Activity In Early Phase Research Articles

Looking through ion channels: recharged concepts in T-cell signaling

It has been suggested that lymphocyte K⁺ and Ca²⁺ channels participate in the early phase of T-cell activation by generating a Ca²⁺ signal to drive T-cell proliferation. Recent biophysical and pharmacological characterization of ion channels responsible for Ca²⁺ entry into cells facilitated a better understanding of the physiological role of these channels. In addition, the simple ‘permissive' function of ion channels in T-cell activation has been challenged recently, by studies indicating that pharmacological tools can selectively inhibit pathological immune reactions, and voltage-gated K⁺ channels might directly participate in the function of the immunological synapse.

Ultraviolet Light Activates NFκB through Translational Inhibition of IκBα Synthesis

UV light induces a delayed and prolonged (3–20 h) activation of NFκB when compared with the immediate and acute (10–90 min) activation of NFκB in response to tumor necrosis factor α treatment. In the early phase (3–12 h) of NFκB activation, UV light reduces inhibitor of NFκB (IκB) through an IκB kinase-independent, but polyubiquitin-dependent, pathway. However, the mechanism for the UV light-induced reduction of IκB and activation of NFκB is not known. In this report, we show that UV light down-regulates the total amount of IκB through decreasing IκB mRNA translation. Our data show that UV light inhibits translation of IκB in wild-type mouse embryo fibroblasts (MEF^S/S) and that this inhibition is prevented in MEF^A/A cells in which the phosphorylation site, Ser-51 in the eukaryotic translation initiation factor 2 α-subunit, is replaced with a non-phosphorylatable Ala (S51A). Our data also show that UV light-induced NFκB activation is delayed in MEF^A/A cells and in an MCF-7 cell line that is stably transfected with a <i>trans</i>-dominant negative mutant protein kinase-like endoplasmic reticulum kinase (PERK). These results suggest that UV light-induced eukaryotic translation initiation factor 2 α-subunit phosphorylation translationally inhibits new IκB synthesis. Without a continuous supply of newly synthesized IκB, the existing IκB is degraded through a polyubiquitin-dependent proteasomal pathway leading to NFκB activation. Based upon our results, we propose a novel mechanism by which UV light regulates early phase NFκB activation by means of an ER-stress-induced translational inhibition pathway.

Camptothecin induced mitochondrial dysfunction leading to programmed cell death in unicellular hemoflagellate Leishmania donovani.

The parasites of the order kinetoplastidae including Leishmania spp. emerge from most ancient phylogenic branches of unicellular eukaryotic lineages. In their life cycle, topoisomerase I plays a significant role in carrying out vital cellular processes. Camptothecin (CPT), an inhibitor of DNA topoisomerase I, induces programmed cell death (PCD) both in the amastigotes and promastigotes form of L. donovani parasites. CPT-induced cellular dysfunction in L. donovani promastigotes is characterized by several cytoplasmic and nuclear features of apoptosis. CPT inhibits cellular respiration that results in mitochondrial hyperpolarization taking place by oligomycin-sensitive F0-F1 ATPase-like protein in leishmanial cells. During the early phase of activation, there is an increase in reactive oxygen species (ROS) inside cells, which causes subsequent elevation in the level of lipid peroxidation and decrease in reducing equivalents like GSH. Endogenous ROS formation and lipid peroxidation cause eventual loss of mitochondrial membrane potential. Furthermore, cytochrome c is released into the cytosol in a manner independent of involvement of CED3/CPP32 group of proteases and unlike mammalian cells it is insensitive to cyclosporin A. These events are followed by activation of both CED3/CPP32 and ICE group of proteases in PCD of Leishmania. Taken together, our study indicates that different biochemical events leading to apoptosis in leishmanial cells provide information that could be exploited to develop newer potential therapeutic targets.

