Low molecular weight kininogen binds to platelets to modulate thrombin-induced platelet activation

The kininogens, high molecular weight kininogen (HK) and low molecular weight kininogen (LK), are multifunctional, single-gene products that contain bradykinin and identical amino-terminal heavy chains. Studies were performed to determine if LK would bind directly to platelets. 125I-LK specifically bound to gel-filtered platelets in the presence of 50 microM Zn2+. HK effectively competed with 125I-LK for the same binding site (Ki = 27 +/- 9 nM, n = 5). Similarly, the Ki for LK inhibition of 125I-LK binding was 12 +/- 1 nM (n = 3). Albumin, fibrinogen, factor XIII, and kallikrein did not inhibit 125I-LK binding to unstimulated platelets. 125I-LK (66 kDa) was not cleaved upon binding to platelets. The binding of 125I-LK to unstimulated platelets was found to be fully reversible by the addition of a 50 molar excess of unlabeled LK at both 10 and 20 min. LK binding to platelets was saturable with an apparent Kd of 27 +/- 2 nM (mean +/- S.E., n = 9) and 647 +/- 147 binding sites/platelet. Both LK and HK at plasma concentrations inhibited thrombin-induced platelet aggregation. LK and HK at about 5% of plasma concentration also inhibited thrombin-induced secretion of both stirred and unstirred platelets. Both kininogens were found to be noncompetitive inhibitors of proteolytically active thrombin binding to platelets. The kininogens did not inhibit D-phenylalanyl-prolyl-arginine chloromethyl ketone-treated thrombin from binding to platelets. These studies indicated that both kininogens have a region on their heavy chain which allows them to bind to platelets. Further, kininogen binding by its heavy chain modulates thrombin activation of platelets since it prevents proteolytically active thrombin from binding to its receptor.     

RalA and RalB differentially regulate development of epithelial tight junctions

Tight junctions (TJs) are structures indispensable to epithelial cells and are responsible for regulation of paracellular diffusion and maintenance of cellular polarity. Although many interactions between TJ constituents have been identified, questions remain concerning how specific functions of TJs are established and regulated. Here we investigated the roles of Ral GTPases and their common effector exocyst complex in the formation of nascent TJs. Unexpectedly, RNA interference-mediated suppression of RalA or RalB caused opposing changes in TJ development. RalA reduction increased paracellular permeability and decreased incorporation of components into TJs, whereas RalB reduction decreased paracellular permeability and increased incorporation of components into TJs. Activities of both Ral GTPases were mediated through the exocyst. Finally, we show that TJ-mediated separation of apical-basal membrane domains is established prior to equilibration of barrier function and that it is unaffected by Ral knockdown or specific composition of TJs.     

A Gi-dependent pathway is required for activation of the small GTPase Rap1B in human platelets

Stimulation of human platelets by cross-linking of the low affinity receptor for immunoglobulin, FcgammaRIIA, caused the rapid activation of the small GTPase Rap1B, as monitored by accumulation of the GTP-bound form of the protein. This process was totally dependent on the action of secreted ADP since it was completely prevented in the presence of either apyrase or creatine phosphate and creatine phosphokinase. Dose-dependent experiments revealed that the inhibitory effect of ADP scavengers was not related to the reduced increase of cytosolic Ca(2+) concentration in stimulated platelets. Activation of Rap1B induced by clustering of FcgammaRIIA was totally suppressed by AR-C69931MX, a specific antagonist of the G(i)-coupled ADP receptor P2Y12, but was not affected by blockade of the G(q)-coupled receptor, P2Y1. Similarly, direct stimulation of platelets with ADP induced the rapid activation of Rap1B. Pharmacological blockade of the P2Y1 receptor totally prevented ADP-induced Ca(2+) mobilization but did not affect activation of Rap1B. By contrast, prevention of ADP binding to the P2Y12 receptor totally suppressed activation of Rap1B without affecting Ca(2+) signaling. In platelets stimulated by cross-linking of FcgammaRIIA, inhibition of Rap1B activation by ADP scavengers could be overcome by the simultaneous recruitment of the G(i)-coupled alpha(2A)-adrenergic receptor by epinephrine. By contrast, serotonin, which binds to a G(q)-coupled receptor, could not restore activation of Rap1B. When tested alone, epinephrine was found to be able to induce GTP binding to Rap1B, whereas serotonin produced only a slight effect. Finally, activation of Rap1B induced by stimulation of the G(q)-coupled thromboxane A(2) receptor by was completely inhibited by ADP scavengers under conditions in which intracellular Ca(2+) mobilization was unaffected. Inhibition of -induced Rap1B activation was also observed upon blockade of the P2Y12 but not of the P2Y1 receptor for ADP. These results demonstrate that stimulation of a G(i)-dependent signaling pathway by either ADP of epinephrine is necessary and sufficient to activate the small GTPase Rap1B.     

