Inmaculada Lopez-Sanchez

GIV/Girdin is a central hub for profibrogenic signalling networks during liver fibrosis

Progressive liver fibrosis is characterized by the deposition of collagen by activated hepatic stellate cells (HSCs). Activation of HSCs is a multiple receptor-driven process in which profibrotic signals are enhanced, and anti-fibrotic pathways are suppressed. Here we report the discovery of a novel signaling platform comprised of G protein subunit, Gαi and GIV, its guanine exchange factor (GEF), which serves as a central hub within the fibrogenic signalling network initiated by diverse classes of receptors. GIV is expressed in the liver after fibrogenic injury and is required for HSC activation. Once expressed, GIV enhances the profibrotic (PI3K-Akt-FoxO1 and TGFβ-SMAD) and inhibits the anti-fibrotic (cAMP-PKA-pCREB) pathways to skew the signalling network in favor of fibrosis, all via activation of Gαi. We also provide evidence that GIV may serve as a biomarker for progression of fibrosis after liver injury and a therapeutic target for arresting and/or reversing HSC activation during liver fibrosis.

Daple is a novel non-receptor GEF required for trimeric G protein activation in Wnt signaling

Wnt signaling is essential for tissue homeostasis and its dysregulation causes cancer. Wnt ligands trigger signaling by activating Frizzled receptors (FZDRs), which belong to the G-protein coupled receptor superfamily. However, the mechanisms of G protein activation in Wnt signaling remain controversial. In this study, we demonstrate that FZDRs activate G proteins and trigger non-canonical Wnt signaling via the Dishevelled-binding protein, Daple. Daple contains a Gα-binding and activating (GBA) motif, which activates Gαi proteins and an adjacent domain that directly binds FZDRs, thereby linking Wnt stimulation to G protein activation. This triggers non-canonical Wnt responses, that is, suppresses the β-catenin/TCF/LEF pathway and tumorigenesis, but enhances PI3K-Akt and Rac1 signals and tumor cell invasiveness. In colorectal cancers, Daple is suppressed during adenoma-to-carcinoma transformation and expressed later in metastasized tumor cells. Thus, Daple activates Gαi and enhances non-canonical Wnt signaling by FZDRs, and its dysregulation can impact both tumor initiation and progression to metastasis. DOI: http://dx.doi.org/10.7554/eLife.07091.001

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

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 GIVs 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.

Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin.

GIV, a guanidine exchange factor for trimeric Gi, contains a unique domain that functions like a SH2 domain. GIVs SH2-like domain binds autophosphorylated RTKs. Binding of GIVs SH2 to RTKs enables the receptors to activate trimeric Gi. Inhibition of GIV:RTK interaction abolishes GIV-dependent Akt enhancement downstream of RTKs.

Cyclin-dependent kinase 5 activates guanine nucleotide exchange factor GIV/Girdin to orchestrate migration–proliferation dichotomy

Significance The guanine nucleotide exchange factor (GEF) GIV/Girdin has previously been shown to trigger noncanonical activation of trimeric G proteins in response to multiple chemical stimuli at the plasma membrane and at various locations within cells. In this work we identified a single phosphoevent and pinpointed cyclin-dependent kinase 5 (CDK5) as the responsible kinase that activates GIVs GEF function and initiates noncanonical G protein signaling via GIV. These studies provide evidence that CDK5 is essential for maximal activation of GIV-GEF in cells and insights into a hitherto unrecognized crosstalk between CDK5 and G proteins via GIV. Such crosstalk may shape migration–proliferation dichotomy during several physiological and pathological scenarios such as wound healing and cancer metastasis. Signals propagated by receptor tyrosine kinases (RTKs) can drive cell migration and proliferation, two cellular processes that do not occur simultaneously—a phenomenon called “migration–proliferation dichotomy.” We previously showed that epidermal growth factor (EGF) signaling is skewed to favor migration over proliferation via noncanonical transactivation of Gαi proteins by the guanine exchange factor (GEF) GIV. However, what turns on GIV-GEF downstream of growth factor RTKs remained unknown. Here we reveal the molecular mechanism by which phosphorylation of GIV by cyclin-dependent kinase 5 (CDK5) triggers GIVs ability to bind and activate Gαi in response to growth factors and modulate downstream signals to establish a dichotomy between migration and proliferation. We show that CDK5 binds and phosphorylates GIV at Ser1674 near its GEF motif. When Ser1674 is phosphorylated, GIV activates Gαi and enhances promigratory Akt signals. Phosphorylated GIV also binds Gαs and enhances endosomal maturation, which shortens the transit time of EGFR through early endosomes, thereby limiting mitogenic MAPK signals. Consequently, this phosphoevent triggers cells to preferentially migrate during wound healing and transmigration of cancer cells. When Ser1674 cannot be phosphorylated, GIV cannot bind either Gαi or Gαs, Akt signaling is suppressed, mitogenic signals are enhanced due to delayed transit time of EGFR through early endosomes, and cells preferentially proliferate. These results illuminate how GIV-GEF is turned on upon receptor activation, adds GIV to the repertoire of CDK5 substrates, and defines a mechanism by which this unusual CDK orchestrates migration–proliferation dichotomy during cancer invasion, wound healing, and development.

