Unmasking of LPA1 receptor-mediated migration response to lysophosphatidic acid by interleukin-1β-induced attenuation of Rho signaling pathways in rat astrocytes

Action mechanism of lipopolysaccharide (LPS), interleukin-1β (IL-1β), and lysophosphatidic acid (LPA) to regulate motility, an important process of astrogliosis, was investigated in rat astrocytes. While LPA exerted no significant effect on the cell migration, the prior treatment of the cells with LPS or IL-1β resulted in the appearance of migration activity in response to LPA. The LPS induction of the migration response to LPA was associated with the production of IL-1β precursor protein and inhibited by the IL-1 receptor antagonist. The IL-1β treatment also allowed LPA to activate Rac1. The LPA-induced Rac1 activation and migration were inhibited by pertussis toxin, a small interfering RNA specific to LPA(1) receptors, and LPA(1) receptor antagonists, including Ki16425. However, the IL-1β treatment had no appreciable effect on LPA(1) receptor mRNA expression and LPA-induced activation of ERK, Akt, and proliferation. The induction of the migration response to LPA by IL-1β was inhibited by a constitutively active RhoA. Moreover, LPA significantly activated RhoA through the LPA(1) receptor in the control cells but not in the IL-1β-treated cells. These results suggest that IL-1β inhibits the LPA(1) receptor-mediated Rho signaling through the IL-1 receptor, thereby disclosing the LPA(1) receptor-mediated G(i) protein/Rac/migration pathway.     

Physical and functional interactions of the lysophosphatidic acid receptors with PDZ domain-containing Rho guanine nucleotide exchange factors (RhoGEFs)

Lysophosphatidic acid (LPA) is a serum-derived phospholipid that induces a variety of biological responses in various cells via heterotrimeric G protein-coupled receptors (GPCRs) including LPA1, LPA2, and LPA3. LPA-induced cytoskeletal changes are mediated by Rho family small GTPases, such as RhoA, Rac1, and Cdc42. One of these small GTPases, RhoA, may be activated via Galpha(12/13)-linked Rho-specific guanine nucleotide exchange factors (RhoGEFs) under LPA stimulation although the detailed mechanisms are poorly understood. Here, we show that the C terminus of LPA1 and LPA2 but not LPA3 interact with the PDZ domains of PDZ domain-containing RhoGEFs, PDZ-RhoGEF, and LARG, which are comprised of PDZ, RGS, Dbl homology (DH), and pleckstrin homology (PH) domains. In LPA1- and LPA2-transfected HEK293 cells, LPA-induced RhoA activation was observed although the C terminus of LPA1 and LPA2 mutants, which failed to interact with the PDZ domains, did not cause LPA-induced RhoA activation. Furthermore, overexpression of the PDZ domains of PDZ domain-containing RhoGEFs served as dominant negative mutants for LPA-induced RhoA activation. Taken together, these results indicate that formation of the LPA receptor/PDZ domain-containing RhoGEF complex plays a pivotal role in LPA-induced RhoA activation.     

Rho-mediated cytoskeletal rearrangement in response to LPA is functionally antagonized by Rac1 and PIP2

G-protein-coupled receptors signal through Rho to induce actin cytoskeletal rearrangement. We previously demonstrated that thrombin stimulates Rho-dependent process retraction and rounding of 1321N1 astrocytoma cells. Surprisingly, while lysophosphatidic acid (LPA) activated RhoA in 1321N1 cells, it failed to produce cell rounding. Thrombin, unlike LPA, decreased Rac1 activity, and activated (GTPase-deficient) Rac1 inhibited thrombin-stimulated cell rounding, while expression of dominant-negative Rac1 promoted LPA-induced rounding. LPA and thrombin receptors appear to differ in coupling to Gi, as LPA but not thrombin-stimulated 1321N1 cell proliferation was pertussis toxin-sensitive. Blocking Gi with pertussis toxin enabled LPA to induce cell rounding and to decrease activated Rac1. These data support the hypothesis that Rac1 and Gi activation antagonize cell rounding. Thrombin and LPA receptors also differentially activated Gq pathways as thrombin but not LPA increased InsP3 formation and reduced phosphatidylinositol 4,5-bisphosphate (PIP2) levels. Microinjection of the plekstrin homology domain of phospholipase C (PLC)delta1, which binds PIP2, enabled LPA to elicit cell rounding, consistent with a requirement for PIP2 reduction. We suggest that Rho-mediated cytoskeletal responses are enhanced by concomitant reductions in cellular levels of PIP2 and Rac1 activation and thus effected only by G-protein-coupled receptors with appropriate subsets of G protein activation.     

