Plexin-B1/RhoGEF-mediated RhoA activation involves the receptor tyrosine kinase ErbB-2

Plexins are widely expressed transmembrane proteins that mediate the effects of semaphorins. The molecular mechanisms of plexin-mediated signal transduction are still rather unclear. Plexin-B1 has recently been shown to mediate activation of RhoA through a stable interaction with the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG. However, it is unclear how the activity of plexin-B1 and its downstream effectors is regulated by its ligand Sema4D. Here, we show that plexin-B family members stably associate with the receptor tyrosine kinase ErbB-2. Binding of Sema4D to plexin-B1 stimulates the intrinsic tyrosine kinase activity of ErbB-2, resulting in the phosphorylation of both plexin-B1 and ErbB-2. A dominant-negative form of ErbB-2 blocks Sema4D-induced RhoA activation as well as axonal growth cone collapse in primary hippocampal neurons. Our data indicate that ErbB-2 is an important component of the plexin-B receptor system and that ErbB-2-mediated phosphorylation of plexin-B1 is critically involved in Sema4D-induced RhoA activation, which underlies cellular phenomena downstream of plexin-B1, including axonal growth cone collapse.     

Thrombin-induced rapid geranylgeranylation of RhoA as an essential process for RhoA activation in endothelial cells

RhoA plays a critical signaling role in thrombin-induced endothelial dysfunction. The possible thrombin regulation of geranylgeranylation, a lipid modification, of unprocessed RhoA and the significance of the geranylgeranylation in RhoA activation in endothelial cells (ECs) are not well understood. The amounts of the unprocessed and geranylgeranylated forms of RhoA in non-stimulated cultured human aortic ECs were 31 +/- 8 and 69 +/- 8% total cellular RhoA, respectively (n = 6, p < 0.0001), as determined by the Triton X-114 partition method. Thrombin-induced rapid conversion of most of the unprocessed RhoA into the geranylgeranylated form within 1 min through stimulating geranylgeranyltransferase I (GGTase I) activity. Thrombin-induced rapid geranylgeranylation was inhibited by acute short term (3 min) pretreatment with atorvastatin as well as by an inhibitor of GGTase I (GGTI-286). Thrombin also rapidly stimulated GTP loading of RhoA, which was blocked by acute pretreatment with either atorvastatin or GGTI-286. These observations indicate the dependence of thrombin stimulation of RhoA on the rapid geranylgeranylation of unprocessed RhoA. Importantly, the addition of geranylgeranylpyrophosphate to ECs pretreated with atorvastatin quickly reversed the atorvastatin inhibition of thrombin stimulation of RhoA. These results suggest that geranylgeranylation of unprocessed RhoA may limit thrombin-induced full activation of RhoA in ECs. Cytoskeleton analysis demonstrated that atorvastatin and GGTI-286 inhibited thrombin-induced stress fiber formation. We provide the evidence that, in thrombin-stimulated ECs, the unprocessed form of RhoA is rapidly geranylgeranylated to become the mature form, which then is converted into GTP-bound active RhoA.     

Requirement for ERK activation in cisplatin-induced apoptosis

Cisplatin activates multiple signal transduction pathways involved in coordinating cellular responses to stress. Here we demonstrate a requirement for extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein kinase family in mediating cisplatin-induced apoptosis of human cervical carcinoma HeLa cells. Cisplatin treatment resulted in dose- and time- dependent activation of ERK. That elevated ERK activity contributed to cell death by cisplatin was supported by several observations: 1) PD98059 and U0126, chemical inhibitors of the MEK/ERK signaling pathway, prevented apoptosis; 2) pretreatment of cells with TPA, an activator of the ERK pathway, enhanced their sensitivity to cisplatin; 3) suramin, a growth factor receptor antagonist that greatly suppressed ERK activation, likewise inhibited cisplatin-induced apoptosis; and, finally, 4) HeLa cell variants selected for cisplatin resistance showed reduced activation of ERK following cisplatin treatment. Cisplatin-induced apoptosis was associated with cytochrome c release and subsequent caspase-3 activation, both of which could be prevented by treatment with the MEK inhibitors. However, the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone protected HeLa cells against apoptosis without affecting ERK activation. Taken together, our findings suggest that ERK activation plays an active role in mediating cisplatin-induced apoptosis of HeLa cells and functions upstream of caspase activation to initiate the apoptotic signal.     

