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

Abstract

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 show that Gib2 directly binds to Gpa1 and Gγ 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.

Keywords: Adenylate Cyclase (Adenylyl Cyclase); Cyclic AMP (cAMP); Fungal Growth and Virulence; G Proteins; Gβ-like/RACK1; Protein Interactive Network; Ras; Signal Transduction.

FIGURE 1.

Gib2 shares high homology with human Gβ-like/RACK1, parasitic L. major LACK1, and S. cerevisiae Asc1 proteins and exists as monomer and dimer. A, multiple sequence alignment was made using ClustalW (v1.83). B, Gib2 is highly homologous to RACK1/LACK1/Asc1 proteins sharing, respectively, 84, 69, and 70% in amino acid sequence similarities. C, Western blotting showing that Gib2 is capable of forming dimeric complexes under normal physiological conditions. hr, time in yeast nitrogen base minimal medium after switching from yeast extract-peptone-dextrose.

FIGURE 2.

Gib2 is involved in growth and pathogenicity of C. neoformans. A, Gib2 is not directly involved in melanin and capsule formation. The upper panel shows the gib2::NAT mutant (var. grubii) and the wild-type strain H99 grown on Niger seed medium agar for 2 days at 30 °C. The lower panel shows cells recovered from mouse brain tissues exhibiting normal sized capsules following India ink staining. B, the growth of the gib2 mutant is reduced at 37 °C in comparison with H99. Partial complementation is achieved in the complemented mutant strain (gib2 GIB2). C, Gib2 has no apparent direct roles in cAMP levels in a transient cAMP assay using the Amersham Biosciences cAMP enzyme immunoassay system (see text). Each cAMP level represents the value estimated from 1 × 10⁶ cells. D, Gib2 is required for full virulence expression. Ten A/JCr mice were infected with the gib2 mutant, H99, and the complemented (gib2 GIB2) strains, respectively. The difference in survival between gib2 and H99 and between gib2 and the complemented strain was statistically significant (p < 0.05), whereas there was no statistically significant difference between H99 and the complemented strain. A, serotype A; D, serotype D.

FIGURE 3.

Gib2 directly interacts with Gpa1 and Gpg2 subunits and with Pde2 through the RAID. A, Gib2 interacts with Gpa1, Gpg1, and Gpg2 in the absence of yeast Ste4. B, schematic representations of C. neoformans PDEs Pde1 and Pde2. Pde2 is 1214 amino acids (aa) long and, similar to Pde4D5, contains a putative RAID between amino acids 280 and 350 (marked by an arrow and enlarged below). Darkly shaded areas indicate identical amino acid residues, and lightly shaded areas show conserved residues. PDEase, phosphodiesterase. C, Gib2 interacts with Pde2 through the putative RAID in a yeast two-hybrid assay. DD indicates dropout medium lacking leucine and tryptophan, and QD indicates dropout medium lacking leucine, tryptophan, histidine, and adenine. 3AT, 3-amino-1,2,4-triazole. 1 mm was used. AD, pGADT7. BD, pGBKT7.

FIGURE 4.

Pde1, but not Pde2, is required for regulation of cAMP levels. A and B, impacts of C. neoformans var. neoformans PDE1 and PDE2 gene disruptions on melanin and capsule formation (cAMP signaling). Cells were spotted on Niger seed agar for melanin and inoculated in DMEM for capsule induction for 3 days at 30 °C, respectively. C, cAMP assay suggests that Pde1, but Pde2, affects cAMP accumulation. Each cAMP level represents the value estimated from 1 × 10⁶ cells.

FIGURE 5.

Ras1 interacts with Cac1 and Gib2. A, C. neoformans var. neoformans (serotype D) (C. n. (D)), var. grubii Cac1 (serotype A) (C. n. (A)), and C. gattii (C. g.) contain putative RA domains that are homologous to those of S. cerevisiae (S. c.) and C. albicans (C. a.). Numbers mark the amino acid locations of the RA domains. B, Ras1 interacts with Cac1 through the conserved RA domain in a yeast two-hybrid assay. C, Ras1 and paralog Ras2 interact with Gib2. D, co-immunoprecipitation confirms the interactions between Gib2 and Ras1, Ras1 and Cac1 (RA domain), and Gib2 and eIF4A. Anti-GST and Anti-Xpress antibodies were used to detect the respective fusion proteins following PAGE and Western blot analysis. E, Gib2 interacts with eukaryotic initiation factor eIF4A homolog. The Y2H assay conditions and media used were the same as that described in Fig. 3. DD indicates dropout medium lacking leucine and tryptophan, and QD indicates dropout medium lacking leucine, tryptophan, histidine, and adenine. co-IP was performed following the previously published method (17). AD, pGADT7; BD, pGBKT7.

FIGURE 6.

Genetic evidence suggests that Gib2 promotes cAMP levels through the Ras1-Cac1 interaction. A, Ras1 negatively regulates melanin formation, whereas Cac1 is required for melanin formation (cAMP signaling) in all of the strains including F4 (gpa1 GAL7-GIB2). B, Ras1 negatively regulates capsule formation in the absence of normal Gpa1 signaling. Induction conditions for melanin and capsule were as the same as those described in Fig. 2.

FIGURE 7.

Ras1 regulates the Cac1-cAMP signaling pathway in C. neoformans. The Ras1 function opposes that of Gpr4/Gpa1. Gpa1, G protein α subunit (8, 62); Gpg1/2, G protein γ subunits 1 and 2 (11); Crg2, cryptococcal regulator of G protein signaling 2 (39, 63); Pkr1 and Pka1, cAMP-dependent protein kinase regulatory and catalytic subunit, respectively (14). TFs denote Nrg1 (60) and other unknown transcriptional factors.

FIGURE 8.

A Gib2 interactive network demonstrates that Gib2 functions as a signal-transducing adaptor protein interconnecting many pathways in C. neoformans. Respective S. cerevisiae proteins homologous to cryptococcal proteins (E-value <1e−5) we found were first identified in the genome of S. cerevisiae (Saccharomyces Genome Database). The S. cerevisiae proteins were then compiled using the STRING database v9.0 for protein-protein interactions using a STRING confidence score over 0.7 to generate an Asc1-protein interactive network (see text). Two interactive networks were generated accordingly: one for C. neoformans Gib2 (A) and another for S. cerevisiae Asc1 (B). Thick lines in A indicate that the connections were experimentally established by us, whereas thin lines indicate the connections derived from those of S. cerevisiae.