Mikel Garcia-Marcos

GIV is a nonreceptor GEF for Gαi with a unique motif that regulates Akt signaling

Heterotrimeric G proteins are molecular switches that control signal transduction. Ligand-occupied, G protein-coupled receptors serve as the canonical guanine nucleotide exchange factors (GEFs) that activate heterotrimeric G proteins. A few unrelated nonreceptor GEFs have also been described, but little or nothing is known about their structure, mechanism of action, or cellular functions in mammals. We have discovered that GIV/Girdin serves as a nonreceptor GEF for Gαi through an evolutionarily conserved motif that shares sequence homology with the synthetic GEF peptide KB-752. Using the available structure of the KB-752·Gαi1 complex as a template, we modeled the Gαi-GIV interface and identified the key residues that are required to form it. Mutation of these key residues disrupts the interaction and impairs Akt enhancement, actin remodeling, and cell migration in cancer cells. Mechanistically, we demonstrate that the GEF motif is capable of activating as well as sequestering the Gα-subunit, thereby enhancing Akt signaling via the Gβγ-PI3K pathway. Recently, GIV has been implicated in cancer metastasis by virtue of its ability to enhance Akt activity and remodel the actin cytoskeleton during cancer invasion. Thus, the novel regulatory motif described here provides the structural and biochemical basis for the prometastatic features of GIV, making the functional disruption of this unique Gαi-GIV interface a promising target for therapy against cancer metastasis.

Activation of Gαi3 triggers cell migration via regulation of GIV

During migration, cells must couple direction sensing to signal transduction and actin remodeling. We previously identified GIV/Girdin as a Gαi3 binding partner. We demonstrate that in mammalian cells Gαi3 controls the functions of GIV during cell migration. We find that Gαi3 preferentially localizes to the leading edge and that cells lacking Gαi3 fail to polarize or migrate. A conformational change induced by association of GIV with Gαi3 promotes Akt-mediated phosphorylation of GIV, resulting in its redistribution to the plasma membrane. Activation of Gαi3 serves as a molecular switch that triggers dissociation of Gβγ and GIV from the Gi3–GIV complex, thereby promoting cell migration by enhancing Akt signaling and actin remodeling. Gαi3–GIV coupling is essential for cell migration during wound healing, macrophage chemotaxis, and tumor cell migration, indicating that the Gαi3–GIV switch serves to link direction sensing from different families of chemotactic receptors to formation of the leading edge during cell migration.

A Gαi-GIV molecular complex binds epidermal growth factor receptor and determines whether cells migrate or proliferate

Migrating cells do not proliferate and vice versa, but the mechanism involved remains unknown. Ghosh et al. reveal how this cellular decision is made by showing that a Gαi–GIV molecular complex interacts with EGF receptor and programs growth factor signaling, triggering migration when assembled and favoring mitosis when assembly is prevented.

A GDI (AGS3) and a GEF (GIV) regulate autophagy by balancing G protein activity and growth factor signals.

This work introduces a nonreceptor GEF for Gαi subunits as a regulator of autophagy. The authors reveal how growth factors reversibly regulate autophagy by a unique mechanism that involves reversible regulation of Gαi3 activity by AGS3, a GDI, and GIV, a GEF, during initiation and reversal of autophagy, respectively.

Hsp70-Bag3 interactions regulate cancer-related signaling networks

Bag3, a nucleotide exchange factor of the heat shock protein Hsp70, has been implicated in cell signaling. Here, we report that Bag3 interacts with the SH3 domain of Src, thereby mediating the effects of Hsp70 on Src signaling. Using several complementary approaches, we established that the Hsp70-Bag3 module is a broad-acting regulator of cancer cell signaling by modulating the activity of the transcription factors NF-κB, FoxM1, Hif1α, the translation regulator HuR, and the cell-cycle regulators p21 and survivin. We also identified a small-molecule inhibitor, YM-1, that disrupts the Hsp70-Bag3 interaction. YM-1 mirrored the effects of Hsp70 depletion on these signaling pathways, and in vivo administration of this drug was sufficient to suppress tumor growth in mice. Overall, our results defined Bag3 as a critical factor in Hsp70-modulated signaling and offered a preclinical proof-of-concept that the Hsp70-Bag3 complex may offer an appealing anticancer target.