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.     

The Rac activator Tiam1 is required for (alpha)3(beta)1-mediated laminin-5 deposition, cell spreading, and cell migration

The Rho-like guanosine triphosphatase Rac1 regulates various signaling pathways, including integrin-mediated adhesion and migration of cells. However, the mechanisms by which integrins signal toward Rac are poorly understood. We show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis 1) is required for the integrin-mediated laminin (LN)-5 deposition, spreading, and migration of keratinocytes. In contrast to wild-type keratinocytes, Tiam1-deficient (Tiam1-/-) keratinocytes are unable to adhere to and spread on a glass substrate because they are unable to deposit their own LN5 substrate. Both Tiam1 and V12Rac1 can rescue the defects of Tiam1-/- keratinocytes, indicating that these deficiencies are caused by impaired Tiam1-mediated Rac activation. Tiam1-/- cells are unable to activate Rac upon alpha3beta1-mediated adhesion to an exogenous LN5 substrate. Moreover, Tiam1 deficiency impairs keratinocyte migration in vitro and reepithelialization of excision wounds in mouse skin. Our studies indicate that Tiam1 is a key molecule in alpha3beta1-mediated activation of Rac, which is essential for proper production and secretion of LN5, a requirement for the spreading and migration of keratinocytes.     

Integrin α6β4 cooperates with LPA signaling to stimulate Rac through AKAP-Lbc-mediated RhoA activation

The α(6)β(4) integrin promotes carcinoma invasion through its ability to promote directed migration and polarization of carcinoma cells. In this study, we explore how the α(6)β(4) integrin cooperates with lysophosphatidic acid (LPA) to activate Rho and Rac small GTPases. Through the use of dominant negative Rho constructs, C3 exotransferase, and Rho kinase inhibitor, we find that Rho is critical for LPA-dependent chemotaxis and lamellae formation. However, utilization of specific Rho isoforms depends on integrin α(6)β(4) expression status. Integrin α(6)β(4)-negative MDA-MB-435 cells utilize only RhoC for motility, whereas integrin α(6)β(4)-expressing cells utilize RhoC but additionally activate and utilize RhoA for LPA-dependent cell motility and lamellae formation. Notably, the activation of RhoA by cooperative LPA and integrin α(6)β(4) signaling requires the Rho guanine nucleotide exchange factor AKAP-Lbc. We also determine that integrin α(6)β(4) cannot activate Rac1 directly but promotes LPA-mediated Rac1 activation that is dependent on RhoA activity and de novo β(1) integrin ligation. Finally, we find that the regulation of Rac1 and RhoA in response to LPA is differentially regulated by phosphodiesterases, PKA, and phosphatidylinositol 3-kinase, thus supporting their spatially distinct compartmentalization. In summary, signaling from integrin α(6)β(4) facilitates LPA-stimulated chemotaxis through preferential activation of RhoA, which, in turn, facilitates activation of Rac1.     

Scaffold Proteins IRSp53 and Spinophilin Regulate Localized Rac Activation by T-lymphocyte Invasion and Metastasis Protein 1 (TIAM1)