Sequential sodium-proton exchange in thrombin-induced human platelets

Thrombin stimulation of human platelets initiates a membrane depolarization attributable to a Na+ influx into, and an alkalinization of, the cytoplasm, both of which follow a similar rapid time scale and thrombin-dose dependence. These responses precede secretion of the contents of the dense granules (serotonin) and, after 1 minute, of lysosomes (beta-glucuronidase). We have evaluated these parameters in the presence of 2H2O in order to determine if the Na+ influx and H+ efflux are sequential or simultaneous. NMR evidence indicates that 2H2O equilibration in rapid, and virtually complete within the 3 min prestimulation platelet equilibration period. In response to an 0.05 U/ml addition of thrombin, the rate of depolarization is 70-80% slower in 2H2O than in H2O. The time to reach maximal depolarization is 5 to 10 seconds longer in 2H2O, the extent of depolarization 60% inhibited, and the pH change 85% inhibited. The serotonin secretion is unaltered, while the beta-glucuronidase secretion is 130-180% enhanced. Dimethylamiloride inhibits the Na+ influx and the pH change completely. These results suggest that the Na+ and H+ fluxes across the plasma membrane are interdependent but neither simultaneous nor electroneutral. Furthermore, granule secretion, previously shown by us to be independent of the existent Na+ gradient, depends on the cytoplasmic K+ and H+ concentrations.     

Ras GTPase-activating protein binds to Akt and is required for its activation

RasGAP (Ras GTPase-activating protein) is a negative regulator as well as a downstream effector of Ras. To identify partners of RasGAP we used it as the bait in a yeast two-hybrid screen. This resulted in discovering its interaction with Akt. Overexpression of RasGAP or a mutant lacking the GTPase-activating domain (nGAP) enhanced phosphorylation and activity of Akt, which was dependent on the upstream integrin-linked kinase. Also, nGAP protected the cells against staurosporin-induced apoptosis through an Akt-dependent pathway. To determine the role of RasGAP in receptor-mediated activation of Akt, we used short hairpin RNA interference to knock out endogenous RasGAP expression. Although this procedure resulted in enhanced Ras activity, it inhibited Akt phosphorylation. Thus, we propose that Ras-GAP interacts with Akt and is necessary for its activation, possibly via integrin-linked kinase-mediated phosphorylation of Ser-473. The data suggest that this effect is independent of Ras activity.     

Triphosphoinositide breakdown and dense body release as the earliest events in thrombin-induced activation of human platelets

The activation by thrombin of human platelets prelabelled with 32P induced a 30-40% decrease in 32P-triphosphoinositides (TPI) in the first 10 sec; the decrease in the other 32P-labelled phosphoinositides occurred by 20-30 sec. At 10 sec., the intensity of these effects was maximum with 0.2-0.4 U/ml thrombin. Under these conditions, 53, 20 and 15% of the dense granule, alpha-granule and lysosome constituents, respectively were released and thromboxane B2 synthesis reached only 10% of its maximum. Together with experiments carried out with chlorpromazine - or PGE1 - treated platelets, our results suggest the existence of a close relationship between TPI-breakdown and dense body release which appear to be the earliest events resulting from the activation of human platelets by thrombin.     

