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 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.     

Vascular endothelial growth factor-stimulated actin reorganization and migration of endothelial cells is regulated via the serine/threonine kinase Akt

Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation, migration, and actin reorganization, all necessary components of an angiogenic response. However, the distinct signal transduction mechanisms leading to each angiogenic phenotype are not known. In this study, we examined the ability of VEGF to stimulate cell migration and actin rearrangement in microvascular endothelial cells infected with adenoviruses encoding beta-galactosidase (beta-gal), activation-deficient Akt (AA-Akt), or constitutively active Akt (myr-Akt). VEGF increased cell migration in cells transduced with beta-gal, whereas AA-Akt blocked VEGF-induced cell locomotion. Interestingly, myr-Akt transduction of bovine lung microvascular endothelial cells stimulated cytokinesis in the absence of VEGF, suggesting that constitutively active Akt, per se, can initiate the process of cell migration. Treatment of beta-gal-infected endothelial cells with an inhibitor of NO synthesis blocked VEGF-induced migration but did not influence migration initiated by myr-Akt. In addition, VEGF stimulated remodeling of the actin cytoskeleton into stress fibers, a response abrogated by infection with dominant-negative Akt, whereas transduction with myr-Akt alone caused profound reorganization of F-actin. Collectively, these data demonstrate that Akt is critically involved in endothelial cell signal transduction mechanisms leading to migration and that the Akt/endothelial NO synthase pathway is necessary for VEGF-stimulated cell migration.     

Rho/Rho激酶信号通路与冠心病

Rho/Rho激酶信号通路是体内普通存在的一条信号转导通路,它通过调节细胞内肌动蛋白骨架的聚合状态参与多种细胞功能,包括细胞收缩、迁移、黏附、增殖、凋亡及基因表达等。Rho/Rho激酶信号通路的异常激活在冠心病的发病机制和病理生理中发挥了重要作用,对此信号转导通路的研究可以为冠心病的预防和治疗提供新的靶点。现就Rho/Rho激酶信号通路的特征及其与冠心病的关系作一综述。     

Sphingosine-1-phosphate and lysophosphatidic acid stimulate endothelial cell migration

Endothelial cell migration is necessary for the formation of new blood vessels. We investigated the effects of 2 lysophospholipid mediators, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), on endothelial cell migration. S1P and LPA stimulated migration of fetal bovine heart endothelial cells (FBHEs) in a 3D-modified Boyden chamber assay with concentrations as low as 15 nmol/L stimulating a 2-fold change and concentrations in the 1- to 2-micromol/L range stimulating 14- to 20-fold changes. S1P specifically stimulated the migration of several endothelial cell strains but did not stimulate the migration of tumor cells or smooth muscle cells. LPA stimulated some endothelial and nonendothelial cell types to migrate. For FBHEs, S1P and LPA were mostly chemokinetic in checkerboard assays. S1P and LPA stimulated extracellular signal-regulated kinase 1/2 phosphorylation and enhanced paxillin localization to focal contacts, with no discernible change in the actin cytoskeleton in FBHEs. To characterize responsible receptor-dependent signaling pathways, we investigated the involvement of G(i), Rho, and phosphoinositide 3-OH kinase in S1P- and LPA-stimulated migration. Although perturbation of all 3 signaling molecules resulted in decreased migration, the mechanisms underlying the decreased migration were different. Pertussis toxin treatment, to target G(i), caused endothelial cells to develop dense bundles of F-actin and distribute paxillin staining to the cell periphery in response to S1P or LPA. Modification of Rho with C3 toxin disrupted the actin cytoskeleton. Inhibition of phosphoinositide 3-OH kinase decreased S1P- or LPA-induced endothelial cell migration with only minor disruption of the actin cytoskeleton. Inhibition of extracellular signal-regulated kinase kinase with PD98059 caused a loss of phosphorylation of extracellular signal-regulated kinase 1/2, similar to pertussis toxin, but only a minimal decrease in migration. These results indicate that S1P and, for some cells, LPA stimulate migration of endothelial cells through a mechanism that likely requires a balance between G(i) and Rho signaling to achieve the cytoskeletal remodeling necessary for cell migration.     

