Gib2, A Novel Gβ-like/RACK1 Homolog, Functions as a Gβ Subunit in cAMP Signaling and Is Essential in Cryptococcus neoformans

Daniel A Palmer,Jill K Thompson,Lie Li,Ashton Prat,Ping Wang

doi:10.1074/jbc.m602768200

Abstract

Canonical G proteins are heterotrimeric, consisting of alpha, beta, and gamma subunits. Despite multiple Galpha subunits functioning in fungi, only a single Gbeta subunit per species has been identified, suggesting that non-conventional G protein signaling exists in this diverse group of eukaryotic organisms. Using the Galpha subunit Gpa1 that functions in cAMP signaling as bait in a two-hybrid screen, we have identified a novel Gbeta-like/RACK1 protein homolog, Gib2, from the human pathogenic fungus Cryptococcus neoformans. Gib2 contains a seven WD-40 repeat motif and is predicted to form a seven-bladed beta propeller structure characteristic of beta transducins. Gib2 is also shown to interact, respectively, with two Ggamma subunit homologs, Gpg1 and Gpg2, similar to the conventional Gbeta subunit Gpb1. In contrast to Gpb1 whose overexpression promotes mating response, overproduction of Gib2 suppresses defects of gpa1 mutation in both melanization and capsule formation, the phenotypes regulated by cAMP signaling and associated with virulence. Furthermore, depletion of Gib2 by antisense suppression results in a severe growth defect, suggesting that Gib2 is essential. Finally, Gib2 is shown to also physically interact with a downstream target of Gpa1-cAMP signaling, Smg1, and the protein kinase C homolog Pkc1, indicating that Gib2 is also a multifunctional RACK1-like protein.

Highlights

G␣s [4, 5]
RACK1 was first found to function as a scaffold protein localizing the activated protein kinase C (PKC) to the insoluble cell fraction (14 –16) and the RACK1 protein interacts with many signal proteins, including the Src tyrosine kinase [17, 18], integrin ␤ subunit [19], phosphodiesterase Pde4D5 [20], and G protein heterotrimeric ␣(t)␤␥ and heterodimeric ␤␥ subunits [21,22,23]
We have recently identified a regulator of the G protein signaling protein, Crg1, as one of the key regulators for mating and virulence in C. neoformans [51, 52] and have shown that Crg1 functions as a GTPase activating protein specific to Gpa2.4 To expand our studies on regulators of G protein signaling, we performed a yeast two-hybrid screen using Gpa1 as bait

Summary

EXPERIMENTAL PROCEDURES

Strains and Media—C. neoformans var. grubii (serotype A) MAT␣ strains H99 and F99 (H99 ura5), MATa strains KN99a and F99a (KN99a ura5), var. neoformans (serotype D), MAT␣ strains JEC21 and JEC43 (JEC21 ura5), MATa strains JEC20, JEC34 (JEC20 ura5), BAC20-1 (gpa ura5), and a laboratory derived diploid strain, RAS009 (a/␣ ade2/ϩ ura5/ura lys1/ϩ lys2/ϩ), were from the laboratory of J. Two-hybrid Interaction—For the yeast two-hybrid screen, GPA1 cDNA was synthesized using primers PW99 (5Ј-AAGGAATTCATGGGCGGCTGTATGTCT-3Ј) and PW100 (5Ј-GAACTGCAGCCTTATAAGATACCAGAGTC-3Ј) and cloned into pGBKT7 (Clontech) at the EcoRI-PstI sites to create pGBKT7-GPA1, expressing the fusion protein GAL4 (BD)Gpa. Overexpression and Suppression of Gib2—The GIB2 gene was inserted into the pCnTel vector containing the C. neoformans galactose inducible GAL7 promoter [48] at the EcoRI site, and the resulting construct (sense orientation) was introduced into JEC43, JEC34, and BAC20-1 (gpa uraϪ) strains by biolistic transformation. The pulldown assay between the GST-Gib fusion protein and C. neoformans Pkc was carried out in buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM Na3VO4, 1 mM NaF, and the protease inhibitor mixture and 0.5 mM phenylmethylsulfonyl fluoride, with or without the PKC activator 200 mM 1-oleoyl2-acetyl-sn-glycerol. Yeast cells carrying both genes were able to grow in SD medium lacking Leu, Trp, His, and Ade, plus 3-aminotrizole that was added to suppress leaky HIS3 expres-

RESULTS

Findings

DISCUSSION