Tyrosine Phosphorylation of the Gα-Interacting Protein GIV Promotes Activation of Phosphoinositide 3-Kinase During Cell Migration

Abstract

GIV (Gα-interacting vesicle-associated protein; also known as Girdin), enhances Akt activation downstream of multiple growth factor– and G-protein–coupled receptors to trigger cell migration and cancer invasion. Here 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 Tyr1764 and Tyr1798 by both receptor and non-receptor tyrosine kinases. These phosphorylation events enabled direct binding of GIV to the N- and C-terminal SH2 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 GIVPI3K interaction a potential therapeutic target within the PI3K-Akt pathway.

Functional characterization of the guanine nucleotide exchange factor (GEF) motif of GIV protein reveals a threshold effect in signaling

“…expression is supported by our results presented here (Fig. S4C) and by previous observations (29) showing that Akt activation is gradually increased in cells of isogenic background from the 21T series that represent different stages of cancer progression and naturally express increasing levels of GIV. However, for a given number of GIV copies, Akt signaing is ultrasensitive to the level of GEF activity.…”

“…Thus, it is tempting to speculate that the ultrasensitive response to GIV's GEF activity is a consequence of this dual effect of activating the G protein and displacing its GDIs; however, we cannot rule out other possibilities such as the existence of regulatory feedback loops and/or synergy with other components of this pathway. For example, it is possible that the ultrasensitive Akt response arises from the cooperative action of GIV's GEF activity and the direct activation of PI3K by tyrosine phosphorylated GIV (29).…”

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

“…Consistent with the central role of GIV's GEF function in the enhancement of EGFR autophosphorylation (9, 30) and PI3K-Akt signaling downstream (7,29), transduction with TAT-GIV-CT-FA inhibited receptor autophosphorylation (as determined by phosphorylation at Y1068 and Y1173 on EGFR tail; ref. Fig.…”

“…The rationale for the design of these peptides is multifactorial: (i) a complete phylogenetic analysis of GIV (17) has revealed that this stretch of GIV's C terminus could be functionally autonomous because it evolved independently of its N terminus (in fish), and both N-and C-termini fused into full length GIV only in birds; (ii) the C terminus contains the GEF and SH2-like domains (Fig. 1B), representing the cross-road between GPCR/G and RTK signaling pathways; (iii) the C terminus of GIV also contains the two critical tyrosines (Y1764 and Y1798) that serve as docking sites for p85α(PI3K) (29); (iv) the coexistence of those tyrosines, the GEF motif, and the SH2-like domain is restricted only to the most complex of eukaryotes, i.e., mammals, and is highly conserved (∼99%) (17,29); (v) biochemical and functional assays (9,25) have convincingly demonstrated that the critical C-terminal domain is necessary and sufficient for GIV to carry out its functions during signal transduction downstream of RTKs; and finally, (vi) biophysical studies (25) have revealed that fluorescent GIV-CT biosensors are effective tools for visualization and manipulation of the fundamental function of GIV in signal transduction, i.e., enabling dynamic association of Gαi with RTKs and noncanonical transactivation of G proteins in cells responding to growth factors. We expressed and purified (∼95-99% purity) the TAT-GIV-CT peptides and confirmed that they were expressed as proteins of expected size by immunoblotting ( Fig.…”

“…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.…”