A circuit for secretion-coupled cellular autonomy in multicellular eukaryotic cells.

Cancers represent complex autonomous systems, displaying self-sufficiency in growth signaling. Autonomous growth is fueled by a cancer cell's ability to "secrete-and-sense" growth factors (GFs): a poorly understood phenomenon. Using an integrated computational and experimental approach, here we dissect the impact of a feedback-coupled GTPase circuit within the secretory pathway that imparts secretion-coupled autonomy. The circuit is assembled when the Ras-superfamily monomeric GTPase Arf1, and the heterotrimeric GTPase Giαßγ and their corresponding GAPs and GEFs are coupled by GIV/Girdin, a protein that is known to fuel aggressive traits in diverse cancers. One forward and two key negative feedback loops within the circuit create closed-loop control, allow the two GTPases to coregulate each other, and convert the expected switch-like behavior of Arf1-dependent secretion into an unexpected dose-response alignment behavior of sensing and secretion. Such behavior translates into cell survival that is self-sustained by stimulus-proportionate secretion. Proteomic studies and protein-protein interaction network analyses pinpoint GFs (e.g., the epidermal GF) as key stimuli for such self-sustenance. Findings highlight how the enhanced coupling of two biological switches in cancer cells is critical for multiscale feedback control to achieve secretion-coupled autonomy of growth factors.

GIV/Girdin and Exo70 Collaboratively Regulate the Mammalian Polarized Exocytic Machinery.

Polarized exocytosis is a fundamental process by which membranes and cargo proteins are delivered to the cell surface with precise spatial control. Although the need for the octameric exocyst complex is conserved from yeast to humans, what imparts spatial control is known only in yeast, i.e., a polarity scaffold called Bem1p. We demonstrate here that the mammalian scaffold protein, GIV/Girdin, fulfills the key criteria and functions of its yeast counterpart Bem1p; both bind Exo70 proteins via similar short-linear interaction motifs, and each prefers its evolutionary counterpart. Selective disruption of the GIV⋅Exo-70 interaction derails the delivery of the metalloprotease MT1-MMP to invadosomes and impairs collagen degradation and haptotaxis through basement membrane matrix. GIV's interacting partners reveal other components of polarized exocytosis in mammals. Findings expose how the exocytic functions aid GIV's pro-metastatic functions and how signal integration via GIV may represent an evolutionary advancement of the exocytic process in mammals.

The chondroitin sulfate moiety mediates thrombomodulin-enhanced adhesion and migration of vascular smooth muscle cells.

BACKGROUND: Thrombomodulin (TM), a transmembrane glycoprotein highly expressed in endothelial cells (ECs), is a potent anticoagulant maintaining circulation homeostasis. Under inflammatory states, TM expression is drastically reduced in ECs while vascular smooth muscle cells (VSMCs) show a robust expression of TM. The functional role of TM in VSMCs remains elusive. METHODS: We examined the role of TM in VSMCs activities in human aortic VSMCs stimulated with platelet-derived growth factor-BB (PDGF-BB). Using rat embryonic aorta-derived A7r5 VSMCs which do not express TM, the role of the chondroitin sulfate (CS) moiety of TM in VSMCs was delineated with cells expressing wild-type TM and the CS-devoid TM mutant. RESULTS: Expression of TM enhanced cell migration and adhesion/spreading onto type I collagen, but had no effect on cell proliferation. Knocking down TM with short hairpin RNA reduced PDGF-stimulated adhesion and migration of human aortic VSMCs. In A7r5 cells, TM-mediated cell adhesion was eradicated by pretreatment with chondroitinase ABC which degrades CS moiety. Furthermore, the TM mutant (TMS490, 492A) devoid of CS moiety failed to increase cell adhesion, spreading or migration. Wild-type TM, but not TMS490, 492A, increased focal adhesion kinase (FAK) activation during cell adhesion, and TM-enhanced cell migration was abolished by a function-blocking anti-integrin ß1 antibody. CONCLUSION: Chondroitin sulfate modification is required for TM-mediated activation of ß1-integrin and FAK, thereby enhancing adhesion and migration activity of VSMCs.

Biochemical, Biophysical and Cellular Techniques to Study the Guanine Nucleotide Exchange Factor, GIV/Girdin.

Canonical signal transduction via heterotrimeric G proteins is spatiotemporally restricted, i.e., triggered exclusively at the plasma membrane, only by agonist activation of G protein-coupled receptors via a finite process that is terminated within a few hundred milliseconds. Recently, a rapidly emerging paradigm has revealed a noncanonical pathway for activation of heterotrimeric G proteins via the nonreceptor guanidine-nucleotide exchange factor, GIV/Girdin. Biochemical, biophysical, and functional studies evaluating this pathway have unraveled its unique properties and distinctive spatiotemporal features. As in the case of any new pathway/paradigm, these studies first required an in-depth optimization of tools/techniques and protocols, governed by rationale and fundamentals unique to the pathway, and more specifically to the large multimodular GIV protein. Here we provide the most up-to-date overview of protocols that have generated most of what we know today about noncanonical G protein activation by GIV and its relevance in health and disease. © 2016 by John Wiley & Sons, Inc.