Glucosamine increases vascular contraction through activation of RhoA/Rho kinase pathway in isolated rat aorta

Diabetes is a well-known independent risk factor for vascular disease. However, its underlying mechanism remains unclear. It has been reported that increased influx of the hexosamine biosynthesis pathway (HBP) induces O-GlcNAcylation of proteins, leading to insulin resistance. In this study, we determined whether or not O-GlcNAc modification of proteins could increase vessel contraction. Using an endothelium-denuded aortic ring, we observed that glucosamine induced OGlcNAcylation of proteins and augmented vessel contraction stimulated by U46619, a thromboxane A(2) agonist, via augmentation of the phosphorylation of MLC(20), MYPT1(Thr855), and CPI17, but not phenylephrine. Pretreatment with OGT inhibitor significantly ameliorated glucosamine-induced vessel constriction. Glucosamine treatment also increased RhoA activity, which was also attenuated by OGT inhibitor. In conclusion, glucosamine, a product of glucose influx via the HBP in a diabetic state, increases vascular contraction, at least in part, through activation of the RhoA/Rho kinase pathway, which may be due to O-GlcNAcylation.     

Mechanism of RhoA/Rho kinase activation in endothelin-1- induced contraction in rabbit basilar artery

This study was undertaken to demonstrate the role of the RhoA/Rho kinase pathway in endothelin-1 (ET-1)-induced contraction of the rabbit basilar artery. Isometric tension and Western blot were used to examine ET-1-induced contraction and RhoA activation. The upstream effect on ET-1-induced RhoA activity was determined by using ET(A) and ET(B) receptor antagonists, protein kinase C (PKC), tyrosine kinase, and phosphatidylinositol-3 kinase inhibitors. The downstream effect of ET-1-induced contraction and RhoA activity was studied in the presence of the Rho kinase inhibitor Y-27632. The effect of Rho kinase inhibitor on ET-1-induced myosin light chain (MLC) phosphorylation was investigated by using urea-glycerol-PAGE immunoblotting. We found 1) ET-1 increased RhoA activity (membrane binding RhoA) in a concentration-dependent manner; 2) ET(A), but not ET(B), receptor antagonist abolished the effect of ET-1 on RhoA activation; 3) phosphodylinositol-3 kinase inhibitor, but not PKC and tyrosine kinase inhibitors, reduced ET-1-induced RhoA activation; 4) Rho kinase inhibitor Y-27632 (10 microM) inhibited ET-1-induced contraction; and 5) ET-1 increased the level of MLC phosphorylation. Rho kinase inhibitor Y-27632 reduced the effect of ET-1 on MLC phosphorylation. This study demonstrated that RhoA/Rho kinase activation is involved in ET-1-induced contraction in the rabbit basilar artery. Phosphodylinositol-3 kinase and MLC might be the upstream and downstream factors of RhoA activation.     

Requirement for protein-tyrosine phosphatase SHP-2 in insulin-induced activation of c-Jun NH(2)-terminal kinase

Mitogen-activated protein kinases, including extracellular signal-regulated kinases and c-Jun NH(2)-terminal kinases (JNKs), are activated by insulin. Although the mechanism by which the insulin receptor activates extracellular signal-regulated kinases is relatively well defined, the pathway that leads to JNK activation is poorly understood. Overexpression of a catalytically inactive mutant (SHP-2C/S) of the protein-tyrosine phosphatase SHP-2 in Rat-1 fibroblasts that also express human insulin receptors has now revealed that activation of JNKs by insulin and epidermal growth factor, but not that by anisomycin or sorbitol, requires SHP-2. A dominant negative mutant (RasN17) of Ha-Ras blocked insulin-induced JNK activation, whereas a dominant negative mutant (RacN17) of Rac1 or a specific inhibitor (LY294002) of phosphoinositide 3-kinase did not, indicating a role for Ras, but not for Rac or phosphoinositide 3-kinase, in this effect. SHP-2C/S markedly inhibited Ras activation in response to insulin without affecting insulin-induced tyrosine phosphorylation of cellular substrates or the dissociation of the Crk-p130(Cas) complex. In contrast, SHP-2C/S did not inhibit activation of JNKs induced by a constitutively active mutant (RasV12) of Ha-Ras. Furthermore, expression of myristoylated SOS, which functions as a potent activator of Ras, induced JNK activation even when SHP-2 was inactivated. These results suggest that SHP-2 contributes to JNK activation in response to insulin by positively regulating the Ras signaling pathway at the same level as, or upstream from, SOS.     

