Functional selectivity of G protein signaling by agonist peptides and thrombin for the protease-activated receptor-1

Thrombin activates protease-activated receptor-1 (PAR-1) by cleavage of the amino terminus to unmask a tethered ligand. Although peptide analogs can activate PAR-1, we show that the functional responses mediated via PAR-1 differ between the agonists. Thrombin caused endothelial monolayer permeability and mobilized intracellular calcium with EC(50) values of 0.1 and 1.7 nm, respectively. The opposite order of activation was observed for agonist peptide (SFLLRN-CONH(2) or TFLLRNKPDK) activation. The addition of inactivated thrombin did not affect agonist peptide signaling, suggesting that the differences in activation mechanisms are intramolecular in origin. Although activation of PAR-1 or PAR-2 by agonist peptides induced calcium mobilization, only PAR-1 activation affected barrier function. Induced barrier permeability is likely to be Galpha(12/13)-mediated as chelation of Galpha(q)-mediated intracellular calcium with BAPTA-AM, pertussis toxin inhibition of Galpha(i/o), or GM6001 inhibition of matrix metalloproteinase had no effect, whereas Y-27632 inhibition of the Galpha(12/13)-mediated Rho kinase abrogated the response. Similarly, calcium mobilization is Galpha(q)-mediated and independent of Galpha(i/o) and Galpha(12/13) because pertussis toxin Y-27632 and had no effect, whereas U-73122 inhibition of phospholipase C-beta blocked the response. It is therefore likely that changes in permeability reflect Galpha(12/13) activation, and changes in calcium reflect Galpha(q) activation, implying that the pharmacological differences between agonists are likely caused by the ability of the receptor to activate Galpha(12/13) or Galpha(q). This functional selectivity was characterized quantitatively by a mathematical model describing each step leading to Rho activation and/or calcium mobilization. This model provides an estimate that peptide activation alters receptor/G protein binding to favor Galpha(q) activation over Galpha(12/13) by approximately 800-fold.     

G alpha 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH(2)-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of the Galpha(12/13)-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of Galpha(12/13), but not by an RGS domain specific for Galpha(q). Constitutively active Galpha(12)- or Galpha(13)-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium. Angiotensin II receptor stimulation rapidly activated Galpha(13), which was completely inhibited by the Galpha(12/13)-specific RGS domain. Furthermore, the Galpha(12/13)-specific but not the Galpha(q)-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin II-induced Rho activation was inhibited by Galpha(12/13)-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not affect angiotensin II-induced JNK activation cascade. These results suggest that Galpha(12/13)-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation.     

Subtype-specific differential regulation of Rho family G proteins and cell migration by the Edg family sphingosine-1-phosphate receptors

One of the striking activities of the Edg family sphingosine-1-phosphate (S1P) receptors includes receptor isotype-specific, bimodal regulatory activity on cell migration. While Edg1 and Edg3 act as typical chemotactic receptors, Edg5 uniquely acts as a chemorepellant receptor. Consistent with this, Edg1 and Edg3, and Edg5 regulate the activity of the Rho family GTPase Rac positively and negatively, respectively. Thus, Edg isotype-specific, differential regulatory activities on Rac seem to be important as mechanisms underlying the bimodal regulation of cell migration by S1P. Edg5-mediated Rac inhibition involves stimulation of Rac-GTPase-activating protein (GAP) activity, rather than inhibition of Rac-guanine nucleotide exchange factor (GEF) activity. Many cell types including vascular smooth muscle and endothelial cells express more than a single S1P receptor isotype. In these cells, it appears that an integration of the Edg isotype-selective, positive and negative signals on cellular Rac activity is a critical determinant for eventual direction of regulation on cell motility by S1P. Physiological and pathological roles for the repulsive activity of Edg5 receptor remain to be clarified.     

