Identification of a novel Rac1-interacting protein involved in membrane ruffling.

The Rac GTP binding proteins are implicated in actin cytoskeleton-membrane interaction in mammalian cells. In fibroblast cells, Rac has been shown to mediate growth factor-induced polymerization of actin to form membrane ruffles and lamellipodia. We report here the isolation of a noval Rac1-interacting protein, POR1. POR1 binds directly to Rac1, and the interaction of POR1 with Rac1 is GTP dependent. A mutation in the Rac1 effector binding loop shown to abolish membrane ruffling also abolishes interaction with POR1. Truncated versions of POR1 inhibit the induction of membrane ruffling by an activated mutant of Rac1, V12Rac1, in quiescent rat embryonic fibroblast REF52 cells. Furthermore, POR1 synergizes with an activated mutant of Ras, V12Ras, in the induction of membrane ruffling. These results suggest a potential role for POR1 in Rac1-mediated signaling pathways.     

Integrin-mediated Signals Regulated by Members of the Rho Family of GTPases

The organization of the actin cytoskeleton can be regulated by soluble factors that trigger signal transduction events involving the Rho family of GTPases. Since adhesive interactions are also capable of organizing the actin-based cytoskeleton, we examined the role of Cdc42-, Rac-, and Rho-dependent signaling pathways in regulating the cytoskeleton during integrin-mediated adhesion and cell spreading using dominant-inhibitory mutants of these GTPases. When Rat1 cells initially adhere to the extracellular matrix protein fibronectin, punctate focal complexes form at the cell periphery. Concomitant with focal complex formation, we observed some phosphorylation of the focal adhesion kinase (FAK) and Src, which occurred independently of Rho family GTPases. However, subsequent phosphorylation of FAK and paxillin occurs in a Rho-dependent manner. Moreover, we found Rho dependence of the assembly of large focal adhesions from which actin stress fibers radiate. Initial adhesion to fibronectin also stimulates membrane ruffling; we show that this ruffling is independent of Rho but is dependent on both Cdc42 and Rac. Furthermore, we observed that Cdc42 controls the integrin-dependent activation of extracellular signal–regulated kinase 2 and of Akt, a kinase whose activity has been demonstrated to be dependent on phosphatidylinositol (PI) 3-kinase. Since Rac-dependent membrane ruffling can be stimulated by PI 3-kinase, it appears that Cdc42, PI 3-kinase, and Rac lie on a distinct pathway that regulates adhesion-induced membrane ruffling. In contrast to the differential regulation of integrin-mediated signaling by Cdc42, Rac, and Rho, we observed that all three GTPases regulate cell spreading, an event that may indirectly control cellular architecture. Therefore, several separable signaling pathways regulated by different members of the Rho family of GTPases converge to control adhesion-dependent changes in the organization of the cytoskeleton, changes that regulate cell morphology and behavior.     

Phosphatidylinositol 3-kinase, Cdc42, and Rac1 act downstream of Ras in integrin-dependent neurite outgrowth in N1E-115 neuroblastoma cells

Ras and Rho family GTPases have been ascribed important roles in signalling pathways determining cellular morphology and growth. Here we investigated the roles of the GTPases Ras, Cdc42, Rac1, and Rho and that of phosphatidylinositol 3-kinase (PI 3-kinase) in the pathway leading from serum starvation to neurite outgrowth in N1E-115 neuroblastoma cells. Serum-starved cells grown on a laminin matrix exhibited integrin-dependent neurite outgrowth. Expression of dominant negative mutants of Ras, PI 3-kinase, Cdc42, or Rac1 all blocked this neurite outgrowth, while constitutively activated mutants of Ras, PI 3-kinase, or Cdc42 were each sufficient to promote outgrowth even in the presence of serum. A Ras(H40C;G12V) double mutant which binds preferentially to PI 3-kinase also promoted neurite formation. Activated Ras(G12V)-induced outgrowth required PI 3-kinase activity, but activated PI 3-kinase-induced outgrowth did not require Ras activity. Although activated Rac1 by itself did not induce neurites, neurite outgrowth induced by activated Cdc42(G12V) was Rac1 dependent. Cdc42(G12V)-induced neurites appeared to lose their normal polarization, almost doubling the average number of neurites produced by a single cell. Outgrowth induced by activated Ras or PI 3-kinase required both Cdc42 and Rac1 activity, but Cdc42(G12V)-induced outgrowth did not need Ras or PI 3-kinase activity. Active Rho(G14V) reduced outgrowth promoted by Ras(G12V). Finally, expression of dominant negative Jun N-terminal kinase or extracellular signal-regulated kinase did not inhibit outgrowth, suggesting these pathways are not essential for this process. Our results suggest a hierarchy of signalling where Ras signals through PI 3-kinase to Cdc42 and Rac1 activation (and Rho inactivation), culminating in neurite outgrowth. Thus, in the absence of serum factors, Ras may initiate cell cycle arrest and terminal differentiation in N1E-115 neuroblastoma cells.     