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

Significance Most common diseases (e.g., cancer, inflammatory disorders, diabetes) are driven by not one, but multiple cell surface receptors that trigger and sustain a pathologic signaling network. The largest fraction of therapeutic agents that target individual receptors/pathways often eventually fail due to the emergence of compensatory mechanisms. Recently, we identified GIV protein as a central platform for receptor cross-talk which integrates signals downstream of a myriad of upstream receptors, and modulates several key pathways within downstream signaling network, all via activation of trimeric G proteins. Here we provide the proof-of-concept that nongenetic exogenous modulation of the GIV-Gi signaling interface using cell-penetrable GIV-derived peptides is an effective strategy to reset pathologic signaling networks downstream of multiple receptors in a diverse array of pathophysiologic conditions. In eukaryotes, receptor tyrosine kinases (RTKs) and trimeric G proteins are two major signaling hubs. Signal transduction via trimeric G proteins has long been believed to be triggered exclusively by G protein-coupled receptors (GPCRs). This paradigm has recently been challenged by several studies on a multimodular signal transducer, Gα-Interacting Vesicle associated protein (GIV/Girdin). We recently demonstrated that GIV’s C terminus (CT) serves as a platform for dynamic association of ligand-activated RTKs with Gαi, and for noncanonical transactivation of G proteins. However, exogenous manipulation of this platform has remained beyond reach. Here we developed cell-permeable GIV-CT peptides by fusing a TAT-peptide transduction domain (TAT-PTD) to the minimal modular elements of GIV that are necessary and sufficient for activation of Gi downstream of RTKs, and used them to engineer signaling networks and alter cell behavior. In the presence of an intact GEF motif, TAT-GIV-CT peptides enhanced diverse processes in which GIV’s GEF function has previously been implicated, e.g., 2D cell migration after scratch-wounding, invasion of cancer cells, and finally, myofibroblast activation and collagen production. Furthermore, topical application of TAT-GIV-CT peptides enhanced the complex, multireceptor-driven process of wound repair in mice in a GEF-dependent manner. Thus, TAT-GIV peptides provide a novel and versatile tool to manipulate Gαi activation downstream of growth factors in a diverse array of pathophysiologic conditions.

Focal adhesions are foci for tyrosine-based signal transduction via GIV/Girdin and G proteins

GIV is a guanine-nucleotide exchange factor and a bona fide metastasis-related protein. It is found, unexpectedly, that focal adhesions are the major foci for GIV-dependent signaling and that GIV modulates integrin-FAK signaling via activation of G proteins. It is also shown how this phenomenon is altered during cancer progression.

Activation of Gαi at the Golgi by GIV/Girdin Imposes Finiteness in Arf1 Signaling

A long-held tenet of heterotrimeric G protein signal transduction is that it is triggered by G protein-coupled receptors (GPCRs) at the PM. Here, we demonstrate that Gi is activated in the Golgi by GIV/Girdin, a non-receptor guanine-nucleotide exchange factor (GEF). GIV-dependent activation of Gi at the Golgi maintains the finiteness of the cyclical activation of ADP-ribosylation factor 1 (Arf1), a fundamental step in vesicle traffic in all eukaryotes. Several interactions with other major components of Golgi trafficking-e.g., active Arf1, its regulator, ArfGAP2/3, and the adaptor protein β-COP-enable GIV to coordinately regulate Arf1 signaling. When the GIV-Gαi pathway is selectively inhibited, levels of GTP-bound Arf1 are elevated and protein transport along the secretory pathway is delayed. These findings define a paradigm in non-canonical G protein signaling at the Golgi, which places GIV-GEF at the crossroads between signals gated by the trimeric G proteins and the Arf family of monomeric GTPases.

Activation of G proteins by GIV-GEF is a pivot point for insulin resistance and sensitivity

A long-held tenet in the field of diabetes is that the tipping point between insulin sensitivity and resistance resides at the level of insulin receptor/insulin receptor substrate–adaptor complexes. Here it is shown that activation of Gαi by GIV/Girdin is a decisive event within the metabolic insulin signaling cascade that reversibly orchestrates insulin sensitivity or resistance.

GIV/girdin binds exocyst subunit-Exo70 and regulates exocytosis of GLUT4 storage vesicles

Insulin resistance (IR) is a metabolic disorder characterized by impaired glucose uptake in response to insulin. The current paradigm for insulin signaling centers upon the insulin receptor (InsR) and its substrate IRS1; the latter is believed to be the chief conduit for post-receptor signaling. We recently demonstrated that GIV, a Guanidine Exchange Factor (GEF) for the trimeric G protein, Gαi, is a major hierarchical conduit for the metabolic insulin response. By virtue of its ability to directly bind the InsR, IRS1 and PI3K, GIV enhances the InsR-IRS1-Akt-AS160 (RabGAP) signaling cascade and cellular glucose uptake via its GEF function. Phosphoinhibition of GIV-GEF by the fatty-acid/PKCθ pathway inhibits the cascade and impairs glucose uptake. Here we show that GIV directly and constitutively binds the exocyst complex subunit Exo-70 and also associates with GLUT4-storage vesicles (GSVs) exclusively upon insulin stimulation. Without GIV or its GEF function, membrane association of Exo-70 as well as exocytosis of GSVs in response to insulin are impaired. Thus, GIV is an essential component within the insulin signaling cascade that couples upstream signal transducers within the InsR and G-Protein signaling cascade to downstream vesicular trafficking events within the exocytic pathway. These findings suggest a role of GIV in coordinating key signaling and trafficking events of metabolic insulin response.