Cyclosporine in thoracic organ transplantation

The discovery of cyclosporine proved to be a breakthrough that helped transform the status of both heart and lung transplantation from experimental to established therapeutic procedures. Cyclosporine inhibits the early phase of T-cell activation and thus of the alloimmune response. It proved to be highly effective in prophylaxis against acute rejection but its use has been limited by dose-related renal toxicity. Consequently, it has generally been used in regimens that combine it with other immunosuppressive agents with the aim of preventing acute rejection while minimising toxicity. For many years ‘triple therapy’ using cyclosporine, azathioprine and corticosteroids was the most commonly used regimen for both heart and lung transplantation. Other agents have now become available that provide more effective prophylaxis against acute rejection than azathioprine; these include, mycophenolate and the TOR inhibitors sirolimus and everolimus. Everolimus has also been shown to inhibit the early phase of cardiac allograft vasculopathy. Unfortunately, the TOR inhibitors also potentiate the nephrotoxicity of cyclosporine. Both mycophenolate and the TOR inhibitors are subject to pharmacokinetic interactions with cyclosporine. We are now entering a new phase of clinical immunosuppression where we need to learn how to best combine the agents that are currently available to provide the safest and most effective immunosuppression for patients undergoing heart or lung transplantation.

A Postmitotic Role for Isl-Class LIM Homeodomain Proteins in the Assignment of Visceral Spinal Motor Neuron Identity

LIM homeobox genes have a prominent role in the regulation of neuronal subtype identity and distinguish motor neuron subclasses in the embryonic spinal cord. We have investigated the role of Isl-class LIM homeodomain proteins in motor neuron diversification using mouse genetic methods. All spinal motor neuron subtypes initially express both Isl1 and Isl2, but Isl2 is rapidly downregulated by visceral motor neurons. Mouse embryos lacking Isl2 function exhibit defects in the migration and axonal projections of thoracic level motor neurons that appear to reflect a cell-autonomous switch from visceral to somatic motor neuron character. Additional genetic mutations that reduce or eliminate both Isl1 and Isl2 activity result in more pronounced defects in visceral motor neuron generation and erode somatic motor neuron character. Thus, an early phase of high Isl expression and activity in newly generated motor neurons permits the diversification of visceral and somatic motor neuron subtypes in the developing spinal cord.

The c-Jun N-terminal Kinase 1 Activity Is Differentially Regulated by Specific Mechanisms during Apoptosis

We show here that JNK1 activity is rapidly up-regulated and prolonged by specific mechanisms during apoptosis induced by paclitaxel- or ginsenoside-Rh2 in SK-HEP-1 cells. The early phase of JNK1 activation is prevented in cells expressing the dominant negative SEK1 mutant, although this JNK1 perturbation does not prevent apoptotic cell death. The later phase of JNK1 activation, which is temporally coincided with caspase-dependent cleavage of JNK1-associated p21(WAF1/CIP1), is efficiently prevented by expressing p21D112N, an uncleavable mutant of p21(WAF1/CIP1) and this perturbation of JNK1 activation results in prevention of apoptosis. The later JNK1 activation and apoptotic progression are also prevented by co-treatments of cells with rottlerin, a PKC-delta inhibitor or z-VAD-fmk, a pan caspase inhibitor. We also provide evidence that apoptotic cell death is significantly promoted in cells expressing JNK1, while this apoptotic cell death is effectively suppressed in cells expressing the dominant negative JNK1 mutant (DN-JNK1) or JBD, a JNK inhibitor protein. Thus, the later phase of JNK1 activation, which is linked to a caspase-dependent mechanism that requires PKC-delta activity, is associated with the induction of apoptosis, while the early JNK1 activation that is associated with a SEK1-mediated mechanism is not directly involved in apoptotic progression.