Coactivation of Rac1 and Cdc42 at lamellipodia and membrane ruffles induced by epidermal growth factor

A major function of Rho-family GTPases is to regulate the organization of the actin cytoskeleton; filopodia, lamellipodia, and stress fiber are regarded as typical phenotypes of the activated Cdc42, Rac, and Rho, respectively. Using probes based on fluorescent resonance energy transfer, we report on the spatiotemporal regulation of Rac1 and Cdc42 at lamellipodia and membrane ruffles. In epidermal growth factor (EGF)-stimulated Cos1 and A431 cells, both Rac1 and Cdc42 were activated diffusely at the plasma membrane, followed by lamellipodial protrusion and membrane ruffling. Although Rac1 activity subsided rapidly, Cdc42 activity was sustained at lamellipodia. A critical role of Cdc42 in these EGF-induced morphological changes was demonstrated as follows. First, phorbol 12-myristate 13-acetate, which activated Rac1 but not Cdc42, could not induce full-grown lamellipodia in Cos1 cells. Second, a GTPase-activating protein for Cdc42, KIAA1204/CdGAP, inhibited lamellipodial protrusion and membrane ruffling without interfering with Rac1 activation. Third, expression of the Cdc42-binding domain of N-WASP inhibited the EGF-induced morphological changes. Therefore, Rac1 and Cdc42 seem to synergistically induce lamellipodia and membrane ruffles in EGF-stimulated Cos1 cells and A431 cells.     

Mutation of tyrosine 960 within the insulin receptor juxtamembrane domain impairs glucose transport but does not inhibit ligand-mediated phosphorylation of insulin receptor substrate-2 in 3T3-L1 adipocytes

CSF-1 is equipotent to insulin in its ability to stimulate 2-[3H]deoxyglucose uptake in 3T3-L1 adipocytes expressing the colony stimulating factor-1 receptor/insulin receptor chimera (CSF1R/IR). However, CSF-1-stimulated glucose uptake and glycogen synthesis is reduced by 50% in comparison to insulin in 3T3-L1 cells expressing a CSF1R/IR mutated at Tyr960 (CSF1R/IRA960). CSF-1-treated adipocytes expressing the CSF1R/IRA960 were impaired in their ability to phosphorylate insulin receptor substrate 1 (IRS-1) but not in their ability to phosphorylate IRS-2. Immunoprecipitation of IRS proteins followed by Western blotting revealed that the intact CSF1R/IR co-precipitates with IRS-2 from CSF-1-treated cells. In contrast, the CSF1R/IRA960 co-precipitates poorly with IRS-2. These observations suggest that Tyr960 is important for interaction of the insulin receptor cytoplasmic domain with IRS-2, but it is not essential to the ability of the insulin receptor tyrosine kinase to use IRS-2 as a substrate. These observations also suggest that in 3T3-L1 adipocytes, tyrosine phosphorylation of IRS-2 by the insulin receptor tyrosine kinase is not sufficient for maximal stimulation of receptor-regulated glucose transport or glycogen synthesis.     

