Rap1b is critical for glycoprotein VI-mediated but not ADP receptor-mediated alpha2beta1 activation.

BACKGROUND: The platelet alpha2beta1 integrin functions as both an adhesion and signaling receptor upon exposure to collagen. Recent studies have indicated that alpha2beta1 function can be activated via inside-out signaling, similar to the prototypical platelet integrin alphaIIbbeta3. However, signaling molecules that regulate alpha2beta1 activation in platelets are not well defined. A strong candidate molecule is the small GTPase Rap1b, the dominant platelet isoform of Rap1, which regulates alphaIIbbeta3 activation. OBJECTIVES: We hypothesized that Rap1b positively regulates alpha2beta1 during agonist-induced platelet activation. METHODS: To test whether Rap1b activates alpha2beta1 downstream of glycoprotein (GP)VI or other platelet receptors, we stimulated platelets purified from Rap1b-/- or wild-type mice with diverse agonists and measured alpha2beta1 activation using fluorescein isothiocyanate-labeled monomeric collagen. We also examined the role of Rap1b in outside-in signaling pathways by analyzing adhesion and spreading of Rap1b-/- or wild-type platelets on monomeric, immobilized collagen. Finally, we monitored the activation status of related Rap GTPases to detect changes in signaling pathways potentially associated with Rap1b-mediated events. RESULTS: Rap1b-/- platelets displayed comparable ADP-induced or thrombin-induced alpha2beta1 activation as wild-type platelets, but reduced convulxin-dependent alpha2beta1 activation. Rap1b-/- platelets exhibited increased spreading on immobilized collagen but similar adhesion to immobilized collagen compared to wild-type platelets. Rap1b-/- platelets also showed Rap1a and Rap2 activation upon agonist stimulation, possibly revealing functional compensation among Rap family members. CONCLUSIONS: Rap1b is required for maximal GPVI-induced but not ADP-induced activation of alpha2beta1 in murine platelets.

G2A is an oncogenic G protein-coupled receptor.

G2A is a heptahelical cell surface protein that has recently been described as a potential tumor suppressor, based on its ability to counteract transformation of pre-B cells and fibroblasts by Bcr-Abl, an oncogenic tyrosine kinase. We have isolated cDNAs encoding G2A in the course of screening libraries for clones that cause oncogenic transformation of NIH3T3 fibroblasts. When expressed at high levels in NIH3T3 cells by retroviral transduction, G2A induced a full range of phenotypes characteristic of oncogenic transformation, including loss of contact inhibition, anchorage-independent survival and proliferation, reduced dependence on serum, and tumorigenicity in mice. When expressed by transfection, G2A greatly enhanced the ability of a weakly oncogenic form of Raf-1 to transform NIH3T3 cells. These results demonstrate that G2A is potently oncogenic both on its own and in cooperation with another oncogene. Expression of G2A in fibroblasts and endothelial cells resulted in changes in cell morphology and cytoskeleton structure that were equivalent to those induced by the G protein subunit Galpha13. Transformation of NIH3T3 cells via G2A expression was completely suppressed by co-expression of LscRGS, a GTPase activating protein that suppresses signaling by Galpha12 and Galpha13. Hyperactivity of Galpha12 or Galpha13 has previously been shown to result in activation of Rho GTPases. G2A expression resulted in activation of Rho, and transformation via G2A was suppressed by a dominant negative form of RhoA. These results indicate that G2A may be directly coupled to Galpha13, and that it is the activation of this Rho-activating Galpha protein which is responsible for the ability of G2A to transform fibroblasts.

Microtubule depolymerization induces stress fibers, focal adhesions, and DNA synthesis via the GTP-binding protein Rho.

Microtubule depolymerization has multiple consequences that include actin stress fiber and focal adhesion assembly, increased tyrosine phosphorylation and DNA synthesis. Similar effects induced by serum, or agents such as lysophosphatidic acid, have previously been shown to be mediated by the GTP-binding protein Rho. We have investigated whether the effects of microtubule depolymerization are similarly mediated by Rho and show that they are blocked by the specific Rho inhibitor, C3 transferase. Because microtubule depolymerization induces these effects in quiescent cells, in which Rho is largely inactive, we conclude that microtubule depolymerization leads to activation of Rho. The activation of Rho in response to microtubule depolymerization and the consequent stimulation of contractility suggest a mechanism by which microtubules may regulate microfilament function in various motile phenomena. These range from growth cone extension to the development of the contractile ring during cytokinesis, in which there are interactions between the microtubule and microfilament systems.

Rho-mediated contractility exposes a cryptic site in fibronectin and induces fibronectin matrix assembly.