The Rac exchange factor Tiam1 is involved in diverse cell functions and signaling pathways through multiple protein interactions, raising the question of how signaling and functional specificity are achieved. We have shown that Tiam1 interactions with different scaffold proteins activate different Rac-dependent pathways by recruiting specific Rac effector proteins, and reasoned that there must be regulatory mechanisms governing each interaction. Fibroblasts express at least two Tiam1-interacting proteins, insulin receptor substrate protein 53 kDa (IRSp53) and spinophilin. We used fluorescent resonance energy transfer (FRET) to measure localized Rac activation associated with IRSp53 and spinophilin complexes in individual fibroblasts to test this hypothesis. Pervanadate or platelet-derived growth factor induced localized Rac activation dependent on Tiam1 and IRSp53. Forskolin or epinephrine induced localized Rac activation dependent on Tiam1 and spinophilin. In spinophilin-deficient cells, Tiam1 co-localized with IRSp53 in response to pervanadate or platelet-derived growth factor. In IRSp53-deficient cells, Tiam1 co-localized with spinophilin in response to forskolin or epinephrine. Total cellular levels of activated Rac were affected only in cells with exogenous Tiam1, and were primarily increased in the membrane fraction. Downstream effects of Rac activation were also stimulus and scaffold-specific. Cell ruffling, spreading, and cell adhesion were dependent on IRSp53, but not spinophilin. Epinephrine decreased IRSp53-dependent adhesion and increased cell migration in a Rac and spinophilin-dependent fashion. These results support the idea that Tiam1 interactions with different scaffold proteins couple distinct upstream signals to localized Rac activation and specific downstream pathways, and suggest that manipulating Tiam1-scaffold interactions can modulate Rac-dependent cellular behaviors.     

Rho-kinase phosphorylates PAR-3 and disrupts PAR complex formation

A polarity complex of PAR-3, PAR-6, and atypical protein kinase C (aPKC) functions in various cell polarization events. PAR-3 directly interacts with Tiam1/Taim2 (STEF), Rac1-specific guanine nucleotide exchange factors, and forms a complex with aPKC-PAR-6-Cdc42*GTP, leading to Rac1 activation. RhoA antagonizes Rac1 in certain types of cells. However, the relationship between RhoA and the PAR complex remains elusive. We found here that Rho-kinase/ROCK/ROK, the effector of RhoA, phosphorylated PAR-3 at Thr833 and thereby disrupted its interaction with aPKC and PAR-6, but not with Tiam2. Phosphorylated PAR-3 was observed in the leading edge, and in central and rear portions of migrating cells having front-rear polarity. Knockdown of PAR-3 by small interfering RNA (siRNA) impaired cell migration, front-rear polarization, and PAR-3-mediated Rac1 activation, which were recovered with siRNA-resistant PAR-3, but not with the phospho-mimic PAR-3 mutant. We propose that RhoA/Rho-kinase inhibits PAR complex formation through PAR-3 phosphorylation, resulting in Rac1 inactivation.     

MEK/ERK regulates adherens junctions and migration through Rac1

Polyamine depletion with the ornithine decarboxylase inhibitor alpha-difluoromethyl ornithine (DFMO), prevents Rac1 activation causing the formation of a thick actin cortex at the cell periphery and inhibits migration of intestinal epithelial cells. In the present study, we demonstrate that MEK activation by EGF increased Rac1 activation, dissociation of intercellular contacts, and migration in both control and polyamine-depleted cells, while U0126, a specific inhibitor of MEK1, prevented disruption of junctions as well as EGF-induced Rac1 activation. Constitutively active MEK1 (CA-MEK) expression altered cell-cell contacts in control and polyamine depleted cells. The expression of constitutively active Rac1 (CA-Rac1) restored beta-catenin to the cell periphery and prevented the formation of actin cortex and caused the appearance of F-actin stress fibers in polyamine-depleted cells. Inhibition of Rac activation by NSC23766, a specific inhibitor of Tiam1, an upstream guanidine nucleotide exchange factor for Rac1, reproduced the beta-catenin localization and actin structure of polyamine-depleted cells. Tiam1 localized more extensively with beta-catenin at the cell periphery in CA-Rac1 cells compared to vector cells. Polyamine depletion decreased the expression of E-cadherin to a greater extent compared to beta-catenin. Subcellular fractionation further confirmed our immuno-localization and western blotting observations. These data suggest that EGF acting through MEK1/ERK to activate Rac1 regulates cell-cell contacts. Thus, decreased migration in polyamine depleted cells may be due to the inhibition of Tiam1 activation of Rac1 and the subsequent decreased expression of beta-catenin and E-cadherin leading to reduced cell-cell contacts.     