Requirement for ERK MAP kinase in mouse preimplantation development

Preimplantation development is a crucial step for successful implantation and pregnancy. Although both compaction and blastocyst formation have been extensively studied, mechanisms regulating the early cell division stages before compaction have remained unclear. Here, we show that extracellular signal regulated kinase (ERK) mitogen-activated protein (MAP) kinase function is required for early embryonic cell division before compaction. Our analysis demonstrates that inhibition of ERK activation in late two-cell-stage embryos leads to a reversible arrest in the G2 phase at the four-cell stage. The G2-arrested four-cell-stage embryos showed weakened cell-cell adhesion as compared with control embryos. Remarkably, microarray analyses showed that most of the programmed changes of upregulated and downregulated gene expression during the four- to eight-cell stages proceeded normally in four-cell-stage-arrested embryos that were subsequently released to resume development; however, the expression profiles of a proportion of genes in these embryos closely paralleled the stages of embryonic rather than normal development. These parallel genes included the genes encoding intercellular adhesion molecules, whose expression appeared to be positively regulated by the ERK pathway. We also show that, whereas ERK inactivation in eight-cell-stage embryos did not lead to cell division arrest, it did cause this arrest when cadherin-mediated cell-cell adhesion was disrupted. These results demonstrate an essential role of ERK function in two-cell to eight-cell-stage embryos, and suggest a loose parallelism between the gene expression programs and the developmental stages before compaction.     

Requirement for the MAP kinase kinase/MAP kinase cascade in Xenopus oocyte maturation.

MAP kinase kinase (MAPKK) has been identified as a protein factor that can induce phosphorylation and activation of inactive MAP kinase in vitro. In this study, we produced an anti-Xenopus MAPKK antibody that can specifically inhibit Xenopus MAPKK activity in vitro. Microinjection of this antibody into immature oocytes prevented progesterone-induced MAP kinase activation. Moreover, progesterone-induced histone H1 kinase activation and germinal vesicle breakdown (GVBD) were inhibited in the oocytes injected previously with this antibody. Furthermore, when a bacterially expressed Mos was introduced into immature oocytes, Mos-induced MAP kinase activation and GVBD were blocked in the oocytes injected with the anti-MAPKK antibody. These results show that MAPKK is responsible for the activation of MAP kinase in vivo and that the MAPKK/MAP kinase cascade plays a pivotal role in the MPF activation during the oocyte maturation process.     

Pregnancy reduces RhoA/Rho kinase and protein kinase C signaling pathways downstream of thromboxane receptor activation in the rat uterine artery

During pregnancy, reduced vascular responses to constrictors contribute to decreased uterine and total vascular resistance. Thromboxane A(2) (TxA(2)) is a potent vasoconstrictor that exerts its actions via diverse signaling pathways, and its biosynthesis increases in preeclampsia. In this study, we hypothesized that maternal vascular responses to TxA(2) will be attenuated via Rho kinase, PKC, p38 MAPK, and ERK1/2 signaling pathways. Isolated ring segments of uterine and small mesenteric arteries from late pregnant (19-21 days) and virgin rats were suspended in a myograph, and isometric force was measured. Pregnancy did not affect uterine and mesenteric artery responses to the TxA(2) analog U-46619 (10(-9)-10(-5) M), but transduction signals associated with these contractions were different between pregnant and nonpregnant rats. Inhibition of Rho kinase (10(-6) M Y-27632) reduced sensitivity to U-46619 in virgin uterine vessels but did not inhibit these contractions in pregnant uterine arteries and had no effect on mesenteric vessels. Treatment of arterial segments with a PKC inhibitor (10(-6) M bisindolylmaleimide I) reduced U-46619-induced contractions in virgin uterine and mesenteric arteries and in pregnant mesenteric arteries. Pregnant uterine arteries, however, were unresponsive to PKC inhibition. Inhibition of ERK1/2 (10(-5) M PD-98059) and p38 MAPK (10(-5) M SB-203580) reduced U46619-induced contractions in nonpregnant vessels and in pregnant uterine and mesenteric vessels. These data suggest that normal pregnancy does not affect uterine and mesenteric contractile responses to TxA(2) but reduces the contribution of Rho kinase and PKC signaling pathways to these contractions in the uterine vasculature. In contrast, the role of ERK1/2 and p38 MAPK in U-46619-induced uterine contractions remains unchanged with pregnancy. TxA(2)-associated transduction signals and its regulators might present potential targets for the development of new treatments for preeclampsia and other pregnancy-associated vascular diseases.     

