascade analogous to the MAP kinase cascade for mating in budding yeast. The second is a nutrient-sensing pathway involving the G protein gpa2, which is a homolog of the S. cerevisiae G protein Gpa2 that regulates filamentous growth (22). In S. pombe, gpa2 activates adenylyl cyclase in response to nutrients, raising cAMP levels and thereby inhibiting mating.

Conclusions. In summary, studies of the yeasts S. cerevisiae and S. pombe, the plant fungal pathogen U. maydis, and the human fungal pathogen C. neoformans have converged to define a novel signaling cascade involving the G protein-coupled receptor Gpr1, a highly conserved G protein, adenylyl cyclase, cAMP, and the cAMP-dependent protein kinase which regulates mating, differentiation, and virulence. The targets of these PKA-regulated pathways remain to be defined, but promise to further our understanding of the conserved signaling pathways regulating differentiation and virulence of diverse yeasts and pathogenic fungi.