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Noncanonical G? Gib2 is a scaffolding protein promoting cAMP signaling through functions of Ras1 and Cac1 proteins in Cryptococcus neoformans.

ABSTRACT: G?-like/RACK1 functions as a key mediator of various pathways and contributes to numerous cellular functions in eukaryotic organisms. In the pathogenic fungus Cryptococcus neoformans, noncanonical G? Gib2 promotes cAMP signaling in cells lacking normal Gpa1 function while displaying versatility in interactions with G? Gpa1, protein kinase Pkc1, and endocytic intersectin Cin1. To elucidate the Gib2 functional mechanism(s), we demonstrate that Gib2 is required for normal growth and virulence. We show that Gib2 directly binds to Gpa1 and G? Gpg1/Gpg2 and that it interacts with phosphodiesterase Pde2 and monomeric GTPase Ras1. Pde2 remains functionally dispensable, but Ras1 is found to associate with adenylyl cyclase Cac1 through the conserved Ras association domain. In addition, the ras1 mutant exhibits normal capsule formation, whereas the ras1 gpa1 mutant displays enhanced capsule formation, and the ras1 gpa1 cac1 mutant is acapsular. Collectively, these findings suggest that Gib2 promotes cAMP levels by relieving an inhibitory function of Ras1 on Cac1 in the absence of Gpa1. In addition, using GST affinity purification combined with mass spectrometry, we identified 47 additional proteins that interact with Gib2. These proteins have putative functions ranging from signal transduction, energy generation, metabolism, and stress response to ribosomal function. After establishing and validating a protein-protein interactive network, we believe Gib2 to be a key adaptor/scaffolding protein that drives the formation of various protein complexes required for growth and virulence. Our study reveals Gib2 as an essential component in deciphering the complexity of regulatory networks that control growth and virulence in C. neoformans.

SUBMITTER: Wang Y

PROVIDER: S-EPMC4007421 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Noncanonical Gβ Gib2 is a scaffolding protein promoting cAMP signaling through functions of Ras1 and Cac1 proteins in Cryptococcus neoformans.

Wang Yanli Y Shen Gui G Gong Jinjun J Shen Danyu D Whittington Amy A Qing Jiang J Treloar Joshua J Boisvert Scott S Zhang Zhengguang Z Yang Cai C Wang Ping P

The Journal of biological chemistry 20140321 18

Gβ-like/RACK1 functions as a key mediator of various pathways and contributes to numerous cellular functions in eukaryotic organisms. In the pathogenic fungus Cryptococcus neoformans, noncanonical Gβ Gib2 promotes cAMP signaling in cells lacking normal Gpa1 function while displaying versatility in interactions with Gα Gpa1, protein kinase Pkc1, and endocytic intersectin Cin1. To elucidate the Gib2 functional mechanism(s), we demonstrate that Gib2 is required for normal growth and virulence. We s ...[more]

PMID: 24659785

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Project description:Cryptococcus neoformans encounters a low oxygen environment when it enters the human host. Here, we show that the conserved Ras1 (a small GTPase) and Cdc24 (the guanine nucleotide exchange factor for Cdc42) play an essential role in cryptococcal growth in hypoxia. Suppressor studies indicate that PTP3 functions epistatically downstream of both RAS1 and CDC24 in regulating hypoxic growth. Ptp3 shares sequence similarity to the family of phosphotyrosine-specific protein phosphatases and the ptp3Δ strain failed to grow in 1% O2. We demonstrate that RAS1, CDC24 and PTP3 function in parallel to regulate thermal tolerance but RAS1 and CDC24 function linearly in regulating hypoxic growth while CDC24 and PTP3 reside in compensatory pathways. The ras1Δ and cdc24Δ strains ceased to grow at 1% O2 and became enlarged but viable single cells. Actin polarization in these cells, however, was normal for up to eight hours after transferring to hypoxic conditions. Double deletions of the genes encoding Rho GTPase Cdc42 and Cdc420, but not of the genes encoding Rac1 and Rac2, caused a slight growth retardation in hypoxia. Furthermore, growth in hypoxia was not affected by the deletion of several central genes functioning in the pathways of cAMP, Hog1, or the two-component like phosphorylation system that are critical in the cryptococcal response to osmotic and genotoxic stresses. Interestingly, although deletion of HOG1 rescued the hypoxic growth defect of ras1Δ, cdc24Δ, and ptp3Δ, Hog1 was not hyperphosphorylated in these three mutants in hypoxic conditions. RNA sequencing analysis indicated that RAS1, CDC24 and PTP3 acted upon the expression of genes involved in ergosterol biosynthesis, chromosome organization, RNA processing and protein translation. Moreover, growth of the wild-type strain under low oxygen conditions was affected by sub-inhibitory concentrations of the compounds that inhibit these biological processes, demonstrating the importance of these biological processes in the cryptococcal hypoxia response.

Dataset Information

Noncanonical G? Gib2 is a scaffolding protein promoting cAMP signaling through functions of Ras1 and Cac1 proteins in Cryptococcus neoformans.

Publications

Noncanonical Gβ Gib2 is a scaffolding protein promoting cAMP signaling through functions of Ras1 and Cac1 proteins in Cryptococcus neoformans.

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