Analysis of Kinase Signaling Pathways Regulating Filamentous Developmentin Saccharomyces cerevisiae and Candida albicans

[摘要] 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, quantitative phosphoproteomics was used in Saccharomyces cerevisiae to identify differentially phosphorylated proteins in kinase-deficient mutant strains surveyed under conditions inducing pseudohyphal growth. The use of stable isotope labeling of amino acids in cell culture (SILAC) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis of phosphopeptide-enriched samples, identified 439 phosphoproteins and 539 novel phosphorylated residues dependent on a pseudohyphal growth kinase. This data set 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, co-localization of the MAPK cascade proteins Kss1p, Ste20p, Fus3p, and also PKA (Tpk2p) with the RNP component Igo1p was observed.Furthermore, Kss1p kinase activity was required for wild-type levels of mRNA localization in P-bodies. Second, the phosphoproteomic data set indicated that a statistically significant set of kinases mediating inositol polyphosphate (IP) signaling undergoes pseudohyphal growth kinase-dependent phosphorylation. Deletion of kinases in the IP synthesis pathway resulted in aberrant pseudohyphal growth, and through metabolic labeling of IP species, striking changes in IP levels under pseudohyphal growth conditions were observed. In particular, a correlation between increased filamentous growth and the presence of high levels of the inositol pyrophosphate produced by Kcs1p, 5PP-IP5 were found. Additionally, interesting changes in IP levels upon the deletion of key kinases involved in the pseudohyphal growth transition were observed. With the relevance of yeast pseudohyphal growth as a model of filamentous development in the pathogen Candida albicans, these studies 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, this work indicated a mechanism where CaCbk1p regulates cross-talk between the RAM and PKA pathways. Taken together, the doctoral research presented here 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.

[发布机构] University of Michigan

[效力级别] inositol polyphosphate

[关键词] filamentous growth;inositol polyphosphate;inositol pyrophosphate;pseudohyphal growth;phosphoproteomic;ribonucleoprotein granules;Molecular;Cellular and Developmental Biology;Science;Molecular, Cellular, and Developmental Biology