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

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.Receptor Tyrosine Kinases (RTK) and G protein coupled receptors (GPCR) are the two most widely studied cell signaling hubs in eukaryotes. For several decades, these two pathways were believed to operate in discrete modes by transducing signals through their respective downstream intermediates; upon ligand stimulation, RTKs propagate the signals to the interior of the cell via adaptor proteins that are recruited to phosphotyrosines on the receptor tail (1), whereas GPCRs, which are 7-transmembrane (TM) receptors with an intrinsic Guanine nucleotide Exchange Factor (GEF) activity recruit and activate G proteins by triggering the exchange of GDP with GTP nucleotide (2). Gathering evidence over time has unraveled a complex cross-talk between these two pathways at multiple tiers (3, 4). For example, transactivation of RTKs by GPCRs via scaffolding proteins, such as β-arrestins (5), is a well documented and widely accepted phenomenon. Numerous studies have also provided evidence to support the reverse concept, i.e., transactivation of heterotrimeric G proteins by growth factors (6). However, it was not until recently that this concept gained traction with the discovery and characterization of Gα-Interacting Vesicle associated protein (GIV; a.k.a. Girdin), an unusual signal transducer that can bind both RTKs and G proteins.GIV is a multimodular (Fig. 1A) signal transducer and a GEF for Gαi (7). Working downstream of a variety of growth factors [EGF (8, 9), IGF (10), VEGF (11), Insulin (7, 12, 13), and PDGFR (14)] GIV modulates (i.e., either enhances or suppresses) a variety of signaling pathways, all via its ability to activate Gαi in the close proximity of ligand-activated RTKs (7). Multiple studies (summarized in Fig. S1; ref. 15) using a selective GEF-deficient GIV mutant (F1685A) have demonstrated that the signaling network downstream of RTKs in cells with wild-type GIV is a mirror image of the network in cells expressing a GEF-deficient mutant GIV. It is because cells can alter (increase or decrease) the levels of GIV mRNA/protein or selectively modulate GIV’s GEF activity to modulate growth factor signaling pathways across a range of intensities (16), we likened GIV to a cellular “rheostat” for signal transduction (17). Consistent with its ability to integrate signals downstream of multiple receptors, GIV modulates growth factor signaling during diverse biological processes (17), e.g., cell migration, chemotaxis (13), invasion (18), development (19), self-renewal (20), apoptosis (14, 21), and autophagy (12). Increasing evidence also supports the clinical significance of GIV-dependent signaling during diverse disease processes (17), e.g., pathologic angiogenesis (11), liver fibrosis (14), nephrotic syndrome (21), vascular repair (22), and tumor metastasis (23).Open in a separate windowFig. 1.Design and purification of cell-permeable TAT-GIV CT peptides. (A, Upper) Schematic representation of the domain organization of GIV. From left to right, the functional domains include a microtubule-binding hook domain (black), a coiled-coil homodimerization domain (yellow), a Gα-binding domain (GBD, blue), a phosphoinositide (PI4P) binding motif (purple), a GEF motif (red), and, finally, a SH2-like domain (red, white, and blue) that is located within the Akt and Actin-binding domains at the extreme C terminus. The numbers denote the amino acids marking the boundaries of each domain. (A, Lower) Schematic showing how GIV’s C-terminal ∼210 amino acids link Gαi to the autophosphorylated cytoplasmic tail of ligand-activated RTKs. Homology model of Gαi3 in complex with GIV aa 1678–1689 (Left) was generated using the structure of the synthetic peptide KB-752 bound to Gαi1 (Protein Data Bank ID 1Y3A) as a template as done previously (7). Green, Gαi3 subunit; red, GIV''s GEF motif. Model of GIV’s SH2-like domain bound to a EGFR-derived phosphotyrosine peptide (purple) corresponding to pTyr1148 and its flanking residues is shown (Right) (30). The acidic, neutral, and basic potentials are displayed in red, white, and blue, respectively. (B) A schematic representation of the modular makeup of cell-permeable TAT-GIV-CT peptides is shown. TAT peptide transduction domain (TAT-PTD) was fused to His and HA tags, and coupled, via a linker (7 residues), to the C terminus of GIV (1660–1870 residues). A GEF-deficient mutant TAT-GIV-CT was generated by substituting a Phe-1685 into an Ala (F1685A). (C and D) Expression and purification of bacterially expressed TAT-GIV CT peptides. The purity and size of TAT-GIV-CT was confirmed by coomassie staining (C) and by immunoblotting (D) with anti-His and anti-GIV-CT antibodies.The molecular mechanisms that govern how GIV influences a diverse range of pathophysiologic processes and how it may couple activation of G protein to multiple receptors have come to light only recently, at least in the context of a numerous RTKs that signal via GIV. GIV-dependent growth factor signaling appears to rely heavily on the unique multimodular nature of its C terminus (CT), within which two unlikely domains coexist: (i) a previously defined GEF motif via which GIV binds and activates Gi (7), and (ii) a newly defined ∼110-aa stretch that folds into a SH2-like domain in the presence of phosphotyrosine ligands; the latter is necessary and sufficient to recognize and bind specific sites of autophosphorylation on the receptor tail (9, 24). Thus, GIV serves as a platform that links RTKs to G proteins within RTK-GIV-Gαi ternary complexes only when both its GEF and SH2-like modules are intact. In the absence of either of these modules, ligand-activated RTKs and Gαi are uncoupled, and the recruitment of Gαi to RTKs and subsequent activation of G proteins is impaired.Although the discovery of coexisting SH2-like and GEF modules in-tandem within GIV-CT supported the idea that GIV’s C terminus has the necessary modular make-up to serve as a platform for convergent signaling downstream of multiple RTKs via G proteins, it was not possible to visualize this platform until recently, when we developed genetically encoded fluorescent biosensors comprised of these two modules within GIV-CT. These biosensors revealed that the evolutionarily conserved C terminus of GIV represents the smallest, functionally autonomous unit that retains most key properties of full length GIV (25), i.e. (i) they can bind and activate Gαi in cells in a GEF dependent manner; (ii) they retain the properties of receptor recruitment and signal transduction characteristic of full length GIV; (iii) they serve as a bona fide platform for assembly of RTK-Gαi complexes at the PM and for noncanonical activation of Gαi in response to growth factors; and (iv) they are sufficient to trigger cell migration/invasion through basement membrane matrix. Thus, comprised of the essential modules (GEF and SH2-like domains), GIV-CT is sufficient for linking G proteins to RTKs, for triggering G protein activation in the vicinity of ligand-activated RTKs, for modulation of growth factor signaling, and for triggering complex cellular processes like cell invasion.Despite the emergence of GIV-CT as the long-sought platform for noncanonical transactivation of G proteins by multiple growth factor RTKs, exogenous manipulation of this platform has remained out of reach. We have developed cell-permeable GIV-CT peptides based on the blueprint of the previously extensively characterized fluorescent GIV-CT biosensors (25), and used them successfully to manipulate the diverse pathophysiologic processes in which GIV has been previously implicated. These findings help validate cell-permeable GIV-CT as a versatile tool for exogenously and selectively engineering noncanonical activation of G proteins in response to growth factors.     

