Protein kinase A operates a molecular switch that governs yeast pseudohyphal differentiation.

Cells possess a network of signal transduction pathways that enable them to sense the environment and respond to different stimuli. Signals from distinct pathways need to be coordinated so that cells can survive, proliferate, and differentiate properly under particular conditions. Signal integration occurs at several levels of signal transduction, including transcriptional control of gene expression, translational regulation, and posttranslational modifications. One example of such integration is the regulation of interleukin-2 gene expression during T-cell activation, in which both the calcineurin and protein kinase C pathways function. The transcription factors NF-AT and AP-1 are controlled by the calcineurin and protein kinase C pathways and bind to a composite site on the interleukin-2 promoter to activate gene expression (9). The coordinately controlled assembly of transcriptional regulators on the interleukin-2 gene promoter ensures expression when both pathways are engaged. In this regard, studies of FLO11 expression in Saccharomyces cerevisiae similarly serve as a paradigm to understand how combinatorial control of gene expression by multiple signals effects a complex physiological process like pseudohyphal differentiation. Pseudohyphal differentiation in diploid cells of the yeast S. cerevisiae occurs in response to nitrogen limitation and the presence of fermentable carbon sources (14, 28). Both a mitogen-activated protein (MAP) kinase pathway and the cyclic AMP (cAMP)-dependent protein kinase A cascade are required for pseudohyphal differentiation (for reviews, see references 13 and 37), and both pathways converge to control expression of the cell wall flocculin Flo11 (35, 39). Flo11 is a glycerol phosphoinositol-anchored cell surface protein that promotes mother-daughter cell adhesion and allows cells to bind to and penetrate growth substrates (21, 23). Flo11 is required for both diploid pseudohyphal differentiation and haploid invasive growth (23, 28, 29, 35, 38, 39), and protein kinase A plays critical roles in both processes. In S. cerevisiae, protein kinase A comprises a single regulatory subunit, Bcy1, and three catalytic subunits, Tpk1, Tpk2, and Tpk3. The Tpk2 catalytic subunit plays a unique positive role and activates pseudohyphal differentiation, whereas the more distantly related Tpk1 and Tpk3 subunits play negative roles, inhibiting filamentous growth (35, 38). Previous genetic studies suggested that Tpk2 activates FLO11 expression by activating Flo8, inhibiting Sfl1, or both (35, 38, 39). Flo8 is a transcriptional activator, and flo8 mutations abolish FLO11 expression and pseudohyphal growth and account for the inability of the common laboratory strain S288C to undergo filamentous growth (22, 35, 39). flo8 mutations block the effects of activated protein kinase A signaling but not activated MAP kinase on FLO11 expression and pseudohyphal differentiation (35, 39). This result suggests that Flo8 acts downstream of the protein kinase A pathway to promote FLO11 expression and filamentous growth. Sfl1 was originally identified as a negative regulator of flocculation in yeast cells (12). The N-terminal region of the Sfl1 protein shows extensive similarity to the DNA-binding domains of the yeast heat shock transcription factor Hsf1 (12) and several other yeast transcription factors (Mga1, Hms2, and Skn7) that enhance pseudohyphal differentiation when overexpressed (25). Sfl1 functions with the Srb/mediator complex of RNA polymerase II holoenzyme to repress gene expression (43). sfl1 mutations enhance FLO11 expression and pseudohyphal growth and restore filamentous growth in tpk2 mutants (38). These results suggest that Sfl1 acts downstream of the protein kinase A pathway and that Tpk2 inactivates Sfl1 to stimulate FLO11 expression and filamentous growth. In this study, we have elucidated the mechanisms by which Flo8 and Sfl1 control FLO11 expression and how Tpk2 controls both transcription factors. We report that Flo8 and Sfl1 are the direct targets of Tpk2 that control FLO11 expression and pseudohyphal growth. Both Flo8 and Sfl1 interact with and are phosphorylated by Tpk2. Flo8 and Sfl1 are both localized to the nucleus, and mutation or activation of Tpk2 had no effect on this localization. The ability of Flo8 and Sfl1 to alter gene expression when targeted to heterologous promoters via fusion to the Gal4 or LexA DNA-binding domains was similarly unaffected by increased or decreased protein kinase A signaling. Flo8 and Sfl1 were both found to act on a 250-bp region of the FLO11 promoter, and both proteins bind to this DNA region in vivo and in vitro. Tpk2 phosphorylates Flo8 and activates its binding to the FLO11 promoter. In contrast, phosphorylation of Sfl1 by Tpk2 inhibits binding to the FLO11 promoter, in accord with a recent report (6). In summary, our studies illustrate how protein kinase A effects a key developmental switch by stimulating an activator and inhibiting a repressor. Similar mechanisms are likely to operate during signal integration and combinatorial transcriptional control of gene expression in other eukaryotic organisms.