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. 1999 Jul;19(7):4874-87.
doi: 10.1128/MCB.19.7.4874.

Cyclic AMP-dependent protein kinase regulates pseudohyphal differentiation in Saccharomyces cerevisiae

X Pan  1 , J Heitman
Affiliations

Cyclic AMP-dependent protein kinase regulates pseudohyphal differentiation in Saccharomyces cerevisiae

X Pan et al. Mol Cell Biol. 1999 Jul.

Abstract

In response to nitrogen starvation, diploid cells of the yeast Saccharomyces cerevisiae differentiate to a filamentous growth form known as pseudohyphal differentiation. Filamentous growth is regulated by elements of the pheromone mitogen-activated protein (MAP) kinase cascade and a second signaling cascade involving the receptor Gpr1, the Galpha protein Gpa2, Ras2, and cyclic AMP (cAMP). We show here that the Gpr1-Gpa2-cAMP pathway signals via the cAMP-dependent protein kinase, protein kinase A (PKA), to regulate pseudohyphal differentiation. Activation of PKA by mutation of the regulatory subunit Bcy1 enhances filamentous growth. Mutation and overexpression of the PKA catalytic subunits reveal that the Tpk2 catalytic subunit activates filamentous growth, whereas the Tpk1 and Tpk3 catalytic subunits inhibit filamentous growth. The PKA pathway regulates unipolar budding and agar invasion, whereas the MAP kinase cascade regulates cell elongation and invasion. Epistasis analysis supports a model in which PKA functions downstream of the Gpr1 receptor and the Gpa2 and Ras2 G proteins. Activation of filamentous growth by PKA does not require the transcription factors Ste12 and Tec1 of the MAP kinase cascade, Phd1, or the PKA targets Msn2 and Msn4. PKA signals pseudohyphal growth, in part, by regulating Flo8-dependent expression of the cell surface flocculin Flo11. In summary, the cAMP-dependent protein kinase plays an intimate positive and negative role in regulating filamentous growth, and these findings may provide insight into the roles of PKA in mating, morphogenesis, and virulence in other yeasts and pathogenic fungi.

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Figures

FIG. 1
FIG. 1
Deletion of the PKA regulatory subunit BCY1 enhances filamentous growth. Homozygous wild-type (MLY61a/α) and Δbcy1/Δbcy1 (XPY1a/α) 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.
FIG. 2
FIG. 2
The tpk2 mutation reduces pseudohyphal growth, whereas tpk1 and tpk3 mutations enhance filamentous growth. Homozygous wild-type (MLY61a/α), Δtpk1/Δtpk1 (XYP4a/α), Δtpk2/Δtpk2 (XPY5a/α), and Δtpk3/Δtpk3 (XPY6a/α) mutant strains were incubated on SLAD medium with 50 or 200 μM ammonium sulfate, as indicated, and incubated for 3 days at 30°C.
FIG. 3
FIG. 3
Epistasis analysis of tpk and bcy1 mutations. (A) The Δtpk2 mutation is epistatic to the Δtpk1 and Δtpk3 mutations. Isogenic wild-type (MLY61a/α) and Δtpk1/Δtpk1 Δtpk2/Δtpk2 (XPY12a/α), Δtpk2/Δtpk2 Δtpk3/Δtpk3 (XPY13a/α), and Δtpk1/Δtpk1 Δtpk3/Δtpk3 (XPY14a/α) mutant diploid strains were incubated on SLAD medium for 3 days at 30°C. (B) The Δbcy1 mutation suppresses the filamentation defect conferred by the tpk2 mutation. Isogenic wild-type (MLY61a/α) and Δbcy1/Δbcy1 (XPY12a/α), Δtpk2/Δtpk2 (XPY5a/α), and Δbcy1/Δbcy1 Δtpk2/Δtpk2 (XPY59a/α) mutant diploid strains were grown on SLAD medium for 3 days at 30°C.
FIG. 4
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/α and assayed for filamentous growth following incubation at 30°C for 3 days on SLAD medium.
FIG. 5
FIG. 5
PKA pathway regulates unipolar budding, while the MAP kinase pathway is required for cell elongation. (A) Isogenic wild-type (MLY61a/α), Δtpk2/Δtpk2 (XPY5a/α), and Δste12/Δste12 (MLY216a/α) 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.
FIG. 6
FIG. 6
Tpk2 acts downstream of Gpa2 and in part downstream of Ras2. (A) The Δtpk2 mutation is epistatic to the activated GPA2Val132 allele. Wild-type (MLY61a/α) and Δtpk2/Δtpk2 (XPY5a/α) 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/α) and Δtpk2/Δtpk2 (XPY5a/α) 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.
FIG. 7
FIG. 7
Activated PKA restores filamentous growth in MAP kinase cascade mutants. Isogenic wild-type (MLY61a/α) and Δste12/Δste12 (MLY216a/α), Δtec1/Δtec1 (MLY183a/α), Δste12/Δste12 Δtec1/Δtec1 (XPY77a/α), Δbcy1/Δbcy1 (XPY1a/α), Δbcy1/Δbcy1 Δste12/Δste12 (XPY69a/α), Δbcy1/Δbcy1 Δtec1/Δtec1 (XPY75a/α), and Δbcy1/Δbcy1 Δste12/Δste12 Δtec1/Δtec1 (XPY76a/α) mutant diploid strains were incubated on SLAD medium at 30°C for 3 days.
FIG. 8
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/α) and Δphd1/Δphd1 (MLY182a/α), Δphd1/Δphd1 Δtec1/Δtec1 (XPY89a/α), Δbcy1/Δbcy1 (XPY1a/α), Δbcy1/Δbcy1 Δphd1/Δphd1 (XPY78a/α), and Δbcy1/Δbcy1 Δphd1/Δphd1 Δtec1/Δtec1 (XPY88a/α) 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/α) and Δtpk2/Δtpk2 mutant (XPY5a/α) diploid strains containing a control plasmid (vector) or the PHD1 overexpression plasmid (pCG68) were incubated on SLAD medium at 30°C for 3 days.
FIG. 9
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/α) and Δflo8/Δflo8 (XPY95a/α), Δflo11/Δflo11 (XPY107a/α), Δbcy1/Δbcy1 (XPY1a/α), Δbcy1/Δbcy1 Δflo8/Δflo8 (XPY99a/α), and Δbcy1/Δbcy1 Δflo11/Δflo11 (XPY119a/α) mutant strains containing a control plasmid and wild-type and Δflo8/Δflo8 and Δflo11/Δflo11 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) (MLY61α) and Δtpk2 (XPY5α), Δflo8 (XPY95α), Δflo11 (XPY107α), Δtec1 (MLY183α), Δbcy1 (XPY1α), Δbcy1 Δflo8 (XPY99α), and Δbcy1 Δtec1 (XPY75α) mutant strains and wild-type and Δflo8 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.
FIG. 10
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 Gα 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.
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