Adiponectin stimulates Rho-mediated actin cytoskeleton remodeling and glucose uptake via APPL1 in primary cardiomyocytes

Objective

Adiponectin is known to confer its cardioprotective effects in obesity and type 2 diabetes, mainly by regulating glucose and fatty acid metabolism in cardiomyocytes. Dynamic actin cytoskeleton remodeling is involved in regulation of multiple biological functions, including glucose uptake. Here we investigated in neonatal cardiomyocytes whether adiponectin induced actin cytoskeleton remodeling and if this played a role in adiponectin-stimulated glucose uptake.

Materials/methods

Primary cardiomyocytes were treated with full-length and globular adiponectin (fAd and gAd, respectively).

Results

Both fAd and gAd increased RhoA activity, phosphorylation of the Rho/ROCK signaling target cofilin and actin polymerization to form filamentous actin as determined by rhodamine–phallodin immunofluorescence and quantitative analysis of filamentous to globular actin ratio. Scanning electron microscopy also demonstrated structural remodeling. Adiponectin stimulated glucose uptake, was significantly abrogated in the presence of inhibitors of actin cytoskeleton remodeling (cytochalasin D) and Rho/ROCK signaling (C3 transferase, Y27632). We showed that adiponectin increased colocalization of actin and APPL1 and that actin remodeling, phosphorylation of AMPK, p38MAPK and cofilin, glucose uptake and oxidation were all attenuated after siRNA-mediated knockdown of APPL1.

Conclusion

We show that adiponectin mediates Rho/ROCK-dependent actin cytoskeleton remodeling to increase glucose uptake and metabolism via APPL1 signaling.     

Src-dependence and pertussis-toxin sensitivity of urokinase receptor-dependent chemotaxis and cytoskeleton reorganization in rat smooth muscle cells.

The catalytically inactive precursor of urokinase-type plasminogen activator (pro-u-PA) induced a chemotactic response in rat smooth muscle cells (RSMC) through binding to the membrane receptor of urokinase (u-PA receptor [u-PAR]). A soluble form of u-PAR activated by chymotrypsin cleavage as well as a peptide located between domain 1 and 2 of u-PAR reproduced the effect of pro-u-PA on cell migration. The chemotactic pro-u-PA effect correlates with a dramatic reorganization of actin cytoskeleton, of adhesion plaques, and with major cell shape changes in RSMC. Pro-u-PA induced a decrease in stress fiber content, membrane ruffling, actin ring formation, and disruption leading to the characteristic elongated cell shape of motile cells with an actin semi-ring located close to the leading edge of cells. u-PAR effects on both chemotaxis and cytoskeleton were sensitive to pertussis toxin and, hence, possibly require G proteins. u-PAR effects are accompanied by a relocation of u-PAR, vitronectin receptor (VNR) alphavbeta3, beta1 integrin subunit, and Src tyrosine kinase to the leading membrane of migrating cells. In conclusion, our data show that pro-u-PA, via binding to u-PAR, controls a signaling pathway, regulated by tyrosine kinases and possibly G proteins, leading to cell cytoskeleton reorganization and cell migration.     