Ligand-dependent inhibition of B16 melanoma cell migration and invasion via endogenous S1P2 G protein-coupled receptor. Requirement of inhibition of cellular RAC activity

We investigated mechanisms for inhibition of B16 melanoma cell migration and invasion by sphingosine-1-phosphate (S1P), which is the ligand for the Edg family G protein-coupled receptors and also implicated as an intracellular second messenger. S1P, dihydro-S1P, and sphingosylphosphorylcholine inhibited B16 cell migration and invasion with the relative potencies expected as S1P2 receptor agonists. The S1P2-selective antagonist JTE013 completely abolished the responses to these agonists. In addition, JTE013 abrogated the inhibition by sphingosine, which is the S1P precursor but not an agonist for S1P receptors, indicating that the sphingosine effects were mediated via S1P2 stimulation, most likely by S1P that was converted from sphingosine. S1P induced inhibition and activation, respectively, of Rac and RhoA in B16 cells, which were abrogated by JTE013. Adenovirus-mediated expression of N17Rac mimicked S1P inhibition of migration, whereas C3 toxin pretreatment, but not Rho kinase inhibitors, reversed the S1P inhibition. Overexpression of S1P2 sensitized, and that of either S1P1 or S1P3 desensitized, B16 cells to S1P inhibition of Rac and migration. In JTE013-pretreated, S1P3-overexpressing B16 cells, S1P stimulated cellular RhoA but failed to inhibit either Rac or migration, indicating that RhoA stimulation itself is not sufficient for inhibition of migration. These results provide compelling evidence that endogenously expressed S1P2 negatively regulates cell motility and invasion through ligand-dependent reciprocal regulation of cellular Rac and RhoA activities. In the presence of JTE013, S1P instead stimulated Rac and migration in B16 cells that overexpress either S1P1 or S1P3, unveiling counteractions between S1P2 and S1P1 or S1P3 chemotactic receptor.     

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.     

Rac activation by lysophosphatidic acid LPA1 receptors through the guanine nucleotide exchange factor Tiam1

Lysophosphatidic acid (LPA) is a serum-borne phospholipid that activates its own G protein-coupled receptors present in numerous cell types. In addition to stimulating cell proliferation, LPA also induces cytoskeletal changes and promotes cell migration in a RhoA- and Rac-dependent manner. Whereas RhoA is activated via Galpha(12/13)-linked Rho-specific guanine nucleotide exchange factors, it is unknown how LPA receptors may signal to Rac. Here we report that the prototypic LPA(1) receptor (previously named Edg2), when expressed in B103 neuroblastoma cells, mediates transient activation of RhoA and robust, prolonged activation of Rac leading to cell spreading, lamellipodia formation, and stimulation of cell migration. LPA-induced Rac activation is inhibited by pertussis toxin and requires phosphoinositide 3-kinase activity. Strikingly, LPA fails to activate Rac in cell types that lack the Rac-specific exchange factor Tiam1; however, enforced expression of Tiam1 restores LPA-induced Rac activation in those cells. Tiam1-deficient cells show enhanced RhoA activation, stress fiber formation, and cell rounding in response to LPA, consistent with Tiam1/Rac counteracting RhoA. We conclude that LPA(1) receptors couple to a G(i)-phosphoinositide 3-kinase-Tiam1 pathway to activate Rac, with consequent suppression of RhoA activity, and thereby stimulate cell spreading and motility.     

The Galpha13-Rho signaling axis is required for SDF-1-induced migration through CXCR4

The CXC chemokine stromal cell-derived factor-1alpha (SDF-1) binds to CXCR4, a seven-transmembrane G protein-coupled receptor that plays a critical role in many physiological processes that involve cell migration and cell fate decisions, ranging from stem cell homing, angiogenesis, and neuronal development to immune cell trafficking. CXCR4 is also implicated in various pathological conditions, including metastatic spread and human immunodeficiency virus infection. Although SDF-1-induced cell migration in CXCR4-expressing cells is sensitive to pertussis toxin treatment, hence involving heterotrimeric G proteins of the G(i) family, whether other G proteins participate in the chemotactic response to SDF-1 is still unknown. In this study, we took advantage of the potent chemotactic activity of SDF-1 in Jurkat T-cells to examine the nature of the heterotrimeric G protein subunits contributing to CXCR4-mediated cell migration. We observed that whereas G(i) and Gbetagamma subunits are involved in SDF-1-induced Rac activation and cell migration, CXCR4 can also stimulate Rho potently leading to the phosphorylation of myosin light chain through the Rho effector, Rho kinase, but independently of G(i). Furthermore, we found that Galpha(13) mediates the activation of Rho by CXCR4 and that the functional activity of both Galpha(13) and Rho is required for directional cell migration in response to SDF-1. Collectively, our data indicate that signaling by CXCR4 to Rho through Galpha(13) contributes to cell migration when stimulated by SDF-1, thus identifying the Galpha(13)-Rho signaling axis as a potential pharmacological target in many human diseases that involve the aberrant function of CXCR4.     