NGF-dependent formation of ruffles in PC12D cells required a different pathway from that for neurite outgrowth

Two signaling pathways, phosphoinositide 3-kinase (PI-3k)/Akt and Ras/MAPK, are major effectors triggered by nerve growth factor (NGF). Rac1, Cdc42 and GSK-3beta are reported to be targets of PI-3k in the signal transduction for neurite outgrowth. Immediately after NGF was added, broad ruffles were observed temporarily around the periphery of PC12 cells prior to neurite growth. As PC12D cells are characterized by a very rapid extension of neurites in response to various agents, the signaling pathways described above were studied in relation to the NGF-induced formation of ruffles and outgrowth of neurites. Wortmannin, an Akt inhibitor (V), and GSK-3beta inhibitor (SB425286) suppressed the neurite growth in NGF-treated cells, but not in dbcAMP-treated cells. The outgrowth of neurites induced by NGF but not by dbcAMP was inhibited with the expression of mutant Ras. But upon the expression of dominant-negative Rac1, cells often extended protrusions, incomplete neurites, lacking F-actin. Intact neurites were observed in cells with dominant-negative Cdc42. These results suggest that NGF-dependent neurite outgrowth occurs via a mechanism involving activation of the Ras/PI-3K/Akt/GSK-3beta pathway, while dbcAMP-dependent neurite growth might be induced in a distinct manner. However, inhibitors for GSK-3beta and PI-3k (wortmannin) did not suppress the NGF-dependent formation of ruffles. In addition, the formation of ruffles was not inhibited by the expression of mutant Ras. On the other hand, it was suppressed by the expression of dominant-negative Rac1 or Cdc42. These results suggest that the NGF-induced ruffling requires activation of Rac1 and Cdc42, but does not require Ras, PI-3k, Akt and GSK-3beta. Taken together, the NGF-dependent formation of ruffles might not require Ras/PI-3k/Akt/GSK-3beta, but these pathways might contribute to the formation of intact neurites due to combined actions including Rac1.     

Activated Rac1 selectively up-regulates the expression of integrin alpha6beta4 and induces cell adhesion and membrane ruffles of nonadherent colon cancer Colo201 cells

Functions of small GTPases in integrin expression were investigated when the interaction of nonadherent human colon carcinoma 201 cells with the extracellular matrix (ECM) was examined. By transfection of the constitutively active form of a small GTPase Rac1, Rac V12, adhesion of cells to the ECM increased with concomitant cell spreading and formation of membrane ruffles. Activated Cdc42 and Cdc42 V12, but not wild-type Rac1, Cdc42, or RhoA, also induced the adhesion and spreading of Colo201 cells. This adhesion is integrin beta4 dependent since an antibody for integrin beta4 inhibited the RacV12-dependent cell adhesion and numbers of adhesive cells on laminin-coated plates exceeded those on collagen- and fibronectin-coated plates. By immunofluorescence, in addition to clustering of integrin molecules, expression of integrin alpha6beta4 on the cell surface of Rac V12- and Cdc42 V12-expressing cells was selectively up-regulated without an increase in biosynthesis of alpha6beta4 integrin. Treatment of Rac V12-expressing cells with wortmannin or LY294002, specific inhibitors of phosphoinositide 3-OH kinase, decreased the up-regulated alpha6beta4 and cell adhesion. In light of this evidence, we propose that the regulation of integrin alpha6beta4 expression induced by Rac1 and Cdc42 may play an important role in cell adhesion and tumorigenesis of colon carcinoma cells.     