Mechanisms of vascular smooth muscle cell migration

Smooth muscle cell migration occurs during vascular development, in response to vascular injury, and during atherogenesis. Many proximal signals and signal transduction pathways activated during migration have been identified, as well as components of the cellular machinery that affect cell movement. In this review, a summary of promigratory and antimigratory molecules belonging to diverse chemical and functional families is presented, along with a summary of key signaling events mediating migration. Extracellular molecules that modulate migration include small biogenic amines, peptide growth factors, cytokines, extracellular matrix components, and drugs used in cardiovascular medicine. Promigratory stimuli activate signal transduction cascades that trigger remodeling of the cytoskeleton, change the adhesiveness of the cell to the matrix, and activate motor proteins. This review focuses on the signaling pathways and effector proteins regulated by promigratory and antimigratory molecules. Prominent pathways include phosphatidylinositol 3-kinases, calcium-dependent protein kinases, Rho-activated protein kinase, p21-activated protein kinases, LIM kinase, and mitogen-activated protein kinases. Important downstream targets include myosin II motors, actin capping and severing proteins, formins, profilin, cofilin, and the actin-related protein-2/3 complex. Actin filament remodeling, focal contact remodeling, and molecular motors are coordinated to cause cells to migrate along gradients of chemical cues, matrix adhesiveness, or matrix stiffness. The result is recruitment of cells to areas where the vessel wall is being remodeled. Vessel wall remodeling can be antagonized by common cardiovascular drugs that act in part by inhibiting vascular smooth muscle cell migration. Several therapeutically important drugs act by inhibiting cell cycle progression, which may reduce the population of migrating cells.     

FRMD4A regulates epithelial polarity by connecting Arf6 activation with the PAR complex

The Par-3/Par-6/aPKC/Cdc42 complex regulates the conversion of primordial adherens junctions (AJs) into belt-like AJs and the formation of linear actin cables during epithelial polarization. However, the mechanisms by which this complex functions are not well elucidated. In the present study, we found that activation of Arf6 is spatiotemporally regulated as a downstream signaling pathway of the Par protein complex. When primordial AJs are formed, Par-3 recruits a scaffolding protein, termed the FERM domain containing 4A (FRMD4A). FRMD4A connects Par-3 and the Arf6 guanine-nucleotide exchange factor (GEF), cytohesin-1. We propose that the Par-3/FRMD4A/cytohesin-1 complex ensures accurate activation of Arf6, a central player in actin cytoskeleton dynamics and membrane trafficking, during junctional remodeling and epithelial polarization.     