Syndecan-4 is required for thrombin-induced migration and proliferation in human vascular smooth muscle cells

Thrombin is a mitogen and chemoattractant for vascular smooth muscle cells (SMCs) and may contribute to vascular lesion formation. We have previously shown that human SMCs, when stimulated with thrombin, release basic fibroblast growth factor (bFGF), causing phosphorylation of FGF receptor-1 (FGFR-1). Treatment with bFGF-neutralizing antibodies (anti-bFGF) or heparin inhibits thrombin-induced DNA synthesis. We concluded that thrombin may stimulate entry into the cell cycle via bFGF release and FGFR-1 activation. In the present study, we demonstrate a requirement for not only FGFR-1 but also syndecan-4, a transmembrane heparan-sulfate proteoglycan. Inhibition of syndecan-4 expression using small interfering RNA (siRNA) resulted in reduced DNA synthesis by human SMCs after stimulation with thrombin (10 nmol/liter). Anti-bFGF antibody, which inhibits DNA synthesis in control cells, had no inhibitory effect when syndecan-4 expression was reduced by siRNA. Thrombin- or bFGF-induced SMC migration, determined in Boyden chamber assays, was reduced in cells treated with syndecan-4 or FGFR-1 siRNA or by anti-bFGF. Thrombin induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in a biphasic pattern. Although thrombin-mediated ERK phosphorylation at 5 min was not affected by syndecan-4 or FGFR-1 siRNA, ERK phosphorylation at later time points was reduced. We conclude that thrombin-released bFGF binds to syndecan-4 and FGFR-1, which is required for thrombin-induced mitogenesis and migration.     

RalA and RalB Proteins Are Ubiquitinated GTPases, and Ubiquitinated RalA Increases Lipid Raft Exposure at the Plasma Membrane

Ras GTPases signal by orchestrating a balance among several effector pathways, of which those driven by the GTPases RalA and RalB are essential to Ras oncogenic functions. RalA and RalB share the same effectors but support different aspects of oncogenesis. One example is the importance of active RalA in anchorage-independent growth and membrane raft trafficking. This study has shown a new post-translational modification of Ral GTPases: nondegradative ubiquitination. RalA (but not RalB) ubiquitination increases in anchorage-independent conditions in a caveolin-dependent manner and when lipid rafts are endocytosed. Forcing RalA mono-ubiquitination (by expressing a protein fusion consisting of ubiquitin fused N-terminally to RalA) leads to RalA enrichment at the plasma membrane and increases raft exposure. This study suggests the existence of an ubiquitination/de-ubiquitination cycle superimposed on the GDP/GTP cycle of RalA, involved in the regulation of RalA activity as well as in membrane raft trafficking.     

A Ral GAP complex links PI 3-kinase/Akt signaling to RalA activation in insulin action

Insulin stimulates glucose transport in muscle and adipose tissue by translocation of glucose transporter 4 (GLUT4) to the plasma membrane. We previously reported that activation of the small GTPase RalA downstream of PI 3-kinase plays a critical role in this process by mobilizing the exocyst complex for GLUT4 vesicle targeting in adipocytes. Here we report the identification and characterization of a Ral GAP complex (RGC) that mediates the activation of RalA downstream of the PI 3-kinase/Akt pathway. The complex is composed of an RGC1 regulatory subunit and an RGC2 catalytic subunit (previously identified as AS250) that directly stimulates the guanosine triphosphate hydrolysis of RalA. Knockdown of RGC proteins leads to increased RalA activity and glucose uptake in adipocytes. Insulin inhibits the GAP complex through Akt2-catalyzed phosphorylation of RGC2 in vitro and in vivo, while activated Akt relieves the inhibitory effect of RGC proteins on RalA activity. The RGC complex thus connects PI 3-kinase/Akt activity to the transport machineries responsible for GLUT4 translocation.     