cGMP-dependent protein kinase phosphorylates and inactivates RhoA

Small GTPase Rho and cGMP/cGMP-dependent protein kinase (cGK) pathways exert opposing effects in specific systems such as vascular contraction and growth. However, the direct interaction between these pathways has remained elusive. We demonstrate that cGK phosphorylates RhoA in vitro at Ser188, the same residue phosphorylated by cAMP-dependent protein kinase. In HeLa cells transfected with constitutively active cGK (C-cGK), stress fiber formation induced by lysophosphatidic acid or V14RhoA was blocked. By contrast, C-cGK failed to inhibit stress fiber formation in cells transfected with mutant RhoA with substitution of Ser188 to Ala. C-cGK did not affect actin reorganization induced by Rac1 or Rho-associated kinase, one of the effectors for RhoA. Furthermore, C-cGK expression inhibited the membrane translocation of RhoA. Collectively, our findings suggest that cGK phosphorylates RhoA at Ser188 and inactivates RhoA signaling. The physiological relevance of the direct interaction between RhoA and cGK awaits further investigation.     

Requirement of vimentin filament assembly for beta3-adrenergic receptor activation of ERK MAP kinase and lipolysis

Catecholamine stimulation of beta-adrenergic receptors (betaAR) in adipocytes activates the cAMP-dependent protein kinase to promote liberation of fatty acids as a fuel source. The adipocyte beta3AR also activates extracellular signal-regulated kinases (ERK)-1 and -2 through direct recruitment and activation of Src kinase. This pathway together with cAMP-dependent protein kinase contributes to maximal beta3AR-stimulated lipolysis. In a search for other molecules that might associate with beta3AR upon agonist stimulation, we identified vimentin using a proteomics approach. Immunoprecipitation of beta3AR from adipocytes in the absence or presence of the beta3AR agonist CL316,243, followed by Western blotting for vimentin confirmed this specific interaction. Since vimentin has also been identified on lipid droplets, the functional consequences of blocking the expression or structural integrity of vimentin intermediate filaments on beta3AR regulation of ERK activation and lipolysis was assessed. Following disruption of intermediate filaments with beta,beta'-iminodipropionitrile, as confirmed by confocal microscopy, beta3AR-stimulated ERK activation was blocked, and lipolysis was reduced by more than 40%. Independently, depletion of vimentin by small hairpin RNA (shRNA) completely inhibited beta3AR-mediated ERK activation and significantly reduced lipolysis. By contrast, disruption of actin-containing microfilaments by cytochalasin D or microtubules by nocodazole had no effect on either lipolysis or ERK activation. These results indicate that vimentin plays an essential role in the signal transduction pathway from beta3AR to the activation ERK and its contribution to lipolysis.     

Requirement for Daxx in mature T-cell proliferation and activation

The protein Daxx promotes Fas-mediated cell death through activation of apoptosis signal-regulating kinase 1, leading to the activation of the MAPKs JNK and p38. Owing to the in utero lethality of daxx-deficient mice, the in vivo role of Daxx has been so far difficult to analyze. We have generated transgenic mice expressing a dominant-negative form of Daxx (Daxx-DN) in the T-cell lineage. We show that Daxx is recruited to the Fas receptor upon FasL engagement and that Daxx-DN expression protects activated T cells from Fas-induced cell death, by preventing the death-inducing signal complex to be properly formed. Normal lymphocyte development and homeostasis are nevertheless observed. Interestingly, we report that both in vitro and in vivo stimulation of Daxx-DN T-lymphocytes leads to increased proliferative T-cell responses. This increased proliferation is associated with a marked increase in tyrosine phosphorylation of LAT and ZAP70 as Daxx-DN favor their recruitment to the T-cell receptor (TCR) complex. These findings identify Daxx as a critical regulator of T-lymphocyte homeostasis by decreasing TCR-induced cell proliferation and by promoting Fas-mediated cell death.     