RhoG GTPase Controls a Pathway That Independently Activates Rac1 and Cdc42Hs

RhoG is a member of the Rho family of GTPases that shares 72% and 62% sequence identity with Rac1 and Cdc42Hs, respectively. We have expressed mutant RhoG proteins fused to the green fluorescent protein and analyzed subsequent changes in cell surface morphology and modifications of cytoskeletal structures. In rat and mouse fibroblasts, green fluorescent protein chimera and endogenous RhoG proteins colocalize according to a tubular cytoplasmic pattern, with perinuclear accumulation and local concentration at the plasma membrane. Constitutively active RhoG proteins produce morphological and cytoskeletal changes similar to those elicited by a simultaneous activation of Rac1 and Cdc42Hs, i.e., the formation of ruffles, lamellipodia, filopodia, and partial loss of stress fibers. In addition, RhoG and Cdc42Hs promote the formation of microvilli at the cell apical membrane. RhoG-dependent events are not mediated through a direct interaction with Rac1 and Cdc42Hs targets such as PAK-1, POR1, or WASP proteins but require endogenous Rac1 and Cdc42Hs activities: coexpression of a dominant negative Rac1 impairs membrane ruffling and lamellipodia but not filopodia or microvilli formation. Conversely, coexpression of a dominant negative Cdc42Hs only blocks microvilli and filopodia, but not membrane ruffling and lamellipodia. Microtubule depolymerization upon nocodazole treatment leads to a loss of RhoG protein from the cell periphery associated with a reversal of the RhoG phenotype, whereas PDGF or bradykinin stimulation of nocodazole-treated cells could still promote Rac1- and Cdc42Hs-dependent cytoskeletal reorganization. Therefore, our data demonstrate that RhoG controls a pathway that requires the microtubule network and activates Rac1 and Cdc42Hs independently of their growth factor signaling pathways.     

Function of the Rho family GTPases in Ras-stimulated Raf activation.

Ras plays an essential role in activation of Raf kinase which is directly responsible for activation of the MEK-ERK kinase pathway. A direct protein-protein interaction between Ras and the N-terminal regulatory domain of Raf is critical for Raf activation. However, association with Ras is not sufficient to activate Raf in vitro, indicating that Ras must activate some other biochemical events leading to activation of Raf. We have observed that RasV12Y32F and RasV12T35S mutants fail to activate Raf, yet retain the ability to interact with Raf. In this report, we showed that RasV12Y32F and RasV12T35S can cooperate with members of the Rho family GTPases to activate Raf while alone the Rho family GTPase is not effective in Raf activation. A dominant negative mutant of Rac or RhoA can block Raf activation by Ras. The effect of Rac or Cdc42 can be substituted by the Pak kinase, which is a direct downstream target of Rac/Cdc42. Furthermore, expression of a kinase inactive mutant of Pak or the N-terminal inhibitory domain of Pak1 can block the effect of Rac or Cdc42. In contrast, Pak appears to play no direct role in relaying the signal from RhoA to Raf, indicating that RhoA utilizes a different mechanism than Rac/Cdc42. Membrane-associated but not cytoplasmic Raf can be activated by Rac or RhoA. Our data support a model by which the Rho family small GTPases play an important role to mediate the activation of Raf by Ras. Ras, at least, has two distinct functions in Raf activation, recruitment of Raf to the plasma membrane by direct binding and stimulation of Raf activating kinases via the Rho family GTPases.     

Serum-activated assembly and membrane translocation of an endogenous Rac1:effector complex

Rho family GTPases (Cdc42, Rac1, and RhoA) function downstream of Ras [1], and in a variety of cellular processes [2]. Studies to examine these functions have not directly linked endogenous protein interactions with specific in vivo functions of Rho GTPases. Here, we show that endogenous Rac1 and two known binding partners, Rho GDP dissociation inhibitor (RhoGDI) and p21-activated kinase (PAK), fractionate as distinct cytosolic complexes. A Rac1:PAK complex is translocated from the cytosol to ruffling membranes upon cell activation by serum. Overexpression of dominant-negative (T17N) Rac1 does not affect the assembly or distribution of this Rac1:PAK complex. This is the first direct evidence of how a specific function of Rac1 is selected by the assembly and membrane translocation of a distinct Rac1:effector complex.     