Regulation of Rac and Cdc42 pathways by G(i) during lysophosphatidic acid-induced cell spreading

The pertussis toxin-sensitive G protein, G(i), has been implicated in lysophosphatidic acid-induced cell mitogenesis and migration, but the mechanisms remain to be detailed. In the present study, we found that pertussis toxin blocks lysophosphatidic acid-induced cell spreading of NIH 3T3 fibroblasts on fibronectin. This prevention of cell spreading was eliminated by the expression of constitutively active mutants of Rho family small GTP-binding proteins, Rac and Cdc42, but not by Rho. In addition, activation of the endogenous forms was suppressed by pertussis toxin, indicating that G(i)-induced cell spreading is mediated through the Rac and Cdc42 pathway. Transfection of constitutively active mutants of G alpha(i) and G alpha(11) and G beta gamma subunits enhanced spreading of pertussis toxin-treated cells. G beta(1) with G gamma(12), a major G gamma form in fibroblasts, was more effective for increasing cell spreading than G beta(1)gamma(2) or G beta(1) plus G gamma(12)S2A, a mutant in which Ser-2, a phosphorylation site for protein kinase C, is replaced with alanine. In addition, a protein kinase C inhibitor diminished G beta(1)gamma(12)-induced cell spreading, suggesting a role for phosphorylation of the protein. These findings indicate that both G alpha(i) and G beta gamma stimulate Rac and Cdc42 pathways with lysophosphatidic acid-induced cell spreading on fibronectin.     

Evidence for transduction of mu but not kappa opioid modulation of extracellular signal-regulated kinase activity by G(z) and G(12) proteins

Chronic treatment with micro or kappa opioid agonists (>/=2 h) inhibits EGF-induced ERK activation in opioid receptor overexpressing COS-7 cells. Although acute mu and kappa opioids activate ERK via a pertussis toxin-sensitive G protein, pertussis toxin insensitivity of the chronic mu (but not kappa) action was observed. Here, we tested several pertussis toxin-insensitive G proteins as candidates to transduce acute and/or chronic opioid modulation of ERK. Overexpressed Galpha(z) (but not Galpha(12)) transduced acute mu (but not kappa) ERK activation in pertussis toxin-treated COS-7 cells. Chronic mu (but not kappa) inhibited EGF stimulation of ERK in pertussis toxin-treated cells overexpressing Galpha(z) or Galpha(12). Transfection of Galpha(13) or Galpha(q) blocked inhibition under the same conditions. Overexpressed interfering and non-interfering Galpha(z) mutants differentially affected mu inhibition of ERK consistent with G(z) transduction. In this and prior studies, Galpha(z) and Galpha(12) immunoreactivity were detected in untransfected COS-7 cells, suggesting that these G proteins may be endogenous mediators of chronic mu inhibitory actions on ERK.     

Activation of protein kinase D3 by signaling through Rac and the alpha subunits of the heterotrimeric G proteins G12 and G13

PKD is the founding member of a novel protein kinase family that also includes PKD2 and PKD3. PKD has been the focus of most studies up to date, but little is known about the mechanisms that mediate PKD3 activation. Here, we show that addition of aluminum fluoride to COS-7 cells cotransfected with PKD3 and Galpha13 or Galpha12 induced PKD3 activation, which was associated with a transient plasma membrane translocation of cytosolic PKD3. Treatment with Clostridium difficile toxin B blocked PKD3 activation induced by either bombesin or by aluminum fluoride-stimulated Galpha12/13 but did not affect Galphaq-induced PKD3 activation. Furthermore, PKD3 immunoprecipitated from cells cotransfected with a constitutively active Rac (RacV12) exhibited a marked increase in PKD3 basal catalytic activity. In contrast, cotransfection with active Rho (RhoQ63L), Cdc42 (Cdc42Q61L), or Ras (RasV12) did not promote PKD3 activation. Expression of either COOH-terminal dominant-negative fragment of Galpha13 or dominant negative Rac (Rac N17) attenuated bombesin-induced PKD3 activation. Treatment with protein kinase C (PKC) inhibitors prevented the increase in PKD3 activity induced by RacV12 and aluminum fluoride-stimulated Galpha12/13. The catalytic activation of PKD3 in response to RacV12, alpha12/13 signaling or bombesin correlated with Ser-731/Ser-735 phosphorylation in the activation loop of this enzyme. Our results indicate that Galpha12/13 and Rac are important components in the signal transduction pathways that mediate bombesin receptor-induced PKD3 activation.     