Microglial activation and increased synthesis of complement component C1q precedes blood–brain barrier dysfunction in rats

A reliable way to visualise the state of microglial activation is to monitor the microglial gene expression profile. Microglia are the only CNS resident cells that synthesise C1q, the recognition sub-component of the classical complement pathway, in vivo. C1q biosynthesis in resting ramified microglia is often low, but it increases dramatically in activated microglia. In this study, the expression of C1q was used to monitor microglial activation at all stages of 3-chloropropanediol-induced neurotoxicity, a new model of blood–brain barrier (BBB) breakdown. In rats, 3-chloropropanediol produces very focused lesions in the brain, characterised by early astrocyte swelling and loss, followed by neuronal death and barrier dysfunction. Using in situ hybridisation, immunohistochemistry, and real-time RT-PCR, we found that increased C1q biosynthesis and microglial activation precede BBB dysfunction by at least 18 and peak 48 h after injection of 3-chloropropanediol, which coincides with the onset of active haemorrhage. Microglial activation is biphasic; an early phase of global activation is followed by a later phase in which microglial activation becomes increasingly focused in the lesions. During the early phase, expression of the pro-inflammatory mediators interleukin-1β (IL1β), tumour necrosis factor α (TNFα) and early growth response-1 (Egr-1) increased in parallel with C1q, but was restricted to the lesions. Expression of C1q (but not IL1β, TNFα or Egr-1) remains high after BBB function is restored, and is accompanied by late up-regulation of the C1q-associated serine proteases, C1r and C1s, suggesting that microglial biosynthesis of the activation complex of the classical pathway may support the removal of cell debris by activation of complement.

Protein kinase Calpha (PKCalpha) acts upstream of PKCtheta to activate IkappaB kinase and NF-kappaB in T lymphocytes.

NF-kappaB is an ubiquitous transcription factor that is a key in the regulation of the immune response and inflammation. T-cell receptor (TCR) cross-linking leads to NF-kappaB activation, an IkappaB kinase (IKK)-dependent process. However, the upstream kinases that regulate IKK activity following TCR activation remain to be fully characterized. Herein, we demonstrate using genetic analysis, pharmacological inhibition, and RNA interference (RNAi) that the conventional protein kinase C (PKC) isoform PKCalpha, but not PKCbeta1, is required for the activation of the IKK complex following T-cell activation triggered by CD3/CD28 cross-linking. We find that in the presence of Ca(2+) influx, the catalytically active PKCalphaA25E induces IKK activity and NF-kappaB-dependent transcription; which is abrogated following the mutations of two aspartates at positions 246 and 248, which are required for Ca(2+) binding to PKCalpha and cell membrane recruitment. Kinetic studies reveal that an early phase (1 to 5 min) of IKK activation following TCR/CD28 cross-linking is PKCalpha dependent and that a later phase (5 to 25 min) of IKK activation is PKCtheta dependent. Activation of IKK- and NF-kappaB-dependent transcription by PKCalphaA25E is abrogated by the PKCtheta inhibitor rottlerin or the expression of the kinase-inactive form of PKCtheta. Taken together, our results suggest that PKCalpha acts upstream of PKCtheta to activate the IKK complex and NF-kappaB in T lymphocytes following TCR activation.

SEK1-dependent JNK1 activation prolongs cell survival during G-Rh2-induced apoptosis

We provide here evidence that c-Jun N-terminal protein kinase 1 (JNK1) activity is differentially up-regulated during apoptosis of SK-HEP-1 cells after treatment with ginsenoside Rh2 (G-Rh2). The G-Rh2-mediated JNK1 activation that occurred for the first 10–30 min was associated with SEK1 activity, but thereafter, the sustained activation was associated not with SEK1 activity, but with proteolytic cleavage of JNK1-associated p21 WAF1/CIP1. Supporting this is that the expression of the dominant negative SEK1 mutant effectively blocked the early JNK1 activation phase but did not alter the sustained activation phase or apoptosis. Furthermore, expression of p21D112N, an uncleavable mutant of p21 WAF1/CIP1, suppressed the later JNK1 activation. Moreover, the stable overexpression of ectopic JNK1 suppressed apoptosis while expression of the dominant negative JNK1 mutant promoted it. We propose that the early SEK1-associated JNK1 activation phase acts to prolong cell survival in response to apoptosis-inducing agents, thereby serving as an intervening checkpoint prior to the commitment to apoptosis.