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.Upon growth factor stimulation, cells initiate signaling cascades favoring either migration or proliferation (migration–proliferation dichotomy) depending on the extracellular environmental cues and/or cellular needs. This dichotomy (also known as “go-or-grow mechanism”) plays a crucial role during a variety of normal and pathophysiologic processes, including development, wound healing, and cancer progression (1–5).Of the multiple molecular mechanisms implicated in the orchestration of migration–proliferation dichotomy, Gα-interacting vesicle associated protein (GIV) (also known as Girdin) is one such player that helps tilt the signaling network to favor migration over proliferation downstream of stimulated growth factor receptors as well as G protein-coupled receptors (GPCRs) (6–12). In the case of EGF stimulation, GIV is recruited to the plasma membrane (PM) where it directly binds ligand-activated EGFR via its SH2-like domain (13) and serves as a platform for the assembly of receptor tyrosine kinase (RTK)-GIV-Gαi complexes and transactivation of Gαi via its guanine exchange factor (GEF) motif (8, 14). Such transactivation of Gi in the vicinity of RTKs at the PM enhances RTK autophosphorylation, prolongs RTK signaling from the PM, enhances PI3K/Akt signals and actin reorganization, and triggers cell migration (7–9, 13). Besides its role in the assembly of RTK-GIV-Gαi complexes at the PM, GIV also assembles EGFR-GIV-Gαs complexes on early endosomes where it facilitates down-regulation of EGFR via endosomal maturation, ensures finiteness of mitogenic signaling from that compartment, and limits cell proliferation (15).Much of the experimental evidence supporting GIV’s ability to skew the phenotypic response toward migration has been generated using a GEF-deficient mutant (F1685A, FA) of GIV, which cannot bind either Gαi at the PM or Gαs on endosomes (8, 15). Without the formation of RTK-GIV-Gαi/s complexes, ligand-activated EGFR spends a shorter time at the cell surface, but takes longer to transit through endosomes due to delayed endosomal maturation. Consequently, cells expressing GIV-FA suppress promigratory PI3K-Akt signals at the PM and enhance mitogenic signals from endosomes to preferentially trigger proliferation (8, 15). Despite the insights gained, the GIV-FA mutant did not illuminate how GIV-GEF may be reversibly switched “on/off” in physiology until recently, when that question was partially answered by the discovery of a key phosphoevent triggered by PKCθ at S1689 on GIV, which turns GIV-GEF “off” (16). However, what turns it “on” upon receptor activation still remained unknown.Here, we identify a key phosphoevent triggered by cyclin-dependent kinase 5 (CDK5) which turns on GIV''s GEF activity by phosphorylating it at Ser1674 and thereby increases GIV''s ability to bind Gαi/s and enhances its ability to activate Gαi. We provide mechanistic insight into how GIV-GEF is activated and also define a previously unidentified substrate by which CDK5 triggers cell migration. Thus, this study illustrates a physiologic event to activate GIV’s GEF function via a promigratory kinase, CDK5, which in turn dictates the orchestration of migration–proliferation dichotomy.     

Gonadotropin-releasing hormone induces actin cytoskeleton remodeling and affects cell migration in a cell-type-specific manner in TSU-Pr1 and DU145 cells

GnRH was first identified as the hypothalamic decapeptide that promotes gonadotropin release from pituitary gonadotropes. Thereafter, direct stimulatory and inhibitory effects of GnRH on cell proliferation were demonstrated in a number of types of primary cultured cells and established cell lines. Recently, the effects of GnRH on cell attachment, cytoskeleton remodeling, and cell migration have also been reported. Thus, the effects of GnRH on various cell activities are of great interest among researchers who study the actions of GnRH. In this study, we demonstrated that GnRH induces actin cytoskeleton remodeling and affects cell migration using two human prostatic carcinoma cell lines, TSU-Pr1 and DU145. In TSU-Pr1, GnRH-I and -II induced the filopodia formation of the cells and promoted cell migration, whereas in DU145, GnRH-I and -II induced the formation of the cells with stress fiber and inhibited cell migration. In our previous studies, we reported the stimulatory and inhibitory effects of GnRH on the cell proliferation of TSU-Pr1 and DU145 cells. This study provides the first evidence for the effects of GnRH on actin cytoskeleton remodeling and cell migration of cells in which cell proliferation was affected by GnRH at the same time. Moreover, we also demonstrated that the same human GnRH receptor subtype, human type I GnRH receptor, is essential for the effects of GnRH-I and -II on actin cytoskeleton remodeling and cell migration in both TSU-Pr1 and DU145 cells using the technique of gene knock-down by RNA interference.     

Estrogen and tamoxifen induce cytoskeletal remodeling and migration in endometrial cancer cells

Much research effort has been directed toward understanding how estrogen [17beta-estradiol (E2)] regulates cell proliferation and motility through the rapid, direct activation of cytoplasmic signaling cascades (i.e. nongenomic signaling). Cell migration is critical to cancer cell invasion and metastasis and involves dynamic filamentous actin cytoskeletal remodeling and disassembly of focal adhesion sites. Although estrogen is recognized to induce cell migration in some model systems, very little information is available regarding the underlying pathways and potential influence of selective estrogen receptor modulators such as 4-hydroxytamoxifen on these processes. Using the human endometrial cancer cell lines Hec 1A and Hec 1B as model systems, we have investigated the effects of E2 and Tam on endometrial nongenomic signaling, cytoskeletal remodeling, and cell motility. Results indicate that both E2 and Tam triggered rapid activation of ERK1/2, c-Src, and focal adhesion kinase signaling pathways and filamentous actin cytoskeletal changes. These changes included dissolution of stress fibers, dynamic actin accumulation at the cell periphery, and formation of lamellipodia, filopodia, and membrane spikes. Longer treatments with either agent induced cell migration in wound healing and Boyden chamber assays. Agent-induced cytoskeletal remodeling and cell migration were blocked by a Src inhibitor. These findings define cytoskeletal remodeling and cell migration as processes regulated by E2 and 4-hydroxytamoxifen nongenomic signaling in endometrial cancer. This new information may serve as the foundation for the development of new clinical therapeutic strategies.     