Many factors influence the assembly of fibronectin into an insoluble fibrillar extracellular matrix. Previous work demonstrated that one component in serum that promotes the assembly of fibronectin is lysophosphatidic acid (Zhang, Q., W.J. Checovich, D.M. Peters, R.M. Albrecht, and D.F. Mosher. 1994. J. Cell Biol. 127:1447-1459). Here we show that C3 transferase, an inhibitor of the low molecular weight GTP-binding protein Rho, blocks the binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment to cells and blocks the assembly of fibronectin into matrix induced by serum or lysophosphatidic acid. Microinjection of recombinant, constitutively active Rho into quiescent Swiss 3T3 cells promotes fibronectin matrix assembly by the injected cells. Investigating the mechanism by which Rho promotes fibronectin polymerization, we have used C3 to determine whether integrin activation is involved. Under conditions where C3 decreases fibronectin assembly we have only detected small changes in the state of integrin activation. However, several inhibitors of cellular contractility, that differ in their mode of action, inhibit cell binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment, decrease fibronectin incorporation into the deoxycholate insoluble matrix, and prevent fibronectin's assembly into fibrils on the cell surface. Because Rho stimulates contractility, these results suggest that Rho-mediated contractility promotes assembly of fibronectin into a fibrillar matrix. One mechanism by which contractility could enhance fibronectin assembly is by tension exposing cryptic self-assembly sites within fibronectin that is being stretched. Exploring this possibility, we have found a monoclonal antibody, L8, that stains fibronectin matrices differentially depending on the state of cell contractility. L8 was previously shown to inhibit fibronectin matrix assembly (Chernousov, M.A., A.I. Faerman, M.G. Frid, O.Y. Printseva, and V.E. Koteliansky. 1987. FEBS (Fed. Eur. Biochem. Soc.) Lett. 217:124-128). When it is used to stain normal cultures that are developing tension, it reveals a matrix indistinguishable from that revealed by polyclonal anti-fibronectin antibodies. However, the staining of fibronectin matrices by L8 is reduced relative to the polyclonal antibody when the contractility of cells is inhibited by C3. We have investigated the consequences of mechanically stretching fibronectin in the absence of cells. Applying a 30-35% stretch to immobilized fibronectin induced binding of soluble fibronectin, 70-kD fibronectin fragment, and L8 monoclonal antibody. Together, these results provide evidence that self-assembly sites within fibronectin are exposed by tension.

Mas oncogene signaling and transformation require the small GTP-binding protein Rac.

The Mas oncogene encodes a novel G-protein-coupled receptor that was identified originally as a transforming protein when overexpressed in NIH 3T3 cells. The mechanism and signaling pathways that mediate Mas transformation have not been determined. We observed that the foci of transformed NIH 3T3 cells caused by Mas were similar to those caused by activated Rho and Rac proteins. Therefore, we determined if Mas signaling and transformation are mediated through activation of a specific Rho family protein. First, we observed that, like activated Rac1, Mas cooperated with activated Raf and caused synergistic transformation of NIH 3T3 cells. Second, both Mas- and Rac1-transformed NIH 3T3 cells retained actin stress fibers and showed enhanced membrane ruffling. Third, like Rac, Mas induced lamellipodium formation in porcine aortic endothelial cells. Fourth, Mas and Rac1 strongly activated the JNK and p38, but not ERK, mitogen-activated protein kinases. Fifth, Mas and Rac1 stimulated transcription from common DNA promoter elements: NF-kappaB, serum response factor (SRF), Jun/ATF-2, and the cyclin D1 promoter. Finally, Mas transformation and some of Mas signaling (SRF and cyclin D1 but not NF-kappaB activation) were blocked by dominant negative Rac1. Taken together, these observations suggest that Mas transformation is mediated in part by activation of Rac-dependent signaling pathways. Thus, Rho family proteins are common mediators of transformation by a diverse variety of oncogene proteins that include Ras, Dbl family, and G-protein-coupled oncogene proteins.

Focal adhesion assembly.

The GTP-binding protein Rho regulates the assembly of focal adhesions and their associated bundles of actin filaments. Two different lines of research have converged to reveal how Rho might regulate assembly of these structures. One approach has been the identification of downstream effectors of Rho, whereas the other has been the exploration of the role of contractility in promoting assembly. It is now apparent that Rho is a key regulator of actomyosin-based contractility in nonmuscle cells and that contractility, combined with adhesion to a rigid substrate, leads to the formation of both stress fibres and focal adhesions.

Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation.

Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events.

Rho-stimulated contractility drives the formation of stress fibers and focal adhesions.

Activated rhoA, a ras-related GTP-binding protein, stimulates the appearance of stress fibers, focal adhesions, and tyrosine phosphorylation in quiescent cells (Ridley, A.J., and A. Hall, 1992. Cell. 70:389-399). The pathway by which rho triggers these events has not been elucidated. Many of the agents that activate rho (e.g., vasopressin, endothelin, lysophosphatidic acid) stimulate the contractility of smooth muscle and other cells. We have investigated whether rho's induction of stress fibers, focal adhesions, and tyrosine phosphorylation is the result of its stimulation of contractility. We demonstrate that stimulation of fibroblasts with lysophosphatidic acid, which activates rho, induces myosin light chain phosphorylation. This precedes the formation of stress fibers and focal adhesions and is accompanied by increased contractility. Inhibition of contractility by several different mechanisms leads to inhibition of rho-induced stress fibers, focal adhesions, and tyrosine phosphorylation. In addition, when contractility is inhibited, integrins disperse from focal adhesions as stress fibers and focal adhesions disassemble. Conversely, upon stimulation of contractility, diffusely distributed integrins are aggregated into focal adhesions. These results suggest that activated rho stimulates contractility, driving the formation of stress fibers and focal adhesions and elevating tyrosine phosphorylation. A model is proposed to account for how contractility could promote these events.