Nitric oxide promotes p53 nuclear retention and sensitizes neuroblastoma cells to apoptosis by ionizing radiation

Nitric oxide (NO) is a potent activator of the p53 tumor suppressor protein. However, the mechanisms underlying p53 activation by NO have not been fully elucidated. We previously reported that a rapid downregulation of Mdm2 by NO may contribute to the early phase of p53 activation. Here we show that NO promotes p53 nuclear retention and inhibits Mdm2-mediated p53 nuclear export. NO induces phosphorylation of p53 on serine 15, which does not require ATM but rather appears to depend on the ATM-related ATR kinase. An ATR-kinase dead mutant or caffeine, which blocks the kinase activity of ATR, effectively abolishes the ability of NO to cause p53 nuclear retention, concomitant with its inhibition of p53 serine 15 phosphorylation. Of note, NO enhances markedly the ability of low-dose ionizing radiation to elicit apoptotic killing of neuroblastoma cells expressing cytoplasmic wild-type p53. These findings imply that, through augmenting p53 nuclear retention, NO can sensitize tumor cells to p53-dependent apoptosis. Thus, NO donors may potentially increase the efficacy of radiotherapy for treatment of certain types of cancer.

Endothelial cell activation by endotoxin involves superoxide/NO-mediated nitration of prostacyclin synthase and thromboxane receptor stimulation.

In bovine coronary artery segments, peroxynitrite inhibits prostacyclin (PGI2) synthase by tyrosine nitration. Using this pharmacological model, we show that a 1 h exposure of bovine coronary artery segments to endotoxin (lipopolysaccharide [LPS]) inhibits the relaxation phase following angiotensin II (Ang II) stimulation and causes a vasospasm that can be suppressed by a thromboxane A2 (TxA2) receptor blocker. In parallel, PGI2 synthesis decreases in favor of prostaglandin E2 formation. Immunoprecipitation and costaining with an anti-nitrotyrosine antibody identified PGI2 synthase as the main nitrated protein in the endothelium. All effects of LPS could be prevented in the presence of the nitric oxide (NO) synthase inhibitor Nomega-mono-methyl-L-arginine and polyethylene-glycolated Cu/Zn- superoxide dismutase. Thus, the early phase of endothelial cell activation in bovine coronary arteries by inflammatory agents proceeds by a protein synthesis-independent priming process for a source of superoxide that we tentatively attribute to xanthine oxidase. Upon receptor activation, Ang II stimulates NO and superoxide production, resulting in a peroxynitrite-mediated nitration and inhibition of PGI2 synthase. The remaining 15-hydroxy-prostaglandin 9,11-endoperoxide (PGH2) first activates the TxA2/PGH2 receptor and then is converted to prostaglandin E2 (PGE2) by smooth muscle cells. PGE2 together with a lack of NO and PGI2 is known to promote the adhesion of white blood cells and their immigration to the inflammatory locus.

A systematic approach for the solubilization of the integral membrane protein lysophospholipid: Acyl-Coa acyltransferase (LAT).

The lysophospholipid∶acyl-CoA acyltransferase (LAT, EC 2.3.1.23) catalyzes the reacylation of lysophospholipids with coenzyme A-activated fatty acids, and is part of the Lands cycle (). The LAT is an important enzyme participating in the rapid turnover of phospholipids thereby: 1) maintaining the membrane lipid composition and the asymmetrical distribution of unsaturated fatty acids within the phospholipids (for review, see refs. ,), and 2) controlling the free arachidonic acid levels (for review, see ref. ). In addition, LAT is thought to play a crucial role in the early phase of T-cell activation by the elevated incorporation of polyunsaturated fatty acids into plasma membrane phospholipids (,). LAT is suggested to be an integral membrane protein, and it is established that there are separate acyltransferases with different specificity toward both lysophospholipids and acyl-CoAs (,).