Rho kinase as a therapeutic target in cardiovascular disease

Rho kinase (ROCK) belongs to the AGC (PKA/PKG/PKC) family of serine/threonine kinases and is a major downstream effector of the small GTPase RhoA. ROCK plays central roles in the organization of the actin cytoskeleton and is involved in a wide range of fundamental cellular functions such as contraction, adhesion, migration, proliferation and gene expression. Two ROCK isoforms, ROCK1 and ROCK2, are assumed to be functionally redundant, based largely on the major common activators, the high degree of homology within the kinase domain and studies from overexpression with kinase constructs and chemical inhibitors (e.g., Y27632 and fasudil), which inhibit both ROCK1 and ROCK2. Extensive experimental and clinical studies support a critical role for the RhoA/ROCK pathway in the vascular bed in the pathogenesis of cardiovascular diseases, in which increased ROCK activity mediates vascular smooth muscle cell hypercontraction, endothelial dysfunction, inflammatory cell recruitment and vascular remodeling. Recent experimental studies, using ROCK inhibitors or genetic mouse models, indicate that the RhoA/ROCK pathway in myocardium contributes to cardiac remodeling induced by ischemic injury or persistent hypertrophic stress, thereby leading to cardiac decompensation and heart failure. This article, based on recent molecular, cellular and animal studies, focuses on the current understanding of ROCK signaling in cardiovascular diseases and in the pathogenesis of heart failure.     

PI3K/Akt/mTOR信号通路与低氧性肺动脉高压研究进展

肺动脉高压是一种预后不良的疾病,在我国因各种肺部慢性疾病引起的低氧性肺动脉高压近年来呈上升发病趋势.磷脂酰肌醇3-激酶(phosphatidylionsitol-3-kinases,PI3K)/蛋白质丝氨酸苏氨酸激酶(protein-serine-threonine kinase,Akt)/哺乳动物雷帕霉素蛋白(mammalian target of rapamycin,mTOR)信号通路作为细胞内重要信号转导通路之一,通过影响细胞的增殖、迁移、凋亡以及蛋白合成、细胞周期等活性参与肺动脉血管重塑以及低氧性肺动脉高压的形成.本文综述了PI3K/Akt/mTOR信号通路在低氧性肺动脉高压中的研究现状,以期为低氧性肺动脉高压的发病机制研究及治疗寻找新的思路.     

A SPIKE1 signaling complex controls actin-dependent cell morphogenesis through the heteromeric WAVE and ARP2/3 complexes

During morphogenesis, the actin cytoskeleton mediates cell-shape change in response to growth signals. In plants, actin filaments organize the cytoplasm in regions of polarized growth, and the filamentous arrays can be highly dynamic. Small GTPase signaling proteins termed Rho of plants (ROP)/RAC control actin polymerization. ROPs cycle between inactive GDP-bound and active GTP-bound forms, and it is the active form that interacts with effector proteins to mediate cytoskeletal rearrangement and cell-shape change. A class of proteins termed guanine nucleotide exchange factors (GEFs) generate GTP-ROP and positively regulate ROP signaling. However, in almost all experimental systems, it has proven difficult to unravel the complex signaling pathways from GEFs to the proteins that nucleate actin filaments. In this article, we show that the DOCK family protein SPIKE1 (SPK1) is a GEF, and that one function of SPK1 is to control actin polymerization via two heteromeric complexes termed WAVE and actin-related protein (ARP) 2/3. The genetic pathway was constructed by using a combination of highly informative spk1 alleles and detailed analyses of spk1, wave, and arp2/3 single and double mutants. Remarkably, we find that in addition to providing GEF activity, SPK1 associates with WAVE complex proteins and may spatially organize signaling. Our results describe a unique regulatory scheme for ARP2/3 regulation in cells, one that can be tested for widespread use in other multicellular organisms.     

Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression

Regulation of the actin-myosin cytoskeleton plays a central role in cell migration and cancer progression. Here, we report the discovery of a cytoskeleton-associated kinase, pseudopodium-enriched atypical kinase 1 (PEAK1). PEAK1 is a 190-kDa nonreceptor tyrosine kinase that localizes to actin filaments and focal adhesions. PEAK1 undergoes Src-induced tyrosine phosphorylation, regulates the p130Cas-Crk-paxillin and Erk signaling pathways, and operates downstream of integrin and epidermal growth factor receptors (EGFR) to control cell spreading, migration, and proliferation. Perturbation of PEAK1 levels in cancer cells alters anchorage-independent growth and tumor progression in mice. Notably, primary and metastatic samples from colon cancer patients display amplified PEAK1 levels in 81% of the cases. Our findings indicate that PEAK1 is an important cytoskeletal regulatory kinase and possible target for anticancer therapy.     