Different metastasis promotive potency of small G-proteins RalA and RalB in in vivo hamster tumor model

Background

Previously we have shown that oncogenic Ha-Ras stimulated in vivo metastasis through RalGEF-Ral signaling. RalA and RalB are highly homologous small G proteins belonging to Ras superfamily. They can be activated by Ras-RalGEF signaling pathway and influence cellular growth and survival, motility, vesicular transport and tumor progression in humans and in animal models. Here we first time compared the influence of RalA and RalB on tumorigenic, invasive and metastatic properties of RSV transformed hamster fibroblasts.

Methods

Retroviral vectors encoding activated forms or effector mutants of RalA or RalB proteins were introduced into the low metastatic HET-SR cell line. Tumor growth and spontaneous metastatic activity (SMA) were evaluated on immunocompetent hamsters after subcutaneous injection of cells. The biological properties of cells, including proliferation, clonogenicity, migration and invasion were determined using MTT, wound healing, colony formation and Boyden chamber assays respectively. Protein expression and phosphorylation was detected by Westen blot analysis. Extracellular proteinases activity was assessed by substrate-specific zymography.

Results

We have showed that although both Ral proteins stimulated SMA, RalB was more effective in metastasis stimulation in vivo as well as in potentiating of directed movement and invasion in vitro. Simultaneous expression of active RalA and RalB didn't give synergetic effect on metastasis formation. RalB activity decreased expression of Caveolin-1, while active RalA stimulated MMP-1 and uPA proteolytic activity, as well as CD24 expression. Both Ral proteins were capable of Cyclin D1 upregulation, JNK1 kinase activation, and stimulation of colony growth and motility. Among three main RalB effectors (RalBP1, exocyst complex and PLD1), PLD1 was essential for RalB-dependent metastasis stimulation.

Conclusions

Presented results are the first data on direct comparison of RalA and RalB impact as well as of RalA/RalB simultaneous expression influence on in vivo cell metastatic activity. We showed that RalB activation significantly more than RalA stimulates SMA. This property correlates with the ability of RalB to stimulate in vitro invasion and serum directed cell movement. We also found that RalB-PLD1 interaction is necessary for the acquisition of RalB-dependent high metastatic cell phenotype. These findings contribute to the identification of molecular mechanisms of metastasis and tumor progression.     

Calmodulin activation of Aurora-A kinase (AURKA) is required during ciliary disassembly and in mitosis

The centrosomal Aurora-A kinase (AURKA) regulates mitotic progression, and overexpression and hyperactivation of AURKA commonly promotes genomic instability in many tumors. Although most studies of AURKA focus on its role in mitosis, some recent work identified unexpected nonmitotic activities of AURKA. Among these, a role for basal body-localized AURKA in regulating ciliary disassembly in interphase cells has highlighted a role in regulating cellular responsiveness to growth factors and mechanical cues. The mechanism of AURKA activation involves interactions with multiple partner proteins and is not well understood, particularly in interphase cells. We show here that AURKA activation at the basal body in ciliary disassembly requires interactions with Ca(2+) and calmodulin (CaM) and that Ca(2+)/CaM are important mediators of the ciliary disassembly process. We also show that Ca(2+)/CaM binding is required for AURKA activation in mitosis and that inhibition of CaM activity reduces interaction between AURKA and its activator, NEDD9. Finally, mutated derivatives of AURKA impaired for CaM binding and/or CaM-dependent activation cause defects in mitotic progression, cytokinesis, and ciliary resorption. These results define Ca(2+)/CaM as important regulators of AURKA activation in mitotic and nonmitotic signaling.     

Distinct roles of RalA and RalB in the progression of cytokinesis are supported by distinct RalGEFs

The Ras family G-proteins RalA and RalB make critical non-overlapping contributions to the generation of a tumorigenic regulatory network, supporting bypass of the normal restraints on both cell proliferation and survival. The Sec6/8 complex, or exocyst, has emerged as a principal direct effector complex for Ral GTPases. Here, we show that RalA and RalB support mitotic progression through mobilization of the exocyst for two spatially and kinetically distinct steps of cytokinesis. RalA is required to tether the exocyst to the cytokinetic furrow in early cytokinesis. RalB is then required for recruitment of the exocyst to the midbody of this bridge to drive abscission and completion of cytokinesis. The collaborative action of RalA and RalB is specified by discrete subcellular compartmentalization and unique pairs of RalGEF proteins that provide inputs from both Ras-family protein-dependent and protein-independent regulatory cues. This suggests that Ral GTPases integrate diverse upstream signals to choreograph multiple roles for the exocyst in mitotic progression.     

