[Show abstract][Hide abstract] ABSTRACT: A long-standing question in the field of signal transduction is to understand the cross-talk between receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major and distinct signaling hubs that control eukaryotic cell behavior. Although stimulation of many RTKs leads to activation of trimeric G proteins, the molecular mechanisms behind this phenomenon remain elusive. We have discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins. Using a combination of homology modeling, protein-protein interaction and kinase assays we demonstrate that a stretch of ∼110 aa within GIV-C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands. Using protein-protein interaction assays we demonstrated that both SH2 and GEF domains of GIV are required for the formation of a ligand-activated ternary complex between GIV, Gαi and growth factor receptors, and for activation of Gαi after growth factor stimulation. Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV, i.e., Akt enhancement, actin remodeling, and cell migration. The mechanistic and structural insights gained herein sheds light on the long standing questions surrounding RTK/G protein cross-talk, sets a novel paradigm, and characterizes a unique pharmacological target for uncoupling GIV-dependent signaling downstream of multiple oncogenic RTKs.
Molecular Biology of the Cell 09/2014; · 4.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: GIV (Gα-interacting vesicle-associated protein; also known as Girdin) enhances Akt activation downstream of multiple growth factor- and G protein (heterotrimeric guanosine 5'-triphosphate-binding protein)-coupled receptors to trigger cell migration and cancer invasion. We demonstrate that GIV is a tyrosine phosphoprotein that directly binds to and activates phosphoinositide 3-kinase (PI3K). Upon ligand stimulation of various receptors, GIV was phosphorylated at tyrosine-1764 and tyrosine-1798 by both receptor and non-receptor tyrosine kinases. These phosphorylation events enabled direct binding of GIV to the amino- and carboxyl-terminal Src homology 2 domains of p85α, a regulatory subunit of PI3K; stabilized receptor association with PI3K; and enhanced PI3K activity at the plasma membrane to trigger cell migration. Tyrosine phosphorylation of GIV and its association with p85α increased during metastatic progression of a breast carcinoma. These results suggest a mechanism by which multiple receptors activate PI3K through tyrosine phosphorylation of GIV, thereby making the GIV-PI3K interaction a potential therapeutic target within the PI3K-Akt pathway.
[Show abstract][Hide abstract] ABSTRACT: Autophagy is the major catabolic process responsible for the removal of aggregated proteins and damaged organelles. Autophagy is regulated by both G proteins and growth factors, but the underlying mechanism of how they are coordinated during initiation and reversal of autophagy is unknown. Using protein-protein interaction assays, G protein enzymology, and morphological analysis, we demonstrate here that Gα-interacting, vesicle-associated protein (GIV, a. k. a. Girdin), a nonreceptor guanine nucleotide exchange factor for Gα(i3), plays a key role in regulating autophagy and that dynamic interplay between Gα(i3), activator of G-protein signaling 3 (AGS3, its guanine nucleotide dissociation inhibitor), and GIV determines whether autophagy is promoted or inhibited. We found that AGS3 directly binds light chain 3 (LC3), recruits Gα(i3) to LC3-positive membranes upon starvation, and promotes autophagy by inhibiting the G protein. Upon growth factor stimulation, GIV disrupts the Gα(i3)-AGS3 complex, releases Gα(i3) from LC3-positive membranes, enhances anti-autophagic signaling pathways, and inhibits autophagy by activating the G protein. These results provide mechanistic insights into how reversible modulation of Gα(i3) activity by AGS3 and GIV maintains the delicate equilibrium between promotion and inhibition of autophagy.
Molecular biology of the cell 02/2011; 22(5):673-86. · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metastasis accounts for the majority of cancer-related deaths. Accurate prediction of metastatic potential of tumors has been elusive, and the search for clinically useful markers continues. We previously reported that GIV/Girdin triggers tumor cell migration by virtue of a C-terminal guanine-nucleotide exchange factor motif that activates Gαi. Here we identify GIV as a metastasis-related protein whose full-length transcript (GIV-fl) is expressed exclusively in highly invasive colon, breast, and pancreatic carcinoma cells and not in their poorly invasive counterparts. A prospective, exploratory biomarker study conducted on a cohort of 56 patients with stage II colorectal cancer revealed a significant correlation between GIV-fl expression in tumor epithelium and shortened metastasis-free survival. Survival rate for patients with GIV-fl-positive tumors is significantly reduced compared with the patients with GIV-fl-negative tumors [P<0.0001; hazard ratio=0.076; CI=0.052-0.30 (95%)]. At the 5-yr mark, survival is 100% in the GIV-fl-negative group and 62 ± 9% (mean±SE; P=6×10(-5)) in the GIV-fl-positive group. Furthermore, GIV-fl expression predicts a risk of mortality independent of the microsatellite stability status, a well-established prognosticator of colorectal cancers. We conclude that GIV-fl is a novel metastasis-related protein and an independent adverse prognosticator that may serve as a useful adjunct to traditional staging strategies in colorectal carcinoma.