Both the ADP receptors P2Y 1 and P2Y 12 , play a role in controlling shape change in human platelets

Two types of ADP receptors, P2Y 1 and P2Y 12 are involved in platelet aggregation. The P2X 1 receptor is also present but its role, in terms of platelet function, is not yet defined. The aim of this study was to establish if the ADP receptors, P2Y 1, P2Y 12 and P2X 1 play a role in controlling platelet shape change (PSC) in human platelets. PSC is an early phase of platelet activation that precedes aggregation. Using a high-resolution channelyzer, PSC was assessed by measuring the median platelet volume (MPV). The P2Y 1 receptor antagonist MRS 2179 (1.06 m 10.25 µmol/l) blocked ADP-induced PSC (by 100%). The median IC 50 was 3.16 µmol/l. MRS 2179 also significantly ( P = 0.01) inhibited PSC induced by the combination of ADP + serotonin (5HT). The P2Y 12 receptor antagonist AR-C69931MX significantly inhibited (at 10s, P = 0.009; 15 s, P = 0.001 and 30 s, P = 0.015) ADP-induced PSC. The P2X 1 receptor antagonist TNP-ATP had no significant effect on ADP- or ADP + 5HT-induced PSC. We conclude that the IC 50 of a P2Y 1 -blocker can be derived because of the high-resolution and reproducibility of the channelyzer technique. In addition to the P2Y 1 purinoceptor, the P2Y 12 receptor appears to be involved in ADP-induced PSC since this process was significantly inhibited by AR-C69931MX. The channelyzer technique may be more reliable than optical aggregometry to assess PSC.

Does sustained downregulation of the TCRzeta chain define the transition from antigen mode to inflammation mode in effector T lymphocytes?

The molecular events that define the early phase of activation and differentiation of effector T cells have been well characterized. Those events promoting the effector function of chronically activated T cells at sites of inflammation are less well understood. Using in vitro and in vivo models, we have explored the effects of the chronic inflammatory process on T-cell differentiation by studying the effects of tumor necrosis factor (TNF) on T-cell activation and effector responses. These studies have revealed that TNF-stimulated T cells resemble rheumatoid arthritis synovial T cells since they express cell surface activation antigens, but are profoundly hyporesponsive to T-cell receptor (TCR) engagement. This may be explained, at least in part, by the observation that TNF selectively targets the expression of the TCRzeta chain. Loss of TCRzeta expression perturbs the assembly, expression and stability of the TCR/CD3 complex, leading to attenuation of membrane proximal tyrosine phosphorylation, intracellular calcium mobilisation and the transcription of cytokine genes upon TCR engagement, when compared with untreated T cells. We set out to explore whether TCRzeta expression could be used to identify chronically activated, hyporesponsive T cells in vivo. Using a FACS-based assay, we have identified subsets of CD3+ T cells expressing low levels of TCRzeta (hereafter termed TCRzetadim cells) in the peripheral blood of healthy donors, as well as patients with inflammatory arthritis. Subsequent experiments revealed that, when compared with TCRzetabright cells, the TCRzetadim population is enriched for cells expressing effector memory cell surface markers. By staining with HLA class I/peptide tetramer complexes, CD8+TCR-zetadim cells are enriched for antigen-specific T cells after stimulation in vitro with specific peptide. While TCRzetadim cells are hyporesponsive to TCR engagement, they retain effector potential, since a significant proportion are TNF-α and IFN-γ producers upon stimulation with phorbol ester and calcium ionophore. In contrast, fewer produce IL-10 when compared with the TCRzetabright subset. These data suggest that TCRzetadim T cells, which are abundant at sites of inflammation, may represent a subset of circulating antigen experienced effector memory cells. Sustained downregulation of TCRzeta may define a checkpoint where the intracellular signals driving T-cell differentiation switch from antigen mode to inflammation mode.

