Nicolas Aznar

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.

Multimodular biosensors reveal a novel platform for activation of G proteins by growth factor receptors.

Significance Long-held tenets in the field of signal transduction are that G proteins are activated exclusively by G protein-coupled receptors and that growth factor receptor tyrosine kinases (RTKs) do not have the wherewithal to do the same. In this work we created fluorescent biosensors derived from the multimodular signal transducer Gα-interacting vesicle-associated protein (GIV), an unusual protein that binds RTKs and activates G proteins, and used them in FRET and bimolecular fluorescent complementation assays to visualize RTK–GIV–G protein signaling complexes directly in single living cells. These studies not only provide evidence that GIV serves as a platform for transactivation of G proteins by growth factor RTKs but also illuminate the spatial and temporal dynamics of such noncanonical G protein signaling. Environmental cues are transmitted to the interior of the cell via a complex network of signaling hubs. Receptor tyrosine kinases (RTKs) and trimeric G proteins are two such major signaling hubs in eukaryotes. Conventionally, canonical signal transduction via trimeric G proteins is thought to be triggered exclusively by G protein-coupled receptors. Here we used molecular engineering to develop modular fluorescent biosensors that exploit the remarkable specificity of bimolecular recognition, i.e., of both G proteins and RTKs, and reveal the workings of a novel platform for activation of G proteins by RTKs in single living cells. Comprised of the unique modular makeup of guanidine exchange factor Gα-interacting vesicle-associated protein (GIV)/girdin, a guanidine exchange factor that links G proteins to a variety of RTKs, these biosensors provide direct evidence that RTK–GIV–Gαi ternary complexes are formed in living cells and that Gαi is transactivated within minutes after growth factor stimulation at the plasma membrane. Thus, GIV-derived biosensors provide a versatile strategy for visualizing, monitoring, and manipulating the dynamic association of Gαi with RTKs for noncanonical transactivation of G proteins in cells and illuminate a fundamental signaling event regulated by GIV during diverse cellular processes and pathophysiologic states.

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.

Heterotrimeric G protein signaling via GIV/Girdin: Breaking the rules of engagement, space, and time

Canonical signal transduction via heterotrimeric G proteins is spatially and temporally restricted, that is, triggered exclusively at the plasma membrane (PM), only by agonist activation of G protein‐coupled receptors (GPCRs) via a process that completes within a few hundred milliseconds. Recently, a rapidly emerging paradigm has revealed a non‐canonical pathway for activation of heterotrimeric G proteins by the non‐receptor guanidine‐nucleotide exchange factor (GEF), GIV/Girdin. This pathway has distinctive temporal and spatial features and an unusual profile of receptor engagement: diverse classes of receptors, not just GPCRs can engage with GIV to trigger such activation. Such activation is spatially and temporally unrestricted, that is, can occur both at the PM and on internal membranes discontinuous with the PM, and can continue for prolonged periods of time. Here, we provide the most complete up‐to‐date review of the molecular mechanisms that govern the unique spatiotemporal aspects of non‐canonical G protein activation by GIV and the relevance of this new paradigm in health and disease.

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.

AMP-activated protein kinase fortifies epithelial tight junctions during energetic stress via its effector GIV/Girdin

Loss of epithelial polarity impacts organ development and function; it is also oncogenic. AMPK, a key sensor of metabolic stress stabilizes cell-cell junctions and maintains epithelial polarity; its activation by Metformin protects the epithelial barrier against stress and suppresses tumorigenesis. How AMPK protects the epithelium remains unknown. Here, we identify GIV/Girdin as a novel effector of AMPK, whose phosphorylation at a single site is both necessary and sufficient for strengthening mammalian epithelial tight junctions and preserving cell polarity and barrier function in the face of energetic stress. Expression of an oncogenic mutant of GIV (cataloged in TCGA) that cannot be phosphorylated by AMPK increased anchorage-independent growth of tumor cells and helped these cells to evade the tumor-suppressive action of Metformin. This work defines a fundamental homeostatic mechanism by which the AMPK-GIV axis reinforces cell junctions against stress-induced collapse and also provides mechanistic insight into the tumor-suppressive action of Metformin. DOI: http://dx.doi.org/10.7554/eLife.20795.001

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

Significance Guanine nucleotide-binding (G) protein α subunit (Gα)-interacting vesicle-associated protein (GIV)/Girdin has previously been shown to serve as a guanine nucleotide exchange factor (GEF) for the Gα activity-inhibiting polypeptide 1 (Gαi) via a conserved motif in its C terminus. Here we show that this motif serves as a guanine nucleotide dissociation inhibitor (GDI) for Gαs. Sequential phosphorylation of two serine residues that flank this motif by two kinases, cyclin-dependent kinase 5 and PKCθ, ensures that GIV exerts its GEF and GDI activities on Gαi and Gαs, respectively, in a temporally and spatially segregated manner. Through its bifunctional role as GEF and GDI, GIV serves as a pleiotropically acting G-protein modulator that integrates, reinforces, and compartmentalizes signals downstream of both growth factors and G proteins and orchestrates migration–proliferation dichotomy. 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.