Inhibition of protein geranylgeranylation and RhoA/RhoA kinase pathway induces apoptosis in human endothelial cells

Geranylgeranylation of RhoA small G-protein is essential for its localization to cell membranes and for its biological functions. Many RhoA effects are mediated by its downstream effector RhoA kinase. The role of protein geranylgeranylation and the RhoA pathway in the regulation of endothelial cell survival has not been elucidated. The hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor lovastatin depletes cellular pools of geranylgeranyl pyrophosphate and farnesol pyrophosphate and thereby inhibits both geranylgeranylation and farnesylation. Human umbilical vein endothelial cells (HUVECs) were exposed to lovastatin (3 microm-30 microm) for 48 h, and cell death was quantitatively determined by cytoplasmic histone-associated DNA fragments as well as caspase-3 activity. The assays showed that lovastatin caused a dose-dependent endothelial cell death. The addition of geranylgeraniol, which restores geranylgeranylation, rescued HUVEC from apoptosis. The geranylgeranyltransferase inhibitor GGTI-298, but not the farnesyltransferase inhibitor FTI-277, induced apoptosis in HUVEC. Cell death was also induced by a blockade of RhoA function by exoenzyme C3. In addition, treatment of HUVEC with the RhoA kinase inhibitors Y-27632 and HA-1077 caused dose-dependent cell death. Y-27632 did not inhibit other well known survival pathways, such as NF-kappa B, ERK, and phosphatidylinositol 3-kinase/Akt. However, there was an increase in p53 protein level concomitant with Y-27632-induced cell death. Unlike the apoptosis induced by TNF-alpha, which occurs only with inhibition of new protein synthesis, apoptosis induced by inhibitors of HMG-CoA reductase, geranylgeranyltransferase, or RhoA kinase was blocked by cycloheximide. Our data indicate that inhibition of protein geranylgeranylation and RhoA pathways induce apoptosis in HUVEC and that induction of p53 or other proapoptotic proteins is required for this process.     

Rap1 is a potent activation signal for leukocyte function-associated antigen 1 distinct from protein kinase C and phosphatidylinositol-3-OH kinase

To identify the intracellular signals which increase the adhesiveness of leukocyte function-associated antigen 1 (LFA-1), we established an assay system for activation-dependent adhesion through LFA-1/intercellular adhesion molecule 1 ICAM-1 using mouse lymphoid cells reconstituted with human LFA-1 and then introduced constitutively active forms of signaling molecules. We found that the phorbol myristate acetate (PMA)-responsive protein kinase C (PKC) isotypes (alpha, betaI, betaII, and delta) or phosphatidylinositol-3-OH kinase (PI 3-kinase) itself activated LFA-1 to bind ICAM-1. H-Ras and Rac activated LFA-1 in a PI 3-kinase-dependent manner, whereas Rho and R-Ras had little effect. Unexpectedly, Rap1 was demonstrated to function as the most potent activator of LFA-1. Distinct from H-Ras and Rac, Rap1 increased the adhesiveness independently of PI 3-kinase, indicating that Rap1 is a novel activation signal for the integrins. Rap1 induced changes in the conformation and affinity of LFA-1 and, interestingly, caused marked LFA-1/ICAM-1-mediated cell aggregation. Furthermore, a dominant negative form of Rap1 (Rap1N17) inhibited T-cell receptor-mediated LFA-1 activation in Jurkat T cells and LFA-1/ICAM-1-dependent cell aggregation upon differentiation of HL-60 cells into macrophages, suggesting that Rap1 is critically involved in physiological processes. These unique functions of Rap1 in controlling cellular adhesion through LFA-1 suggest a pivotal role as an immunological regulator.