Lipid phosphate phosphatase-1 regulates lysophosphatidate-induced fibroblast migration by controlling phospholipase D2-dependent phosphatidate generation

Lysophosphatidate (LPA) stimulates cell migration and division through a family of G-protein-coupled receptors. Lipid phosphate phosphatase-1 (LPP1) regulates the degradation of extracellular LPA as well as the intracellular accumulation of lipid phosphates. Here we show that increasing the catalytic activity of LPP1 decreased the pertussis toxin-sensitive stimulation of fibroblast migration by LPA and an LPA-receptor agonist that could not be dephosphorylated. Conversely, knockdown of endogenous LPP1 activity increased LPA-induced migration. However, LPP1 did not affect PDGF- or endothelin-induced migration of fibroblasts in Transwell chamber and "wound healing" assays. Thus, in addition to degrading exogenous LPA, LPP1 controls signaling downstream of LPA receptors. Consistent with this conclusion, LPP1 expression decreased phospholipase D (PLD) stimulation by LPA and PDGF, and phosphatidate accumulation. This LPP1 effect was upstream of PLD activation in addition to the possible metabolism of phosphatidate to diacylglycerol. PLD(2) activation was necessary for LPA-, but not PDGF-induced migration. Increased LPP1 expression also decreased the LPA-, but not the PDGF-induced activation of important proteins involved in fibroblast migration. These included decreased LPA-induced activation of ERK and Rho, and the basal activities of Rac and Cdc42. However, ERK and Rho activation were not downstream targets of LPA-induced PLD(2) activity. We conclude that the intracellular actions of LPP1 play important functions in regulating LPA-induced fibroblast migration through PLD2. LPP1 also controls PDGF-induced phosphatidate formation. These results shed new light on the roles of LPP1 in controlling wound healing and the growth and metastasis of tumors.     

Tiam1 interaction with the PAR complex promotes talin-mediated Rac1 activation during polarized cell migration

Migrating cells acquire front-rear polarity with a leading edge and a trailing tail for directional movement. The Rac exchange factor Tiam1 participates in polarized cell migration with the PAR complex of PAR3, PAR6, and atypical protein kinase C. However, it remains largely unknown how Tiam1 is regulated and contributes to the establishment of polarity in migrating cells. We show here that Tiam1 interacts directly with talin, which binds and activates integrins to mediate their signaling. Tiam1 accumulated at adhesions in a manner dependent on talin and the PAR complex. The interactions of talin with Tiam1 and the PAR complex were required for adhesion-induced Rac1 activation, cell spreading, and migration toward integrin substrates. Furthermore, Tiam1 acted with talin to regulate adhesion turnover. Thus, we propose that Tiam1, with the PAR complex, binds to integrins through talin and, together with the PAR complex, thereby regulates Rac1 activity and adhesion turnover for polarized migration.     

The N-terminal DH-PH domain of Trio induces cell spreading and migration by regulating lamellipodia dynamics in a Rac1-dependent fashion

The guanine-nucleotide exchange factor Trio encodes two DH-PH domains that catalyze nucleotide exchange on Rac1, RhoG and RhoA. The N-terminal DH-PH domain is known to activate Rac1 and RhoG, whereas the C-terminal DH-PH domain can activate RhoA. The current study shows that the N-terminal DH-PH domain, upon expression in HeLa cells, activates Rac1 and RhoG independently from each other. In addition, we show that the flanking SH3 domain binds to the proline-rich region of the C-terminus of Rac1, but not of RhoG. However, this SH3 domain is not required for Rac1 or RhoG GDP-GTP exchange. Rescue experiments in Trio-shRNA-expressing cells showed that the N-terminal DH-PH domain of Trio, but not the C-terminal DH-PH domain, restored fibronectin-mediated cell spreading and migration defects that are observed in Trio-silenced cells. Kymograph analysis revealed that the N-terminal DH-PH domain, independent of its SH3 domain, controls the dynamics of lamellipodia. Using siRNA against Rac1 or RhoG, we found that Trio-D1-induced lamellipodia formation required Rac1 but not RhoG expression. Together, we conclude that the GEF Trio is responsible for lamellipodia formation through its N-terminal DH-PH domain in a Rac1-dependent manner during fibronectin-mediated spreading and migration.     

