Yeast pseudohyphal growth is a stress response characterized by elongated cell morphology, exaggerated polarized growth, and increased cell-cell adhesion. The signaling network that regulates the formation of pseudohyphal filaments has been the subject of intense research interest, as filament formation is required for virulence in numerous pathogenic fungi. Pseudohyphal growth is regulated through highly conserved kinase pathways, encompassing MAPK/ERK, PKA, and AMPK signaling modules; however, the full scope of these pathways has not been elucidated fully. To address this knowledge gap, we used quantitative phosphoproteomics in Saccharomyces cerevisiae to identify differentially phosphorylated proteins in kinase deficient mutant strains surveyed under conditions inducing pseudohyphal growth. By SILAC labeling and LC-MS/MS analysis of phosphopeptide-enriched samples, we identified 439 phosphoproteins and 539 novel phosphorylated residues dependent on a pseudohyphal growth kinase. These data sets yielded two interesting results. First, the identified phosphoprotein set was significantly enriched for ribonucleoprotein (RNP) granule components, including P-bodies and stress granules. Through fluorescence microscopy of GFP-chimeras, we observe co-localization of the MAPK cascade proteins Kss1p, Ste20p, Fus3p, and also PKA (Tpk2p) with the RNP component Igo1p. Furthermore, I find that Kss1p kinase activity is required for wild type levels of mRNA localization in P-bodies. Second, our phosphoproteomic data set indicates that a statistically significant set of kinases mediating inositol polyphosphate (IP) signaling undergoes pseudohyphal growth kinase-dependent phosphorylation. We find that deletion of kinases in the IP synthesis pathway results in aberrant pseudohyphal growth, and through metabolic labeling of IP species, I observe striking changes in IP levels under pseudohyphal growth conditions. In particular, we saw a correlation between increased filamentous growth and the presence of high levels of the inositol pyrophosphate produce by Kcs1p, 5PP-IP5. Additionally, I observe interesting changes in IP levels upon the deletion of key kinases involved in the pseudohyphal growth transition. With the relevance of yeast pseudohyphal growth as a model of filamentous development in the pathogen Candida albicans, my doctoral studies have also investigated the role of the C. albicans cell wall integrity kinase Cbk1p in morphogenesis during hyphal development. In collaborative studies with Damian Krysan’s group at the University of Rochester, my work indicates a mechanism where CaCbk1p regulates cross-talk between the RAM and PKA pathways. Taken together, my doctoral research provides insight into the mechanisms through which conserved kinase signaling networks regulate filamentous growth, with particular relevance in understanding the mechanisms controlling RNP dynamics and regulated IP biogenesis.

Mentor: Anuj Kumar, Professor