Expression of GIV/Girdin, a metastasis-related protein, predicts patient survival in colon cancer

Metastasis accounts for the majority of cancer‐related deaths. Accurate prediction of meta‐static 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‐nucle‐otide exchange factor motif that activates Gai. 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 colo‐rectal cancers. We conclude that GIV‐fl is a novel metastasis‐related protein and an independent adverse prognos‐ticator that may serve as a useful adjunct to traditional staging strategies in colorectal carcinoma.—Garcia‐Marcos, M., Jung, B. H., Ear, J., Cabrera, B., Carethers, J. M., Ghosh, P. Expression of GIV/Girdin, a metastasis‐related protein, predicts patient survival in colon cancer. FASEB J. 25, 590–599 (2011). www.fasebj.org

A Structural Determinant That Renders Gαi Sensitive to Activation by GIV/Girdin Is Required to Promote Cell Migration

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 Gαi3 necessary for its regulation by GIV/girdin, a guanine-nucleotide exchange factor (GEF) that activates Gαi subunits. Using G protein activity and in vitro pulldown assays we demonstrate that Gαi3 is a better substrate for GIV than the highly homologous Gαo. We identified Trp-258 in the Gαi subunit as a novel structural determinant for GIV binding by comparing GIV binding to Gαi3/Gαo chimeras. Mutation of Trp-258 to the corresponding Phe in Gαo decreased GIV binding in vitro and in cultured cells but did not perturb interaction with other Gα-binding partners, i.e. Gβγ, AGS3 (a guanine nucleotide dissociation inhibitor), GAIP/RGS19 (a GTPase-activating protein), and LPAR1 (a G protein-coupled receptor). Activation of Gαi3 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 Gαi3 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 Gαi3 by GIV is essential for biological functions associated with Gαi3 activation. In conclusion, we have discovered a novel structural determinant on Gαi that plays a key role in defining the selectivity and efficiency of the GEF activity of GIV on Gαi and that represents an attractive target site for designing small molecules to disrupt the Gαi-GIV interface for therapeutic purposes.

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

GIV links ligand stimulation of various receptors to downstream activation of a kinase involved in cell migration. GIVing Migration a Boost Phosphoinositide 3-kinase (PI3K) can promote cell migration, and the activation of PI3K occurs downstream of ligand binding to several cell surface receptors. Lin et al. show that the guanine nucleotide exchange factor GIV (Gα-interacting vesicle-associated protein) may link ligand stimulation of these receptors to activation of PI3K. GIV was tyrosine phosphorylated by various receptor and non-receptor tyrosine kinases, and these phosphorylation events enabled GIV to bind to a regulatory subunit of PI3K, increase PI3K activity at the plasma membrane, and promote cell migration. The metastatic and invasive extent of a breast carcinoma was positively correlated to tyrosine phosphorylation of GIV and its association with the regulatory subunit of PI3K. Thus, manipulations that prevent or decrease the tyrosine phosphorylation of GIV could potentially be used to slow the progression of invasive cancers. 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.

GIV/Girdin is a rheostat that fine-tunes growth factor signals during tumor progression

GIV/Girdin is a multidomain signaling molecule that enhances PI3K-Akt signals downstream of both G protein-coupled and growth factor receptors. We previously reported that GIV triggers cell migration via its C-terminal guanine-nucleotide exchange factor (GEF) motif that activates Gαi. Recently we discovered that GIVs C-terminus directly interacts with the epidermal growth factor receptor (EGFR), and when its GEF function is intact, a Gαi-GIV-EGFR signaling complex assembles. By coupling G proteins to growth factor receptors, GIV is uniquely poised to intercept the incoming receptor-initiated signals and modulate them via G protein intermediates. Subsequent work has revealed that expression of the highly specialized C-terminus of GIV undergoes a bipartite dysregulation during oncogenesis-full length GIV with an intact C-terminus is expressed at levels ~20–50-fold above normal in highly invasive cancer cells and metastatic tumors, but its C-terminus is truncated by alternative splicing in poorly invasive cancer cells and non-invasive tumors. The consequences of such dysregulation on graded signal transduction and cellular phenotypes in the normal epithelium and its implication during tumor progression are discussed herein. Based on the fact that GIV grades incoming signals initiated by ligand-activated receptors by linking them to cyclical activation of G proteins, we propose that GIV is a molecular rheostat for signal transduction.

GIV/ Girdin transmits signals from multiple receptors by triggering trimeric G protein activation

Activation of trimeric G proteins has been traditionally viewed as the exclusive job of G protein-coupled receptors (GPCRs). This view has been challenged by the discovery of non-receptor activators of trimeric G proteins. Among them, GIV (a.k.a. Girdin) is the first for which a guanine nucleotide exchange factor (GEF) activity has been unequivocally associated with a well defined motif. Here we discuss how GIV assembles alternative signaling pathways by sensing cues from various classes of surface receptors and relaying them via G protein activation. We also describe the dysregulation of this mechanism in disease and how its targeting holds promise for novel therapeutics.