GIV/Girdin (Gα-interacting, Vesicle-associated Protein/Girdin) Creates a Positive Feedback Loop That Potentiates Outside-in Integrin Signaling in Cancer Cells

Abstract

Activation of the tyrosine kinase focal adhesion kinase (FAK) upon cell stimulation by the extracellular matrix initiates integrin outside-in signaling. FAK is directly recruited to active integrins, which enhances its kinase activity and triggers downstream signaling like activation of PI3K. We recently described that G␣-interacting, vesicle-associated protein (GIV), a protein up-regulated in metastatic cancers, is also required for outside-in integrin signaling. More specifically, we found that GIV is a non-receptor guanine nucleotide exchange factor that activates trimeric G proteins in response to integrin stimulation to enhance PI3K signaling and tumor cell migration. In contrast, previous reports have established that GIV is involved in phosphotyrosine (Tyr(P))-based signaling in response to growth factor stimulation; i.e. GIV phosphorylation at Tyr-1764 and Tyr-1798 recruits and activates PI3K. Here we show that phosphorylation of GIV at Tyr-1764/Tyr-1798 is also required to enhance PI3K-Akt signaling and tumor cell migration in response to integrin stimulation, indicating that GIV functions in Tyr(P)-dependent integrin signaling. Unexpectedly, we found that activation of FAK, an upstream component of the integrin Tyr(P) signaling cascade, was diminished in GIV-depleted cells, suggesting that GIV is required to establish a positive feedback loop that enhances integrin-FAK signaling. Mechanistically, we demonstrate that this feedback activation of FAK depends on both guanine nucleotide exchange factor and Tyr(P) GIV signaling as well as on their convergence point, PI3K. Taken together, our results provide novel mechanistic insights into how GIV promotes proinvasive cancer cell behavior by working as a signal-amplifying platform at the crossroads of trimeric G protein and Tyr(P) signaling.

GIV•Kindlin interaction is required for Kindlin-Mediated Integrin Recognition and Activation

“…As for the impact of the GIV•K2 interaction on GIV biology, findings showcased here, together with our understanding of how GIV modulates integrin/FAK signaling via G protein intermediates (Leyme et al, 2016;Leyme et al, 2015;Lopez-Sanchez et al, 2015), provide a more complete mechanistic insight into the roles of GIV in both early and late steps of integrin signaling [Fig 7A]. Because integrin signaling aids cancer growth, metastasis, and drug resistance, the signaling interfaces assembled by GIV (GIV•K2 and GIV•Gαi) provide new strategies for targeting the integrin pathway.…”

“…Next we asked how the newly defined GIV•kindlin-2 interaction impact cellular phenotypes. Prior studies have shown that GIV is required for signal amplification downstream of ligand-activated β 1-integrins via its ability to directly bind and activate Gαi and Class 1 PI3-kinase; the readouts used were cell adhesion and spreading, haptotaxis, activation of the PI3K→Akt, FAK→pY1764GIV and RhoA→myosin light chain (MLC2) signaling axes, the degree of maturation of FAs with sequential recruitment of paxillin and vinculin proteins and activation of FAK (Leyme et al, 2016;Leyme et al, 2015;Lopez-Sanchez et al, 2015). If these downstream events were dependent on GIV's ability to bind ligand-activated β 1-integrins, we hypothesized that uncoupling GIV from β 1-integrins will impair them all.…”

“…Taken together, these findings implicate the GIV•K2 interaction in some of the earliest 'upstream' events that begin within nascent FAs, i.e., enhanced recruitment of K2 to active β 1, formation of GIV•K2•β1integrin complexes, integrin clustering and activation. Consequently, a myriad of 'downstream' events within mature FAs are also derailed, e.g., their number and size, phosphoactivation of MLC, Akt and FAK and the activation of a previously defined feed-forward loop [FAK↔pYGIV↔PI3K↔FAK; (Leyme et al, 2016;Lopez-Sanchez et al, 2015)]. It is certainly possible that some of the post-receptor activation pathway/downstream signaling in PGxA mutant cells may reflect some of the impact of GIV's ability to bind the PTB domain of Tensin.…”

“…Besides talin and kindlin, we (Lopez-Sanchez et al, 2015) and others (Leyme et al, 2016;Leyme et al, 2015) have recently reported that exposing cells to ECM also triggers the tyrosine phosphorylation of another adaptor protein, GIV and that focal adhesions (FAs) serve as the major hubs for tyr-based mechanochemical signaling via GIV. GIV is a guanine nucleotide exchange factor (GEF) for trimeric GTPase, Gi (Garcia-Marcos et al, 2009;Kalogriopoulos et al, 2019) and is an actin remodeler (Enomoto et al, 2005).…”

“…These signals and cellular phenotypes are further reinforced via a forward-feedback loop in which activated FAK phosphorylates GIV and that such phosphorylation further enhances PI3K-Akt signaling, the integrity of FAs, cell adhesion and motility (Leyme et al, 2016;Lopez-Sanchez et al, 2015). Spatially restricted signaling via tyrosine phosphorylated GIV at the FAs is enhanced during cancer metastasis (Midde et al, 2018).…”