The survival kinase Mirk/dyrk1B is activated through Rac1-MKK3 signaling

The serine/threonine kinase Mirk/dyrk1B is activated in several solid tumors where it mediates cell survival, but the mechanism by which Mirk is activated in tumors is unknown. We now demonstrate that Mirk is activated as a kinase by signaling from Rac1 to the mitogen-activated protein kinase kinase MKK3. Rac is a Ras superfamily GTPase that, when activated, functions downstream of Ras oncoproteins to promote cell survival, transformation, and membrane ruffling. The constitutively active mutant Rac1QL activated Mirk in several cell types through MKK3, which in turn activated Mirk by phosphorylation. Dominant negative Rac1, dominant negative MKK3, and knockdown of MKK3 by RNA interference inhibited the kinase activity of co-expressed Mirk. E-cadherin ligation in confluent Madin-Darby canine kidney (MDCK) epithelial cells is known to transiently activate Rac1. Mirk was activated by endogenous Rac1 following E-cadherin ligation in confluent MDCK epithelial cells, whereas treatment of confluent MDCK cells with an Rac1 inhibitor decreased Mirk activity. Disruption of cadherin ligation by EGTA or prevention of cadherin ligation by maintenance of cells at subconfluent density blocked activation of Mirk. Engagement of cadherin molecules on subconfluent cells by an E-cadherin/Fc chimeric molecule transiently activated both Rac1 and Mirk with a similar time course. Rac activity is up-regulated in many human tumors and mediates survival signals, which enable tumor cells to evade apoptosis. This study characterizes a new anti-apoptotic signaling pathway that connects Rac1 with a novel downstream effector, Mirk kinase, which has recently been demonstrated to mediate survival in human tumors.     

The C-terminal domain of Rac1 contains two motifs that control targeting and signaling specificity

Rho-like GTPases control a wide range of cellular functions such as integrin- and cadherin-mediated adhesion, cell motility, and gene expression. The hypervariable C-terminal domain of these GTPases has been implicated in membrane association and effector binding. We found that cell-permeable peptides, encoding the C termini of Rac1, Rac2, RhoA, and Cdc42, interfere with GTPase signaling in a specific fashion in a variety of cellular models. Pull-down assays showed that the C terminus of Rac1 does not associate to either RhoGDI or to Pak. In contrast, the C terminus of Rac1 (but not Rac2 or Cdc42) binds to phosphatidylinositol 4,5-phosphate kinase (PIP5K) via amino acids 185-187 (RKR). Moreover, Rac1 associates to the adapter protein Crk via the N-terminal Src homology 3 (SH3) domain of Crk and the proline-rich stretch in the Rac1 C terminus. These differential interactions mediate Rac1 localization, as well as Rac1 signaling, toward membrane ruffling, cell-cell adhesion, and migration. These data show that the C-terminal, hypervariable domain of Rac1 encodes two distinct binding motifs for signaling proteins and regulates intracellular targeting and differential signaling in a unique and non-redundant fashion.     

IpgB1 is a novel Shigella effector protein involved in bacterial invasion of host cells. Its activity to promote membrane ruffling via Rac1 and Cdc42 activation

Shigella, the causative agent of bacillary dysentery, is capable of inducing the large scale membrane ruffling required for the bacterial invasion of host cells. Shigella secrete a subset of effectors via the type III secretion system (TTSS) into the host cells to induce membrane ruffling. Here, we show that IpgB1 is secreted via the TTSS into epithelial cells and plays a major role in producing membrane ruffles via stimulation of Rac1 and Cdc42 activities, thus promoting bacterial invasion of epithelial cells. The invasiveness of the ipgB1 mutant was decreased to less than 50% of the wild-type level (100%) in a gentamicin protection or plaque forming assay. HeLa cells infected with the wild-type or a IpgB1-hyperproducing strain developed membrane ruffles, with the invasiveness and the scale of membrane ruffles being comparable with the level of IpgB1 production in bacteria. Upon expression of EGFP-IpgB1 in HeLa cells, large membrane ruffles are extended, where the EGFP-IpgB1 was predominantly associated with the cytoplasmic membrane. The IpgB1-mediated formation of ruffles was significantly diminished by expressing Rac1 small interfering RNA and Cdc42 small interfering RNA or by treatment with GGTI-298, an inhibitor of the geranylgeranylation of Rho GTPases. When IpgB1 was expressed in host cells or wild-type Shigella-infected host cells, Rac1 and Cdc42 were activated. The results thus indicate that IpgB1 is a novel Shigella effector involved in bacterial invasion of epithelial cells via the activation of Rho GTPases.     