PI3K/Akt信号通路调节心脏功能的研究进展

PI3K/Akt是调节心脏功能的一条重要的信号转导通路,主要通过血管内皮生长因子介导血管生成、抑制心肌细胞凋亡、重构心室、促进细胞能量代谢等方面调节心脏功能。研究发现PI3K/Akt信号通路在内分泌疾病、肾病、肝纤维化、肿瘤及心血管疾病的发生、发展中起着重要作用。本文就PI3K/Akt信号通路调节心脏功能的分子机制作一阐述。     

G protein beta gamma subunit-dependent Rac-guanine nucleotide exchange activity of Ras-GRF1/CDC25(Mm)

Ras-GRF1 has been implicated as a Ras-specific guanine nucleotide exchange factor (GEF), which mediates calcium- and muscarinic receptor-triggered signals in the brain. Although a Dbl homology domain known as a motif conserved among GEFs that target Rho family GTP-binding proteins exists in Ras-GRF1, GEF activity toward Rho family proteins has not been observed. Here we show that Ras-GRF1 exhibits Rac1-specific GEF activity when recovered from cells overexpressing G protein beta gamma subunits (Gbeta gamma). Substitution of conserved amino acids within the Dbl homology domain abolished this activity. Activation of the Rac pathway in the cell was further evidenced by synergistic activation of the stress kinase JNK1 by Ras-GRF1 and Gbeta gamma, which is sensitive to inhibitory action of dominant-negative Rac1(17N). In addition, association of Ras-GRF1 with Rac1(17N) was demonstrated by coimmunoprecipitation. Evidence for the involvement of tyrosine kinase(s) in Gbeta gamma-mediated induction of Rac1-specific GEF activity was provided by the use of specific inhibitors. These results suggest a role of Ras-GRF1 for regulating Rac-dependent as well as Ras-dependent signaling pathways, particularly in the brain functions.     

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

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.The ability of cells to respond and adapt to external signals is achieved through the concerted action of several receptors and regulatory proteins. Receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) are the two most widely studied cell signaling hubs in eukaryotes. Canonical RTK signaling begins with ligand binding to the ectodomain of the receptor, leading to receptor dimerization followed by autophosphorylation of the tyrosine residues on the cytoplasmic tail and propagation of the signals to the interior of the cell via adaptor proteins (1). Canonical G protein signaling begins with ligand binding to GPCRs, which are seven transmembrane receptors with an intrinsic guanine nucleotide exchange factor (GEF) activity that enables G protein recruitment and subsequent activation through the exchange of GDP for the GTP nucleotide (2). For several decades these two pathways were believed to operate in a selective and discrete mode by transducing signals through their respective downstream intermediates. However, mounting evidence over time has unfolded a complex array of cross-talk between these two pathways, so that activated receptors from one pathway transactivate the other pathway either directly by activating the receptors (3) or indirectly by activating the downstream adaptor proteins (4). A well-documented and widely accepted phenomenon is transactivation of RTKs by GPCRs via scaffolding proteins such as β-arrestins (5). However, the reverse concept, i.e., transactivation of trimeric G proteins by RTKs, remains controversial. Despite numerous clues supporting the concept that growth factors trigger the activation of heterotrimeric G proteins (6), the fundamental question as to how such trigger occurs in cells remains poorly understood. This poorly understood concept is met with skepticism because there is no evidence that G proteins and ligand-activated RTKs come within close proximity in cells or that RTKs or any member of the growing family of signal-transducing adaptors used by RTKs can serve as GEFs. Some of these unanswered questions are being clarified by the discovery and characterization of Gα-interacting vesicle-associated protein (GIV; also known as “girdin”), an unusual signal transducer that can bind both RTKs and G proteins.GIV is a multimodular signal transducer (Fig. 1A) and a GEF for Gαi (7). Working downstream of a variety of growth factors [EGF (8, 9), IGF (10), VEGF (11), insulin (7, 12, 13), and PDGF receptor (14)], GIV enhances PI3K-Akt activity, links Akt signaling to actin cytoskeleton remodeling, and triggers cell migration, all via activation of Gαi (7). Because cells can modulate incoming growth factor signals from multiple RTKs by altering the cellular levels of GIV or selectively modulating its GEF function, we likened GIV to a rheostat by which cells tune incoming signals up or down (15). Consistent with its ability to signal downstream of a variety of receptors, GIV modulates growth factor signaling during diverse biological processes (15), e.g., cell migration, chemotaxis (13), invasion (16), development (17), self-renewal (18), apoptosis (19, 20), and autophagy (12). Additionally, evidence gathered by us and others has demonstrated the clinical significance of GIV-dependent signaling during diverse disease processes, e.g., pathologic angiogenesis (11), liver fibrosis (19), diabetes (21), nephrotic syndrome (20), vascular repair (22), and tumor metastasis across a variety of cancers [gastric (23), esophageal (24), prostate (16), breast (10, 25, 26), colon (27), and glioblastoma (18)].Fig. 1.Design and characterization of GIV-derived biosensors. (A, Upper) Schematic of various known functional domains of GIV. (Lower) The unique modular make-up of GIV''s CT with in-tandem coexistence of a GEF domain (red) that activates Gαi (gray), ...Despite the accumulating information on the biological and clinical significance of GIV, how it may couple to multiple RTKs remained unknown until recently. Protein interaction assays showed that GIV''s C terminus (CT) directly binds autophosphorylated cytoplasmic tails of multiple RTKs (9). Homology modeling, sequence analysis, side-chain substitution, and limited proteolysis showed that an ∼110-aa stretch within GIV''s CT folds into a Scr homology 2 (SH2)-like domain and is necessary and sufficient to recognize and bind phosphotyrosine peptides (28) (Fig. 1A) and recruit Gαi to RTKs. The discovery of coexisting SH2-like and GEF domains in tandem within the GIV CT (Fig. 1A) supported the idea that GIV''s CT has the necessary modular make-up to serve as a platform for linking G proteins to multiple RTKs. However, the in vitro and standard biochemical assays used thus far have failed to provide direct in cellulo evidence that GIV assembles RTK–GIV–Gαi ternary complexes, and if it does, when and where this assembly might occur, what might be the consequences of such assembly on G protein signaling, and how such signaling compares with the dynamics of canonical GPCR-driven G protein signaling. Such evidence would provide insights into the fundamental mechanisms that define GIV''s role at the cross-roads of RTK and G protein signaling pathways in diverse pathophysiologic processes. Such findings also will imply that the evolutionarily conserved CT of GIV serves as the long-sought modular platform for transactivation of G proteins downstream of growth factors, a phenomenon that has been observed and reported by several groups over the past few decades (6).     