The FASEB Journal 10/2010; 25(2):590-9. · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cells respond to growth factors by either migrating or proliferating, but not both at the same time, a phenomenon termed migration-proliferation dichotomy. The underlying mechanism of this phenomenon has remained unknown. We demonstrate here that Galpha(i) protein and GIV, its nonreceptor guanine nucleotide exchange factor (GEF), program EGF receptor (EGFR) signaling and orchestrate this dichotomy. GIV directly interacts with EGFR, and when its GEF function is intact, a Galpha(i)-GIV-EGFR signaling complex assembles, EGFR autophosphorylation is enhanced, and the receptor's association with the plasma membrane (PM) is prolonged. Accordingly, PM-based motogenic signals (PI3-kinase-Akt and PLCgamma1) are amplified, and cell migration is triggered. In cells expressing a GEF-deficient mutant, the Galphai-GIV-EGFR signaling complex is not assembled, EGFR autophosphorylation is reduced, the receptor's association with endosomes is prolonged, mitogenic signals (ERK 1/2, Src, and STAT5) are amplified, and cell proliferation is triggered. In rapidly growing, poorly motile breast and colon cancer cells and in noninvasive colorectal carcinomas in situ in which EGFR signaling favors mitosis over motility, a GEF-deficient splice variant of GIV was identified. In slow growing, highly motile cancer cells and late invasive carcinomas, GIV is highly expressed and has an intact GEF motif. Thus, inclusion or exclusion of GIV's GEF motif, which activates Galphai, modulates EGFR signaling, generates migration-proliferation dichotomy, and most likely influences cancer progression.
Molecular biology of the cell 05/2010; 21(13):2338-54. · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although several non-receptor activators of heterotrimeric G proteins have been identified, the structural features of G proteins that determine their interaction with such activators and the subsequent biological effects are poorly understood. Here we investigated the structural determinants in G alpha(i3) necessary for its regulation by GIV/girdin, a guanine-nucleotide exchange factor (GEF) that activates G alpha(i) subunits. Using G protein activity and in vitro pulldown assays we demonstrate that G alpha(i3) is a better substrate for GIV than the highly homologous G alpha(o). We identified Trp-258 in the G alpha(i) subunit as a novel structural determinant for GIV binding by comparing GIV binding to G alpha(i3)/G alpha(o) chimeras. Mutation of Trp-258 to the corresponding Phe in G alpha(o) decreased GIV binding in vitro and in cultured cells but did not perturb interaction with other G alpha-binding partners, i.e. G betagamma, AGS3 (a guanine nucleotide dissociation inhibitor), GAIP/RGS19 (a GTPase-activating protein), and LPAR1 (a G protein-coupled receptor). Activation of G alpha(i3) by GIV was also dramatically reduced when Trp-258 was replaced with Tyr, Leu, Ser, His, Asp, or Ala, highlighting that Trp is required for maximal activation. Moreover, when mutant G alpha(i3) W258F was expressed in HeLa cells they failed to undergo cell migration and to enhance Akt signaling after growth factor or G protein-coupled receptor stimulation. Thus activation of G alpha(i3) by GIV is essential for biological functions associated with G alpha(i3) activation. In conclusion, we have discovered a novel structural determinant on G alpha(i) that plays a key role in defining the selectivity and efficiency of the GEF activity of GIV on G alpha(i) and that represents an attractive target site for designing small molecules to disrupt the G alpha(i)-GIV interface for therapeutic purposes.
Journal of Biological Chemistry 02/2010; 285(17):12765-77. · 4.60 Impact Factor