Activated Rac1 Selectively Up-regulates the Expression of Integrin α6β4 and Induces Cell Adhesion and Membrane Ruffles of Nonadherent Colon Cancer Colo201 Cells

Functions of small GTPases in integrin expression were investigated when the interaction of nonadherent human colon carcinoma 201 cells with the extracellular matrix (ECM) was examined. By transfection of the constitutively active form of a small GTPase Rac1, Rac V12, adhesion of cells to the ECM increased with concomitant cell spreading and formation of membrane ruffles. Activated Cdc42 and Cdc42 V12, but not wild-type Rac1, Cdc42, or RhoA, also induced the adhesion and spreading of Colo201 cells. This adhesion is integrin β4 dependent since an antibody for integrin β4 inhibited the RacV12-dependent cell adhesion and numbers of adhesive cells on laminin-coated plates exceeded those on collagen- and fibronectin-coated plates. By immunofluorescence, in addition to clustering of integrin molecules, expression of integrin α6β4 on the cell surface of Rac V12- and Cdc42 V12-expressing cells was selectively up-regulated without an increase in biosynthesis of α6β4 integrin. Treatment of Rac V12-expressing cells with wortmannin or LY294002, specific inhibitors of phosphoinositide 3-OH kinase, decreased the up-regulated α6β4 and cell adhesion. In light of this evidence, we propose that the regulation of integrin α6β4 expression induced by Rac1 and Cdc42 may play an important role in cell adhesion and tumorigenesis of colon carcinoma cells.     

Ras-GAP Controls Rho-Mediated Cytoskeletal Reorganization through Its SH3 Domain

Proteins of the Ras superfamily, Ras, Rac, Rho, and Cdc42, control the remodelling of the cortical actin cytoskeleton following growth factor stimulation. A major regulator of Ras, Ras-GAP, contains several structural motifs, including an SH3 domain and two SH2 domains, and there is evidence that they harbor a signalling function. We have previously described a monoclonal antibody to the SH3 domain of Ras-GAP which blocks Ras signalling in Xenopus oocytes. We now show that microinjection of this antibody into Swiss 3T3 cells prevents the formation of actin stress fibers stimulated by growth factors or activated Ras, but not membrane ruffling. This inhibition is bypassed by coinjection of activated Rho, suggesting that the Ras-GAP SH3 domain is necessary for endogenous Rho activation. In agreement, the antibody blocks lysophosphatidic acid-induced neurite retraction in differentiated PC12 cells. Furthermore, we demonstrate that microinjection of full-length Ras-GAP triggers stress fiber polymerization in fibroblasts in an SH3-dependent manner, strongly suggesting an effector function besides its role as a Ras downregulator. These results support the idea that Ras-GAP connects the Ras and Rho pathways and, therefore, regulates the actin cytoskeleton through a mechanism which probably does not involve p190 Rho-GAP.     

Rich, a rho GTPase-activating protein domain-containing protein involved in signaling by Cdc42 and Rac1.

A previously unidentified Rho GTPase-activating protein (GAP) domain-containing protein was found in a yeast two-hybrid screen for cDNAs encoding proteins binding to the Src homology 3 domain of Cdc42-interacting protein 4 (CIP4). The protein was named RICH-1 (RhoGAP interacting with CIP4 homologues), and, in addition to the RhoGAP domain, it contained an N-terminal domain with endophilin homology and a C-terminal proline-rich domain. Transient transfections of RICH-1 indicated that it bound to CIP4 in vivo, as shown by co-immunoprecipitation experiments, as well as co-localization assays. In vitro assays demonstrated that the RhoGAP domain of RICH-1 catalyzed GTP hydrolysis on Cdc42 and Rac1, but not on RhoA. Ectopic expression of the RhoGAP domain as well as the full-length protein interfered with platelet-derived growth factor BB-induced membrane ruffling, but not with serum-induced stress fiber formation, further emphasizing the notion that, in vivo, RICH-1 is a GAP for Cdc42 and Rac1.     