Regulation of MT1-MMP and MMP-2 by leptin in cardiac fibroblasts involves Rho/ROCK-dependent actin cytoskeletal reorganization and leads to enhanced cell migration

Altered leptin action has been implicated in the pathophysiology of heart failure in obesity, a hallmark of which is extracellular matrix remodeling. Here, we characterize the direct influence of leptin on matrix metalloproteinase (MMP) activity in primary adult rat cardiac fibroblasts and focus on elucidating the molecular mechanisms responsible. Leptin increased expression and cell surface localization of membrane type 1 (MT1)-MMP, measured by cell surface biotinylation assay and antibody-based colorimetric detection of an exofacial epitope in intact cells. Coimmunoprecipitation analysis showed that leptin also induced the formation of a cluster of differentiation 44/MT1-MMP complex. Qualitative analysis using rhodamine-conjugated phalloidin immunofluorescence indicated that leptin stimulated actin cytoskeletal reorganization and enhanced stress fiber formation. Hence, we analyzed activation of Ras homolog gene family (Rho), member A GTPase activity and found a rapid increase in response to leptin that corresponded with increased phosphorylation of cofilin. Quantitative analysis of cytoskeleton reorganization upon separation of globular and filamentous actin by differential centrifugation confirmed the significant increase in filamentous to globular actin ratio in response to leptin, which was prevented by pharmacological inhibition of Rho (C3 transferase) or its downstream effector kinase Rho-associated coiled-coil-forming protein kinase (ROCK) (Y-27632). Inhibition of Rho or ROCK also attenuated leptin-stimulated increases in cell surface MT1-MMP content. Pro-MMP-2 is a known MT1-MMP substrate, and we observed that enhanced cell surface MT1-MMP in response to leptin resulted in enhanced extracellular activation of pro-MMP-2 measured by gelatin zymography, which was again attenuated by inhibition of Rho or ROCK. Using wound scratch assays, we observed enhanced cell migration, but not proliferation, measured by 5-bromo2'-deoxy-uridine incorporation, in response to leptin, again via a Rho-dependent signaling mechanism. Our results suggest that leptin regulates myocardial matrix remodeling by regulating the cell surface localization of MT1-MMP in adult cardiac fibroblasts via Rho/ROCK-dependent actin cytoskeleton reorganization. Subsequent pro-MMP-2 activation then contributes to stimulation of cell migration.