Differential involvement of Galpha12 and Galpha13 in receptor-mediated stress fiber formation.

The ubiquitously expressed heterotrimeric guanine nucleotide-binding proteins (G-proteins) G12 and G13 have been shown to activate the small GTPase Rho. Rho stimulation leads to a rapid remodeling of the actin cytoskeleton and subsequent stress fiber formation. We investigated the involvement of G12 or G13 in stress fiber formation induced through a variety of Gq/G11-coupled receptors. Using fibroblast cell lines derived from wild-type and Galphaq/Galpha11-deficient mice, we show that agonist-dependent activation of the endogenous receptors for thrombin or lysophosphatidic acid and of the heterologously expressed bradykinin B2, vasopressin V1A, endothelin ETA, and serotonin 5-HT2C receptors induced stress fiber formation in either the presence or absence of Galphaq/Galpha11. Stress fiber assembly induced through the muscarinic M1 and the metabotropic glutamate subtype 1alpha receptors was dependent on Gq/G11 proteins. The activation of the Gq/G11-coupled endothelin ETB and angiotensin AT1A receptors failed to induce stress fiber formation. Lysophosphatidic acid, B2, and 5-HT2C receptor-mediated stress fiber formation was dependent on Galpha13 and involved epidermal growth factor (EGF) receptors, whereas thrombin, ETA, and V1A receptors induced stress fiber accumulation via Galpha12 in an EGF receptor-independent manner. Our data demonstrate that many Gq/G11-coupled receptors induce stress fiber assembly in the absence of Galphaq and Galpha11 and that this involves either a Galpha12 or a Galpha13/EGF receptor-mediated pathway.     

Antagonistic regulation of neurite morphology through Gq/G11 and G12/G13

The induction of neurite retraction and growth cone collapse via G-protein-coupled receptors is involved in developmental as well as regenerative processes. The role of individual G-protein-mediated signaling processes in the regulation of neurite morphology is still incompletely understood. Using primary neurons from brains lacking Galpha(q)/Galpha(11) or Galpha(12)/Galpha(13), we show here that G(12)/G(13)-mediated signaling is absolutely required for neurite retraction and growth cone collapse induced by the blood-borne factors lysophosphatidic acid and thrombin. Interestingly, the effects of lysophosphatidic acid were mediated mainly by G(13), whereas thrombin effects required G(12). Surprisingly, lack of Galpha(q)/Galpha(11) resulted in overshooting responses to both stimuli, indicating that G(q)/G(11)-mediated signaling most likely via activation of Rac antagonizes the effects of G(12)/G(13).     

G13-dependent activation of MAPK by thyrotropin

Stimulation of the thyrotropin receptor (TSHR) activates G proteins of all four subfamilies (G(s), G(i/o), G(q/11), and G(12/13)). Whereas G(s)/cAMP-dependent cellular responses upon TSHR stimulation are well established, other signaling pathways are less characterized. We evaluated TSH-elicited cellular responses in human follicular thyroid carcinoma cells stably expressing the TSHR and in primary, nonneoplastic human thyrocytes. In these cellular models, stimulation with TSH caused activation of p44/42 MAPK and subsequent induction of c-Fos. MAPK stimulation occurred independently of G(s), G(i/o), and G(q/11) signaling. Dominant negative constructs of G(12) or G(13) as well as shRNA-mediated suppression of Galpha(12) or Galpha(13) revealed that MAPK activation was dependent on G(13) but not on G(12) signaling. Furthermore, G(13)-dependent transactivation of the epidermal growth factor receptor was necessary for MAPK activation in follicular carcinoma cells, whereas EGFR was not involved in MAPK activation in nonneoplastic primary thyrocytes. The use of bacterial inhibitors of monomeric GTPases revealed that MAPK activation proceeded independently of Rho proteins but was clostridial toxin B-sensitive, suggesting involvement of Cdc42 or Rac. Thus, our data shed new light on cAMP-independent TSHR signaling and identify the first G(13)-dependent TSHR signaling pathway in human thyrocytes.     