Cytoplasmic Ca2+ is necessary for thrombin-induced platelet activation

alpha-Thrombin induces a dose-dependent rapid transient increase in platelet cytosolic Ca2+ levels, coming solely from intracellular stores, since EGTA has no effect. In contrast, the post-stimulation equilibrium [Ca2+]in depends upon an influx from the extracellular milieu, and is lower in the presence of EGTA. We measured the Ca2+ transient (with Indo-1, 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2'-amino-5'-methylp henoxy)- ethane-N,N,N',N'-tetraacetic acid), cytosolic alkalinization (with BCECF, 2',7-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein), membrane depolarization (with diS-C3-(5), 3,3'-dipropylthiodi-carbocyanide iodide), and degranulation (by beta-glucuronidase release) induced in washed human platelets by 9 nM thrombin in the absence or presence of extracellular or intracellular Ca2+ chelating agents (EGTA and BAPTA, 5,5'-dimethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, respectively). Platelets loaded simultaneously with 2 microM Indo-1 and 15 microM BAPTA (each as the acetoxymethyl ester) before addition of thrombin exhibited no cytoplasmic Ca2+ transient or alkalinization, no depolarization or degranulation. Replenishment of such cells with extracellular CaCl2 restored resting [Ca2+]in. Upon stimulation with 9 nM thrombin these replenished platelets exhibited no Ca2+ transient, and a slow gradual increase in [Ca2+]in from extracellular stores, a slow alkalinization and depolarization, and partial degranulation, all abolished by extracellular EGTA. Thus thrombin-induced platelet activation exhibits a biphasic Ca2+ requirement: the initial transient increase in [Ca2+]in comes from intracellular stores only, while the later steps of depolarization, alkalinization, and degranulation can proceed, albeit more slowly, if only extracellular Ca2+ is available.     

Membrane depolarization inhibits thrombin-induced calcium influx and aggregation in human platelets.

The relationship between thrombin-evoked changes in intracellular calcium concentration [( Ca2+]i) and aggregation was examined in Indo-1-loaded human platelets. The stimulus-induced intracellular calcium release and external calcium influx, as well as platelet aggregation, were studied in the same cell preparation. A close correlation between the sustained high [Ca2+]i level, depending on calcium entry, and the aggregation response was found. Gramicidin, at a concentration high enough to induce membrane depolarization, strongly inhibited the calcium influx and aggregation, but did not influence the thrombin-induced intracellular calcium release. We conclude that calcium influx through depolarization-inhibited calcium channels is a prerequisite of thrombin-induced platelet aggregation.     

Microfilament assembly is required for antigen-receptor-mediated activation of human B lymphocytes

The mechanisms responsible for initiating the conversion of globular to filamentous actin (assembly) after stimulation of B lymphocytes and the role of these cytoskeletal changes in cell activation are incompletely understood. We investigated the molecular basis of the signals leading to actin polymerization and concentrated on the involvement of guanosine triphosphate (GTP)-binding regulatory proteins, and protein kinase C (PKC). In addition, we related these early events to later events in B-cell activation, including cell proliferation. Cross-linking the Ag receptor with Staphylococcus aureus Cowan I (SAC) or anti-IgM antibodies, or stimulation of PKC with phorbol ester induced a time- and concentration-dependent increase in the filamentous actin content of B cells. Inhibition or depletion of PKC resulted in decreased actin assembly induced by anti-IgM, SAC, and PMA, suggesting that the signal for polymerization is generated distally to PKC activation. Pertussis toxin pretreatment inhibited the responses to anti-IgM and SAC but not PMA, and direct stimulation of permeabilized cells with GTP gamma S induced microfilament assembly, indicating the involvement of a GTP-binding protein for receptor-mediated events. Disruption of actin polymerization with botulinum C2 toxin or cytochalasin D inhibited the assembly of actin and [3H]TdR incorporation induced by all stimuli. We conclude that human B cell activation by receptor-mediated stimuli results in actin polymerization by signaling pathways coupled to GTP-binding proteins. These changes in the cytoskeleton may be involved in the transduction of messages leading to responses such as proliferation in B lymphocytes.     