Collapsin response mediator protein switches RhoA and Rac1 morphology in N1E-115 neuroblastoma cells and is regulated by Rho kinase

The formation and directional guidance of neurites involves dynamic regulation of Rho family GTPases. Rac and Cdc42 promote neurite outgrowth, whereas Rho activation causes neurite retraction. Here we describe a role for collapsin response mediator protein (Crmp-2), a neuronal protein implicated in axonal outgrowth and a component of the semaphorin 3A pathway, in switching GTPase signaling when expressed in combination with either dominant active Rac or Rho. In neuroblastoma N1E-115 cells, co-expression of Crmp-2 with dominant active RhoA V14 induced Rac morphology, cell spreading and ruffling (and the formation of neurites). Conversely, co-expression of Crmp-2 with dominant active Rac1 V12 inhibited Rac morphology, and in cells already expressing Rac1 V12, Crmp-2 caused localized peripheral collapse, involving Rho (and Cdc42) activation. Rho kinase was a pivotal regulator of Crmp-2; Crmp-2 phosphorylation was required for Crmp-2/Rac1 V12 inhibition, but not Crmp-2/RhoA V14 induction, of Rac morphology. Thus Crmp-2, regulated by Rho kinase, promotes outgrowth and collapse in response to active Rho and Rac, respectively, reversing their usual morphological effects and providing a mechanism for dynamic modulation of growth cone guidance.     

Invasion of T-lymphoma cells: cooperation between Rho family GTPases and lysophospholipid receptor signaling.

Rho-like GTPases orchestrate distinct cytoskeletal changes in response to receptor stimulation. Invasion of T-lymphoma cells into a fibroblast monolayer is induced by Tiam1, an activator of the Rho-like GTPase Rac, and by constitutively active V12Rac1. Here we show that activated V12Cdc42 can also induce invasion of T-lymphoma cells. Activated RhoA potentiates invasion, but fails by itself to mimic Rac and Cdc42. However, invasion is inhibited by the Rho-inactivating C3 transferase. Thus, RhoA is required but not sufficient for invasion. Invasion of T-lymphoma cells is critically dependent on the presence of serum. Serum can be replaced by the serum-borne lipids lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) (10(-7)-10(-6) M), which act on distinct G protein-linked receptors to activate RhoA and phospholipase C (PLC)-Ca2+ signaling. LPA- and S1P-induced invasion is preceded by Rho-dependent F-actin redistribution and pseudopodia formation. However, expression of both V14RhoA and V12Rac1 does not bypass the LPA/S1P requirement for invasion, indicating involvement of an additional signaling pathway independent of RhoA. The PLC inhibitor U-73122, but not the inactive analog U-73343, abolishes invasion. Our results indicate that T-lymphoma invasion is driven by Tiam1/Rac or Cdc42 activation, and is dependent on LPA/S1P receptor-mediated RhoA and PLC signaling pathways which lead to pseudopod formation and enhanced infiltration.     

Regulation of the protein kinase Raf-1 by oncogenic Ras through phosphatidylinositol 3-kinase, Cdc42/Rac and Pak

Activation of the protein kinase Raf-1 is a complex process involving association with the GTP-bound form of Ras (Ras-GTP), membrane translocation and both serine/threonine and tyrosine phosphorylation (reviewed in [1]). We have reported previously that p21-activated kinase 3 (Pak3) upregulates Raf-1 through direct phosphorylation on Ser338 [2]. Here, we investigated the origin of the signal for Pak-mediated Raf-1 activation by examining the role of the small GTPase Cdc42, Rac and Ras, and of phosphatidylinositol (PI) 3-kinase. Pak3 acted synergistically with either Cdc42V12 or Rac1V12 to stimulate the activities of Raf-1, Raf-CX, a membrane-localized Raf-1 mutant, and Raf-1 mutants defective in Ras binding. Raf-1 mutants defective in Ras binding were also readily activated by RasV12. This indirect activation of Raf-1 by Ras was blocked by a dominant-negative mutant of Pak, implicating an alternative Ras effector pathway in Pak-mediated Raf-1 activation. Subsequently, we show that Pak-mediated Raf-1 activation is upregulated by both RasV12C40, a selective activator of PI 3-kinase, and p110-CX, a constitutively active PI 3-kinase. In addition, p85Delta, a mutant of the PI 3-kinase regulatory subunit, inhibited the stimulated activity of Raf-1. Pharmacological inhibitors of PI 3-kinase also blocked both activation and Ser338 phosphorylation of Raf-1 induced by epidermal growth factor (EGF). Thus, Raf-1 activation by Ras is achieved through a combination of both physical interaction and indirect mechanisms involving the activation of a second Ras effector, PI 3-kinase, which directs Pak-mediated regulatory phosphorylation of Raf-1.