Focal adhesions, contractility, and signaling.

Focal adhesions are sites of tight adhesion to the underlying extracellular matrix developed by cells in culture. They provided a structural link between the actin cytoskeleton and the extracellular matrix and are regions of signal transduction that relate to growth control. The assembly of focal adhesions is regulated by the GTP-binding protein Rho. Rho stimulates contractility which, in cells that are tightly adherent to the substrate, generates isometric tension. In turn, this leads to the bundling of actin filaments and the aggregation of integrins (extracellular matrix receptors) in the plane of the membrane. The aggregation of integrins activates the focal adhesion kinase and leads to the assembly of a multicomponent signaling complex.

Tyrosine phosphorylation is involved in reorganization of the actin cytoskeleton in response to serum or LPA stimulation.

Tyrosine phosphorylation is known to regulate the formation of focal adhesions in cells adhering to extracellular matrix (ECM). We have investigated the possible involvement of tyrosine phosphorylation and the focal adhesion kinase (FAK) in the cytoskeletal changes induced by serum or lysophosphatidic acid (LPA) in quiescent Swiss 3T3 fibroblasts. As shown previously by others, quiescent cells stimulated with serum or LPA reveal a rapid reappearance of focal adhesions and stress fibers. Here we show that this is accompanied by an increase in phosphotyrosine in focal adhesions and specifically an increase in the tyrosine phosphorylation of FAK. The LPA-stimulated reappearance of focal adhesions and stress fibers is blocked by inhibitors of phospholipase C but not by pertussis toxin (PTX), indicating that this LPA signaling pathway is mediated by phospholipase C activation and does not involve PTX-sensitive G proteins. In the absence of serum or LPA, these cytoskeletal effects and the tyrosine phosphorylation of FAK can be mimicked by sodium orthovanadate in conjunction with hydrogen peroxide, agents that inhibit protein tyrosine phosphatases and thereby elevate levels of phosphotyrosine. Two tyrosine kinase inhibitors, erbstatin and genistein block both the serum-induced tyrosine phosphorylation of FAK and the assembly of focal adhesions and stress fibers. Two other tyrosine kinase inhibitors, tyrphostins 47 and 25, previously shown to inhibit FAK, failed to prevent FAK phosphorylation or the reassembly of focal adhesions and stress fibers in response to serum. However, these inhibitors did prevent FAK phosphorylation and cytoskeletal assembly in response to lysophosphatidic acid (LPA), one component of serum previously shown to stimulate assembly of focal adhesions and stress fibers. Our findings suggest that the response to serum is complex and that although FAK phosphorylation is important, other tyrosine kinases may also be involved.

Dbl and Vav mediate transformation via mitogen-activated protein kinase pathways that are distinct from those activated by oncogenic Ras.

Vav and Dbl are members of a novel class of oncogene proteins that share significant sequence identity in a approximately 250-amino-acid domain, designated the Dbl homology domain. Although Dbl functions as a guanine nucleotide exchange factor (GEF) and activator of Rho family proteins, recent evidence has demonstrated that Vav functions as a GEF for Ras proteins. Thus, transformation by Vav and Dbl may be a consequence of constitutive activation of Ras and Rho proteins, respectively. To address this possibility, we have compared the transforming activities of Vav and Dbl with that of the Ras GEF, GRF/CDC25. As expected, GRF-transformed cells exhibited the same reduction in actin stress fibers and focal adhesions as Ras-transformed cells. In contrast, Vav- and Dbl-transformed cells showed the same well-developed stress fibers and focal adhesions observed in normal or RhoA(63L)-transformed NIH 3T3 cells. Furthermore, neither Vav- or Dbl-transformed cells exhibited the elevated levels of Ras-GTP (60%) observed with GRF-transformed cells. Finally, GRF, but not Vav or Dbl, induced transcriptional activation from Ras-responsive DNA elements (ets/AP-1, fos promoter, and kappa B). However, like Ras- and GRF-transformed cells, both Vav- and Dbl-transformed cells exhibited constitutively activated mitogen-activated protein kinases (MAPKs) (primarily p42MAPK/ERK2). Since kinase-deficient forms of p42MAPK/ERK2 and p44MAPK/ERK1 inhibited Dbl transformation, MAPK activation may be an important component of its transforming activity. Taken together, our observations indicate that Vav and Dbl transformation is not a consequence of Ras activation and instead may involve the constitutive activation of MAPKs.