p85 beta-PIX is required for cell motility through phosphorylations of focal adhesion kinase and p38 MAP kinase

Lysophosphatidic acid (LPA) mediates diverse biological responses, including cell migration, through the activation of G-protein-coupled receptors. Recently, we have shown that LPA stimulates p21-activated kinase (PAK) that is critical for focal adhesion kinase (FAK) phosphorylation and cell motility. Here, we provide the direct evidence that p85 beta-PIX is required for cell motility of NIH-3T3 cells by LPA through FAK and p38 MAP kinase phosphorylations. LPA induced p85 beta-PIX binding to FAK in NIH-3T3 cells that was inhibited by pretreatment of the cells with phosphoinositide 3-kinase inhibitor, LY294002. Furthermore, the similar inhibition of the complex formation was also observed, when the cells were transfected with either p85 beta-PIX mutant that cannot bind GIT or dominant negative mutants of Rac1 (N17Rac1) and PAK (PAK-PID). Transfection of the cells with specific p85 beta-PIX siRNA led to drastic inhibition of LPA-induced FAK phosphorylation, peripheral redistribution of p85 beta-PIX with FAK and GIT1, and cell motility. p85 beta-PIX was also required for p38 MAP kinase phosphorylation induced by LPA. Finally, dominant negative mutant of Rho (N19Rho)-transfected cells did not affect PAK activation, while the cells stably transfected with p85 beta-PIX siRNA or N17Rac1 showed the reduction of LPA-induced PAK activation. Taken together, the present data suggest that p85 beta-PIX, located downstream of Rac1, is a key regulator for the activations of FAK or p38 MAP kinase and plays a pivotal role in focal complex formation and cell motility induced by LPA.     

The Guanine Nucleotide Exchange Factor Tiam1 Affects Neuronal Morphology; Opposing Roles for the Small GTPases Rac and Rho

The invasion-inducing T-lymphoma invasion and metastasis 1 (Tiam1) protein functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. Differentiation-dependent expression of Tiam1 in the developing brain suggests a role for this GEF and its effector Rac1 in the control of neuronal morphology. Here we show that overexpression of Tiam1 induces cell spreading and affects neurite outgrowth in N1E-115 neuroblastoma cells. These effects are Rac-dependent and strongly promoted by laminin. Overexpression of Tiam1 recruits the α6β1 integrin, a laminin receptor, to specific adhesive contacts at the cell periphery, which are different from focal contacts. Cells overexpressing Tiam1 no longer respond to lysophosphatidic acid– induced neurite retraction and cell rounding, processes mediated by Rho, suggesting that Tiam1-induced activation of Rac antagonizes Rho signaling. This inhibition can be overcome by coexpression of constitutively active RhoA, which may indicate that regulation occurs at the level of Rho or upstream. Conversely, neurite formation induced by Tiam1 or Rac1 is further promoted by inactivating Rho. These results demonstrate that Rac- and Rho-mediated pathways oppose each other during neurite formation and that a balance between these pathways determines neuronal morphology. Furthermore, our data underscore the potential role of Tiam1 as a specific regulator of Rac during neurite formation and illustrate the importance of reciprocal interactions between the cytoskeleton and the extracellular matrix during this process.     