G protein G alpha q/11 and G alpha i1,2 are activated by pancreastatin receptors in rat liver: studies with GTP-gamma 35S and azido-GTP-alpha-32P

In the liver, pancreastatin exerts a glycogenolytic effect through interaction with specific receptors, followed by activation of phospholipase C and guanylate cyclase. Pancreastatin receptor seems to be coupled to two different G protein systems: a pertussis toxin-insensitive G protein that mediates activation of phospholipase C, and a pertussis toxin sensitive G protein that mediates the cyclic GMP production. The aim of this study was to identify the specific G protein subtypes coupling pancreastatin receptors in rat liver membranes. GTP binding was determined by using gamma-35S-GTP; specific anti-G protein alpha subtype sera were used to block the effect of pancreastatin receptor activation. Activation of G proteins was demonstrated by the incorporation of the photoreactive GTP analogue 8-azido-alpha-32P-GTP into liver membranes and into specific immunoprecipitates of different Galpha subunits from soluble rat liver membranes. Pancreastatin stimulation of rat liver membranes increases the binding of gamma-35S-GTP in a time- and dose-dependent manner. Activation of the soluble receptors still led to the pancreastatin dose-dependent stimulation of gamma-35S-GTP binding. Besides, WGA semipurified receptors also stimulates GTP binding. The binding was inhibited by treatment with anti-Galphaq/11 (85%) and anti-Galphai1,2 (15%) sera, whereas anti-Galphao,i3 serum failed to affect the binding. Finally, pancreastatin stimulates GTP photolabeling of particulate membranes. Moreover, it specifically increased the incorporation of 8-azido-alpha-32P-GTP into Galphaq/11 and Galpha, but not into Galphao,i3 from soluble rat liver membranes. In conclusion, pancreastatin stimulation of rat liver membranes led to the activation of Galphaq/11 and Galphai1,2 proteins. These results suggest that Galphaq/11 and Galphai1,2 may play a functional role in the signaling of pancreastatin receptor by mediating the production of IP3 and cGMP respectively.     

Inhibitory regulation of Rac activation, membrane ruffling, and cell migration by the G protein-coupled sphingosine-1-phosphate receptor EDG5 but not EDG1 or EDG3

Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid that induces a variety of biological responses in diverse cell types. Many, if not all, of these responses are mediated by members of the EDG (endothelial differentiation gene) family G protein-coupled receptors EDG1, EDG3, and EDG5 (AGR16). Among prominent activities of S1P is the regulation of cell motility; S1P stimulates or inhibits cell motility depending on cell types. In the present study, we provide evidence for EDG subtype-specific, contrasting regulation of cell motility and cellular Rac activity. In CHO cells expressing EDG1 or EDG3 (EDG1 cells or EDG3 cells, respectively) S1P as well as insulin-like growth factor I (IGF I) induced chemotaxis and membrane ruffling in phosphoinositide (PI) 3-kinase- and Rac-dependent manners. Both S1P and IGF I induced a biphasic increase in the amount of the GTP-bound active form of Rac. In CHO cells expressing EDG5 (EDG5 cells), IGF I similarly stimulated cell migration; however, in contrast to what was found for EDG1 and EDG3 cells, S1P did not stimulate migration but totally abolished IGF I-directed chemotaxis and membrane ruffling, in a manner dependent on a concentration gradient of S1P. In EDG5 cells, S1P stimulated PI 3-kinase activity as it did in EDG1 cells but inhibited the basal Rac activity and totally abolished IGF I-induced Rac activation, which involved stimulation of Rac-GTPase-activating protein activity rather than inhibition of Rac-guanine nucleotide exchange activity. S1P induced comparable increases in the amounts of GTP-RhoA in EDG3 and EDG5 cells. Neither S1P nor IGF I increased the amount of GTP-bound Cdc42. However, expression of N(17)-Cdc42, but not N(19)-RhoA, suppressed S1P- and IGF I-directed chemotaxis, suggesting a requirement for basal Cdc42 activity for chemotaxis. Taken together, the present results demonstrate that EDG5 is the first example of a hitherto-unrecognized type of receptors that negatively regulate Rac activity, thereby inhibiting cell migration and membrane ruffling.     