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

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.

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

GIV/Girdin is a multimodular signal transducer and a bona fide metastasis-related protein. As a guanidine exchange factor (GEF), GIV modulates signals initiated by growth factors (chemical signals) by activating the G protein Gαi. Here we report that mechanical signals triggered by the extracellular matrix (ECM) also converge on GIV-GEF via ß1 integrins and that focal adhesions (FAs) serve as the major hubs for mechanochemical signaling via GIV. GIV interacts with focal adhesion kinase (FAK) and ligand-activated ß1 integrins. Phosphorylation of GIV by FAK enhances PI3K-Akt signaling, the integrity of FAs, increases cell-ECM adhesion, and triggers ECM-induced cell motility. Activation of Gαi by GIV-GEF further potentiates FAK-GIV-PI3K-Akt signaling at the FAs. Spatially restricted signaling via tyrosine phosphorylated GIV at the FAs is enhanced during cancer metastasis. Thus GIV-GEF serves as a unifying platform for integration and amplification of adhesion (mechanical) and growth factor (chemical) signals during cancer progression.

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

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 GIV's 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.

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 ß-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.

Ets-1 mediates platelet-derived growth factor-BB-induced thrombomodulin expression in human vascular smooth muscle cells.

AIMS: Thrombomodulin (TM), a potent anticoagulant, is not detected in quiescent vascular smooth muscle cells (VSMCs). In diseased vessels, VSMC expresses TM, but the mechanisms are unclear. This study examined molecular mechanisms for TM expression in VSMCs. METHODS AND RESULTS: Platelet-derived growth factor-BB (PDGF-BB) induced TM expression in cultured human aortic VSMCs. PDGF-induced TM is functional in activating protein C. TM induction was eliminated by inhibitors of Src kinase, phosphatidylinositol 3-kinase (PI3-kinase), and mammalian target of rapamycin (mTOR) and by expressing dominant-negative Akt while expressing active Akt-stimulated TM expression. PDGF-BB activated the TM promoter, and the deletion of a sequence segment -394/-255 drastically reduced TM promoter activity. Transcription factor E26 transformation-specific sequence-1 (Ets-1) was upregulated by PDGF-BB in a PI3-kinase- and mTOR-dependent manner. RNA interference of Ets-1 inhibited PDGF induction of TM, and overexpressing Ets-1 increased TM expression. Chromatin immunoprecipitation and electrophoretic mobility shift assay detected increased Ets-1 binding to the TM promoter after PDGF treatment. Following carotid artery ligation of C57/BL6 mice, PDGF-BB and TM were co-expressed in the media and neointima. CONCLUSION: In VSMCs, PDGF-BB stimulates TM expression that is mainly mediated by Ets-1 via the Src kinase/PI3-kinase/Akt/mTOR signalling pathway. Furthermore, PDGF-BB may regulate TM expression in VSMCs during vascular remodelling.

Reactive oxygen species and ERK 1/2 mediate monocyte chemotactic protein-1-stimulated smooth muscle cell migration.

Monocyte chemotactic protein-1 (MCP-1), a potent chemoattractant for monocytes, is thought to play a major role in atherosclerosis, but whether its atherogenic effects involve the direct modulation of vascular smooth muscle cell (SMC) functions remains unclear. This study examined the effects of MCP-1 on the migration of cultured A7r5 SMCs and the signaling pathways involved. Addition of recombinant MCP-1 stimulated SMC migration in modified Boyden chambers coated with type I collagen in a concentration-dependent manner, with 10(-9) M being maximally effective. Using untreated A7r5 cells, two MCP-1 receptors, CCR2 and CCR4, were detected and MCP-1 secretion was significantly increased by stimulation with platelet-derived growth factor. MCP-1-stimulated A7r5 migration was completely blocked by the NAD(P)H oxidase inhibitor, diphenylene iodonium (DPI), and dose-dependently inhibited by polyethylene glycol-conjugated superoxide dismutase (PEG-SOD), suggesting a role for reactive oxygen species (ROS) in this process. During MCP-1 stimulation, ROS production increased rapidly, then gradually decayed over 60 min, and this effect was markedly decreased by pretreatment with DPI or PEG-SOD. Interestingly, U0126 and PD98059, which inhibit activation of extracellular signal-regulated kinases 1/2 (ERK 1/2), significantly inhibited MCP-1-activated ROS generation. Furthermore, transfection of an active mutant of MEK1 (ERK 1/2 kinase) markedly increased superoxide production in rat aortic smooth muscle cells, as detected by dihydroethydium staining, suggesting that ERK 1/2 activation stimulates ROS generation. ERK 1/2 activation was increased for at least 30 min in cells incubated with MCP-1, and this effect was abolished by U0126 or DPI pretreatment. These results demonstrate that MCP-1 is a chemoattractant for SMCs and that MCP-1-stimulated migration requires both ROS production and ERK 1/2 activation in a positive activation loop, which may contribute to the atherogenic effects of MCP-1.