MAGI-3 regulates LPA-induced activation of Erk and RhoA

Lysophosphatidic acids (LPA) exert multiple biological effects through specific G protein-coupled receptors. The LPA-activated receptor subtype LPA(2) contains a carboxyl-terminal motif that allows interaction with PDZ domain-containing proteins, such as NHERF2 and PDZ-RhoGEF. To identify additional interacting partners of LPA(2), the LPA(2) carboxyl-terminus was used to screen a proteomic array of PDZ domains. In addition to the previously identified NHERF2, several additional LPA(2)-interacting PDZ domains were found. These included MAGI-2, MAGI-3 and neurabin. In the present work, we demonstrate the specific interaction between LPA(2) and MAGI-3, and the effects of MAGI-3 in colon cancer cells using SW480 as a cell model. MAGI-3 specifically bound to LPA(2), but not to LPA(1) and LPA(3). This interaction was mediated via the fifth PDZ domain of MAGI-3 interacting with the carboxyl-terminal 4 amino acids of LPA(2), and mutational alteration of the carboxyl-terminal sequences of LPA(2) severely attenuated its ability to bind MAGI-3. LPA(2) also associated with MAGI-3 in cells as determined by co-affinity purification. Overexpression of MAGI-3 in SW480 cells showed no apparent effect on LPA-induced activation of Erk and Akt. In contrast, silencing of MAGI-3 expression by siRNA drastically inhibited LPA-induced Erk activation, suggesting that the lack of an effect by overexpression was due to the high endogenous MAGI-3 level in these cells. Previous studies have shown that the cellular signaling elicited by LPA results in activation of the small GTPase RhoA by Galpha(12/13) - as well as Galpha(q)-dependent pathways. Overexpression of MAGI-3 stimulated LPA-induced RhoA activation, whereas silencing of MAGI-3 by siRNA resulted in a small but statistically significant decrease in RhoA activation. These results demonstrate that MAGI-3 interacts directly with LPA(2) and regulates the ability of LPA(2) to activate Erk and RhoA.     

Rho-dependent, Rho kinase-independent inhibitory regulation of Rac and cell migration by LPA1 receptor in Gi-inactivated CHO cells

Lysophosphatidic acid (LPA) is a major serum lysophospholipid that stimulates cell migration in diverse cell types including ovarian cancer cells. We report here that in the absence of Gi function, LPA induces inhibition, rather than stimulation, of cellular Rac activity, lamellipodium formation, and cell migration in response to insulin like growth factor I (IGF-I) in Chinese hamster ovary (CHO) cells, which solely express LPA1 as a LPA receptor. The inhibitory effects of LPA are abrogated by the expression of either Galpha13 C-terminal peptide or C3 toxin pretreatment, but not a Rho kinase inhibitor. Without PTX pretreatment, LPA stimulates Rac and cell migration yet similarly activates Rho, indicating that Rho activation by itself is not sufficient for inhibition of cell migration. Conversely, the expression of a dominant negative Rac mutant sufficiently mimics the LPA inhibition of cell migration. LPA inhibits IGF I-induced Akt activation by only 40% in a manner dependent on Rho kinase. These results demonstrate that inhibition of Gi function converts LPA regulation on Rac and cell migration to an inhibitory mode, which is mediated by G13 and Rho but not Rho kinase, and raise a possibility of Gi as a new therapeutic target for LPA-dependent tumor progression.     

Tiam1 is recruited to β1-integrin complexes by 14-3-3ζ where it mediates integrin-induced Rac1 activation and motility

14-3-3 is an adaptor protein that localizes to the leading edge of spreading cells, returning to the cytoplasm as spreading ceases. Previously, we showed that integrin-induced Rac1 activation and spreading were inhibited by sequestration of 14-3-3ζ and restored by its overexpression. Here, we determined whether 14-3-3 mediates integrin signaling by localizing a guanine nucleotide exchange factor (GEF) to Rac1-activating integrin complexes. We showed that GST-14-3-3ζ recruited the Rac1-GEF, Tiam1, from cell lysates through Tiam1 residues 1-182 (N(1-182) Tiam1). The physiological relevance of this interaction was examined in serum-starved Hela cells plated on fibronectin. Both Tiam1 and N(1-182) Tiam1 were recruited to 14-3-3-containing β1-integrin complexes, as shown by co-localization and co-immunoprecipitation. Integrin-induced Rac1 activation was inhibited when Tiam1 was depleted with siRNA or by overexpression of catalytically inactive N(1-182) Tiam1, which was incorporated into 14-3-3/β1-integrin complexes and inhibited spreading in a manner that was overcome by constitutively active Rac1. Integrin-induced Rac1 activation, spreading, and migration were also inhibited by overexpression of 14-3-3ζ S58D, which was unable to recruit Tiam1 from lysates, co-immunoprecipitate with Tiam1, or mediate its incorporation into β1-integrin complexes. Taken together, these findings suggest a previously unrecognized mechanism of integrin-induced Rac1 activation in which 14-3-3 dimers localize Tiam1 to integrin complexes, where it mediates integrin-dependent Rac1 activation, thus initiating motility-inducing pathways. Moreover, since Tiam1 is recruited to other sites of localized Rac1 activation through its PH-CC-EX domain, the present findings show that a mechanism involving its N-terminal 182 residues is utilized to recruit Tiam1 to motility-inducing integrin complexes.     