Galpha13 stimulates cell migration through cortactin-interacting protein Hax-1

Galpha13, the alpha-subunit of the heterotrimeric G protein G13, has been shown to stimulate cell migration in addition to inducing oncogenic transformation. Cta, a Drosophila ortholog of G13, has been shown to be critical for cell migration leading to the ventral furrow formation in Drosophila embryos. Loss of Galpha13 has been shown to disrupt cell migration associated with angiogenesis in developing mouse embryos. Whereas these observations point to the vital role of G13-orthologs in regulating cell migration, widely across the species barrier, the mechanism by which Galpha13 couples to cytoskeleton and cell migration is largely unknown. Here we show that Galpha13 physically interacts with Hax-1, a cytoskeleton-associated, cortactin-interacting intracellular protein, and this interaction is required for Galpha13-stimulated cell migration. Hax-1 interaction is specific to Galpha13, and this interaction is more pronounced with the mutationally or functionally activated form of Galpha13 as compared with the wild-type Galpha13. Expression of Hax-1 reduces the formation of actin stress fibers and focal adhesion complexes in Galpha13-expressing NIH3T3 cells. Coexpression of Hax-1 also attenuates Galpha(13)-stimulated activity of Rho while potentiating Galpha13-stimulated activity of Rac. The presence of a quadnary complex consisting of Galpha13, Hax-1, Rac, and cortactin indicates the role of Hax-1 in tethering Galpha13 to the cytoskeletal component(s) involved in cell movement. Whereas the expression of Hax-1 potentiates Galpha13-mediated cell movement, silencing of endogenous Hax-1 with Hax-1-specific small interfering RNAs drastically reduces Galpha13-mediated cell migration. These findings, along with the observation that Hax-1 is overexpressed in metastatic tumors and tumor cell lines, suggest a novel role for the association of oncogenic Galpha13 and Hax-1 in tumor metastasis.     

Budding yeast Cdc5 phosphorylates Net1 and assists Cdc14 release from the nucleolus

Lysophosphatidic acid (LPA) has been shown to be a potent mitogen for vascular smooth muscle cells. Src-dependent transactivation of receptor tyrosine kinases has been previously demonstrated to mediate LPA-induced activation of MAP kinase ERK1/2. Furthermore, generation of reactive oxygen species (ROS) by LPA is also known to contribute to MAP kinase activation. Rho family small G-proteins Rac and Cdc42, and their immediate downstream effector p21-activated kinase (PAK), have been demonstrated to mediate important effects on the cytoskeleton that are relevant for cell migration and proliferation. In the present report we evaluated stimulation of PAK by LPA in rat aortic vascular smooth muscle cells (VSMC) by PAK immunocomplex MBP in-gel kinase assay. LPA increased PAK activity 3-fold, peaking at 5 min and showing sustained activation up to 45 min. Inhibition of tyrosine kinases by pretreatment of VSMC with genistein or specific inhibition of Src by PP1 greatly diminished LPA-induced PAK activation, whereas specific inhibition of PDFG- and EGF receptor kinase by tyrphostin AG1296 and AG1478 had no effect. Furthermore, inhibition of Galpha(i) by pertussis toxin and inhibition of NADH/NADPH oxidase by diphenylene iodonium also diminished LPA-induced stimulation of PAK. This is the first study to demonstrate that LPA activates PAK. In VSMC, PAK activation by LPA is mediated by Galpha(i) and is dependent on Src, whereas EGF- or PDGF receptor transactivation are not involved. Furthermore, generation of ROS is required for LPA-induced activation of PAK.     

Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets

Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the alpha-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Galphaq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72(syk) and stimulation of pp60(c-src) as well as in phosphorylation of myosin light chain (MLC) in Galphaq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Galphaq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase-mediated regulation of MLC phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase-dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change.