Calmodulin in human serum and the specific release of calmodulin from calmodulin-rich platelets

Calmodulin(CaM)-like activity was detected in human serum and foetal calf serum, with an activity i0 times more than that detectable in plasma. Serum CaM was largely accounted for by release from human platelets as confirmed by both radioimmunoassay and sodium-dodecyl-sulphate/polyacrylamide-gel electrophoresis of CaM partially purified from platelet releasate obtained in response to thrombin. Lactate dehydrogenase release was unaffected by thrombin. Platelet CaM content was very variable (1.3 to 11.3 pg/mg protein; n=15). It is suggested that intact platelets are rich in CaM and that release of CaM during preparation explains the variation in CaM content reported here and in the literature.     

Calmodulin is required for cell-cycle progression during G1 and mitosis.

In order to examine the consequences of a transient increase or decrease in intracellular calmodulin (CaM) levels, two bovine-papilloma-virus (BPV)-based expression vectors capable of inducibly synthesizing CaM sense (BPV-MCM) or anti-sense (BPV-CaMAS) RNA have been constructed and used to stably transform mouse C127 cells. Upon addition of Zn2+, cells containing the BPV-MCM vector have transiently increased CaM mRNA and protein levels. Cells carrying the BPV-CaMAS vector transiently produce CaM anti-sense RNA resulting in a significant decrease in intracellular CaM concentration. Increased CaM caused a transient acceleration of proliferation, while the anti-sense RNA induced decrease in CaM caused a transient cell cycle arrest. Flow cytometric analysis showed that progression through G1 and mitosis was affected by changes in CaM levels. These data indicate that CaM levels may limit the rate of cell-cycle progression under normal conditions of growth.     

NADPH oxidase activation is required for migration by LIGHT in human monocytes

Homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus (HSV) glycoprotein D (gD) for herpes virus entry mediator (HVEM; TR2) (LIGHT), a ligand of herpes virus entry mediator (HVEM), increased reactive oxygen species (ROS) and enhanced the destruction of bacteria in human monocytes. In this study, rhLIGHT was found to increase the expression of the chemokine receptors, chemokine receptor 1 (CCR1) and CCR2, as well as to accelerate the migration activity of human monocytes. Additionally, rhLIGHT was found to increase ROS via NADPH oxidase p47^phox phosphorylation, which was found to be required for LIGHT-induced NF-κB activation, CCR1 and CCR2 expression, migration and IL-8 and TNF-α production. Taken together, these results indicate that NADPH oxidase activation is required for rhLIGHT-induced migration in human monocytes.     

Resonance assignments for the RLIP76 Ral binding domain in its free form and in complex with the small G protein RalB

We report ¹H and ¹⁵N resonance assignments for the free Ral binding domain of RLIP76 (393–446) and the ¹H, ¹⁵N and ¹³C resonance assignments for the RLIP76 Ral binding domain in complex with the active conformation of RalB. The BMRB accession code for free RLIP76 is 15524 and in complex with RalB is 15525.     

pH sensitivity of the thrombin-induced rise in fructose 2,6-bisphosphate content of human platelets

The stimulation of human platelets with thrombin results in a rapid and sustained increase in the fructose 2,6-bisphosphate content which may play an important role in the potentiation of glycolytic flux induced by the agonist. The investigation of the effect of pH on thrombin-induced rise in platelet fructose 2,6-bisphosphate content is reported here. The results indicate that the early intracellular alkalinization which follows platelet stimulation may contribute to mediate the positive effect of thrombin on the regulatory metabolite.