G alpha 13 signals via p115RhoGEF cascades regulating JNK1 and primitive endoderm formation

The heterotrimeric G-protein G(13) mediates the formation of primitive endoderm from mouse P19 embryonal carcinoma cells in response to retinoic acid, signaling to the level of activation of c-Jun N-terminal kinase. The signal linkage map from MEKK1/MEKK4 to MEK1/MKK4 to JNK is obligate in this G alpha(13)-mediated pathway, whereas that between G alpha(13) and MEKKs is not known. The overall pathway to primitive endoderm formation was shown to be inhibited by treatment with Clostridium botulinum C3 exotoxin, a specific inactivator of RhoA family members. Constitutively active G alpha(13) was found to activate RhoA as well as Cdc42 and Rac1 in these cells. Although constitutively active Cdc42, Rac1, and RhoA all can activate JNK1, only the RhoA mutant was able to promote formation of primitive endoderm, mimicking expression of the constitutively activated G alpha(13). Expression of the constitutively active mutant form of p115RhoGEF (guanine nucleotide exchange factor) was found to activate RhoA and JNK1 activities. Expression of the dominant negative p115RhoGEF was able to inhibit activation of both RhoA and JNK1 in response to either retinoic acid or the expression of a constitutively activated mutant of G alpha(13). Expression of the dominant negative mutants of RhoA as well as those of either Cdc42 or Rac1, but not Ras, attenuated G alpha(13)-stimulated as well as retinoic acid-stimulated activation of all three of these small molecular weight GTPases, suggesting complex interrelationships among the three GTPases in this pathway. The formation of primitive endoderm in response to retinoic acid also could be blocked by expression of dominant negative mutants of RhoA, Cdc42, or Rac1. Thus, the signal propagated from G alpha(13) to JNK requires activation of p115RhoGEF cascades, including p115RhoGEF itself, RhoA, Cdc42, and Rac1. In a concerted effort, RhoA in tandem with Cdc42 and Rac1 activates the MEKK1/4, MEK1/MKK4, and JNK cascade, thereby stimulating formation of primitive endoderm.     

Activation of p21-activated kinase 1 is required for lysophosphatidic acid-induced focal adhesion kinase phosphorylation and cell motility in human melanoma A2058 cells.

Lysophosphatidic acid (LPA), one of the naturally occurring phospholipids, stimulates cell motility through the activation of Rho family members, but the signaling mechanisms remain to be elucidated. In the present study, we investigated the roles of p21-activated kinase 1 (PAK1) on LPA-induced focal adhesion kinase (FAK) phosphorylation and cell motility. Treatment of human melanoma cells A2058 with LPA increased phosphorylation and activation of PAK1, which was blocked by treatment with pertussis toxin and by inhibition of phosphoinositide 3-kinase (PI3K) with an inhibitor LY294002 or by overexpression of catalytically inactive mutant of PI3Kgamma, indicating that LPA-induced PAK1 activation was mediated via a Gi protein and the PI3Kgamma signaling pathway. In addition, we demonstrated that Rac1/Cdc42 signals acted as upstream effector molecules of LPA-induced PAK activation. However, Rho-associated kinase, MAP kinase kinase 1/2 or phospholipase C might not be involved in LPA-induced PAK1 activation or cell motility stimulation. Furthermore, PAK1 was necessary for FAK phosphorylation by LPA, which might cause cell migration, as transfection of the kinase deficient mutant of PAK1 or PAK auto-inhibitory domain significantly abrogated LPA-induced FAK phosphorylation. Taken together, these findings strongly indicated that PAK1 activation was necessary for LPA-induced cell motility and FAK phosphorylation that might be mediated by sequential activation of Gi protein, PI3Kgamma and Rac1/Cdc42.