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

Garcia‐Marcos, Mikel; Kietrsunthorn, Patrick; Pavlova, Yelena; Adia, Michelle A.; Ghosh, Pradipta; Farquhar, Marilyn G.

doi:10.1073/pnas.1120538109

Cited by 48 publications

(55 citation statements)

References 30 publications

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“…The major finding in this work is the identification of GIV as a GDI for Gαs. Together with its previously well-characterized ability to bind Gαi (1,25,26), GIV joins several other receptor GEFs, i.e., GPCRs (27)(28)(29)(30), that can dually couple to both Gi and Gs, two G proteins with opposing effects on adenylyl cyclase and the generation of cellular cAMP. In all instances in which dual coupling of GPCRs to Gi and Gs has been reported, ligand stimulation leads to the activation of both G proteins.…”

Section: Discussionmentioning

confidence: 99%

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

Gupta

Bhandari

Leyme

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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We previously showed that guanine nucleotide-binding (G) protein α subunit (Gα)-interacting vesicle-associated protein (GIV), a guanine-nucleotide exchange factor (GEF), transactivates Gα activity-inhibiting polypeptide 1 (Gαi) proteins in response to growth factors, such as EGF, using a short C-terminal motif. Subsequent work demonstrated that GIV also binds Gαs and that inactive Gαs promotes maturation of endosomes and shuts down mitogenic MAPK–ERK1/2 signals from endosomes. However, the mechanism and consequences of dual coupling of GIV to two G proteins, Gαi and Gαs, remained unknown. Here we report that GIV is a bifunctional modulator of G proteins; it serves as a guanine nucleotide dissociation inhibitor (GDI) for Gαs using the same motif that allows it to serve as a GEF for Gαi. Upon EGF stimulation, GIV modulates Gαi and Gαs sequentially: first, a key phosphomodification favors the assembly of GIV–Gαi complexes and activates GIV’s GEF function; then a second phosphomodification terminates GIV’s GEF function, triggers the assembly of GIV–Gαs complexes, and activates GIV’s GDI function. By comparing WT and GIV mutants, we demonstrate that GIV inhibits Gαs activity in cells responding to EGF. Consequently, the cAMP→PKA→cAMP response element-binding protein signaling axis is inhibited, the transit time of EGF receptor through early endosomes are accelerated, mitogenic MAPK–ERK1/2 signals are rapidly terminated, and proliferation is suppressed. These insights define a paradigm in G-protein signaling in which a pleiotropically acting modulator uses the same motif both to activate and to inhibit G proteins. Our findings also illuminate how such modulation of two opposing Gα proteins integrates downstream signals and cellular responses.

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Section: Discussionmentioning

confidence: 99%

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

Gupta

Bhandari

Leyme

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Protein-protein interactions were determined by pulldown and immunoprecipitation assays as described previously (2,5,11,12).…”

Section: Methodsmentioning

confidence: 99%

“…The C-terminally located GEF motif has emerged as a key modulator of GIV's functions. Cells expressing a GEF-deficient F1685A (FA) mutant display a phenotype strikingly different from those that express the wild-type (WT) protein in that they fail to activate Gi (6) or couple Gi to ligand-activated RTKs (2), maximally enhance PI3K-Akt signaling in response to growth factors or ligands for GPCRs (2,6,8,12), generate actin stress fibers (6), trigger cell migration (2), or arrest autophagy (5). Instead, they enhance the MAPK/ERK pathway (2), display increased cortical actin (6), and trigger mitosis (2).…”

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confidence: 99%

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

López-Sánchez¹,

Garcia‐Marcos

Mittal³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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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.

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“…For this, we depleted endogenous GIV from MDA-MB-231 cells using RNAi (previously validated GIV shRNA2; ϳ95% efficiency (17)) followed by ectopic expression of RNAi-resistant GIV WT or GIV YF mutant. For comparison, we also expressed GIV F1685A (FA), a mutant that disrupts the GEF activity of GIV (21,23) and blunts Akt activation in response to integrin stimulation (17). Importantly, it has been validated previously (39) that the FA and YF mutants affect specifically GEF activity and tyrosine phosphorylation, respectively, and that these two functions of GIV function independently of each other.…”

Section: Giv Phosphorylation At Tyrosines 1764/1798 Facilitatesmentioning

confidence: 99%

“…GIV belongs to an emerging group of atypical G protein activators called non-receptor GEFs (33)(34)(35)(36)(37)(38), which mimic the action of GPCRs but are cytoplasmic factors instead of transmembrane receptors. The GEF activity of GIV is associated with a defined G␣-binding and -activating motif of ϳ30 amino acids located in its C-terminal region (21,23) (Fig. 1), and disabling the GEF activity of this motif by site-directed mutagenesis inhibits PI3K activation downstream of GPCRs, RTKs, and integrins (17,18).…”

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confidence: 99%

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

Leyme

Marivin

Garcia‐Marcos

2016

Journal of Biological Chemistry

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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.

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

Cited by 48 publications

References 30 publications

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

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

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

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

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