Protein kinase C-theta (PKCθ) phosphorylates and inhibits the guanine exchange factor, GIV/Girdin

Abstract

Gα-interacting, vesicle-associated protein (GIV/Girdin) is a multidomain signal transducer that enhances PI3K-Akt signals downstream of both G-protein–coupled receptors and growth factor receptor tyrosine kinases during diverse biological processes and cancer metastasis. Mechanistically, GIV serves as a non-receptor guanine nucleotide exchange factor (GEF) that enhances PI3K signals by activating trimeric G proteins, Gαi1/2/3. Site-directed mutations in GIV’s GEF motif disrupt its ability to bind or activate Gi and abrogate PI3K-Akt signals; however, nothing is known about how GIV’s GEF function is regulated. Here we report that PKCθ, a novel protein kinase C, down-regulates GIV’s GEF function by phosphorylating Ser(S)1689 located within GIV’s GEF motif. We demonstrate that PKCθ specifically binds and phosphorylates GIV at S1689, and this phosphoevent abolishes GIV’s ability to bind and activate Gαi. HeLa cells stably expressing the phosphomimetic mutant of GIV, GIV-S1689→D, are phenotypically identical to those expressing the GEF-deficient F1685A mutant: Actin stress fibers are decreased and cell migration is inhibited whereas cell proliferation is triggered, and Akt (a.k.a. protein kinase B, PKB) activation is impaired downstream of both the lysophosphatidic acid receptor, a G-protein–coupled receptor, and the insulin receptor, a receptor tyrosine kinase. These findings indicate that phosphorylation of GIV by PKCθ inhibits GIV's GEF function and generates a unique negative feedback loop for downregulating the GIV-Gi axis of prometastatic signaling downstream of multiple ligand-activated receptors. This phosphoevent constitutes the only regulatory pathway described for terminating signaling by any of the growing family of nonreceptor GEFs that modulate G-protein activity.

Therapeutic effects of cell-permeant peptides that activate G proteins downstream of growth factors

“…To analyze the effects of TAT-GIV-CT peptide on cells, we first optimized cellular uptake of these peptides in HeLa cells. We chose to study HeLa cells because this is a well accepted model system and has been extensively used to characterize the role of GIV in our prior work (16,18,29,30). Incubation of HeLa cells with 400-800 nM TAT-GIV-CT peptides for 30 min resulted in efficient uptake (∼90-100% cells by immunofluorescence) with no observed toxicity (Fig.…”

“…15) using a selective GEF-deficient GIV mutant (F1685A) have demonstrated that the signaling network downstream of RTKs in cells with wild-type GIV is a mirror image of the network in cells expressing a GEF-deficient mutant GIV. It is because cells can alter (increase or decrease) the levels of GIV mRNA/protein or selectively modulate GIV's GEF activity to modulate growth factor signaling pathways across a range of intensities (16), we likened GIV to a cellular "rheostat" for signal transduction (17). Consistent with its ability to integrate signals downstream of multiple receptors, GIV modulates growth factor signaling during diverse biological processes (17), e.g., cell migration, chemotaxis (13), invasion (18), development (19), self-renewal (20), apoptosis (14,21), and autophagy (12).…”

GIV/Girdin activates Gαi and inhibits Gαs via the same motif

“…5). However, selective ablation of GIV's GBA motif not only disables cells' ability to migrate in response to a stimulus but also results in an unexpected gain in phenotype, i.e., cells begin to proliferate (11)(12)(13). How GIV's GBA motif actively suppresses cell proliferation remained unclear.…”

The GAPs, GEFs, GDIs and…now, GEMs: New kids on the heterotrimeric G protein signaling block

“…They showed that in the case of the prototypical GEM GIV, sequential phosphorylation of 2 Ser residues that flank the bifunctional GEF/GDI motif on GIV by 2 kinases, CDK5 35 and PKCu, 36 ensures that GIV exerts its GEF and GDI activities on Gai and Gas, respectively, in a temporally and spatially segregated manner (Fig. 2).…”