Molecular Mechanisms for Lipopolysaccharide-induced Biphasic Activation of Nuclear Factor-κB (NF-κB)

The nuclear factor-kappaB (NF-kappaB) is an important transcription factor necessary for initiating and sustaining inflammatory and immune reactions. The inducers of NF-kappaB are well characterized, but the molecular mechanisms underlying multiple in vivo NF-kappaB activation processes are poorly understood. The injection of lipopolysaccharide resulted in a biphasic activation of NF-kappaB during the 18-h observation period in various organs of mice. The early and late phases of NF-kappaB activation occurred at 0.5-2 h and 8-12 h, respectively. Platelet-activating factor, which is released in response to lipopolysaccharide injection, was responsible for the activation of the early phase of NF-kappaB. The early NF-kappaB activity led to the induction of proinflammatory cytokines, tumor necrosis factor (TNF), and interleukin (IL)-1beta, which are known to be efficient inducers of NF-kappaB. Using the TNF knockout and IL-1 receptor knockout mice, we found that TNF and IL-1beta had a role in the second phase activation of NF-kappaB. These cytokines did promote the synthesis of platelet-activating factor, which in turn induced the secondary activation of NF-kappaB. These observations describe a novel autoregulatory molecular mechanism for the biphasic activation of NF-kappaB.

Involvement of Tachykinin NK1 and NK2 Receptors in Changes in Lung Mechanics and Airway Microvascular Leakage during the Early Phase of Endotoxemia in Guinea Pigs

We investigated the role of tachykinins in airway neurogenic responses occurring in the early phase of endotoxemia. Forty-eight anesthetized guinea pigs were evenly divided into six groups pretreated with either saline vehicle, CP-96,345 (a tachykinin NK₁ receptor antagonist), SR-48,968 (a tachykinin NK₂ receptor antagonist) or CP-96,345 and SR-48,968 in combination. Animals then received an intravenous injection of either saline (the vehicle for endotoxin) or endotoxin (30 mg/kg). Total lung resistance (R_L) and dynamic lung compliance (C_dyn) were continuously measured before and 30 min after administration of saline or endotoxin. Airway microvascular leakage was assessed at the end of the observation period. Endotoxin significantly increased R_L and decreased C_dyn 10 min after intravenous endotoxin injection. Plasma extravasation significantly increased in the trachea, main bronchi and intrapulmonary airways with endotoxin administration. These changes in lung mechanics were abolished by SR-48,968, but were unaffected by CP-96,345. The plasma extravasation was largely attenuated by CP-96,345 and/or SR-48,968. We conclude that (1) endogenous tachykinins play an important role in producing changes in lung mechanics and airway microvascular leakage during the early phase of endotoxemia and (2) activation of tachykinin NK₂ receptors is responsible for the former response, while activation of both tachykinin NK₁ and NK₂ receptors is involved in the latter response.

Involvement of tachykinin NK(1) and NK(2) receptors in changes in lung mechanics and airway microvascular leakage during the early phase of endotoxemia in Guinea pigs.

We investigated the role of tachykinins in airway neurogenic responses occurring in the early phase of endotoxemia. Forty-eight anesthetized guinea pigs were evenly divided into six groups pretreated with either saline vehicle, CP-96,345 (a tachykinin NK(1) receptor antagonist), SR-48,968 (a tachykinin NK(2) receptor antagonist) or CP-96,345 and SR-48,968 in combination. Animals then received an intravenous injection of either saline (the vehicle for endotoxin) or endotoxin (30 mg/kg). Total lung resistance (R(L)) and dynamic lung compliance (C(dyn)) were continuously measured before and 30 min after administration of saline or endotoxin. Airway microvascular leakage was assessed at the end of the observation period. Endotoxin significantly increased R(L) and decreased C(dyn) 10 min after intravenous endotoxin injection. Plasma extravasation significantly increased in the trachea, main bronchi and intrapulmonary airways with endotoxin administration. These changes in lung mechanics were abolished by SR-48,968, but were unaffected by CP-96,345. The plasma extravasation was largely attenuated by CP-96,345 and/or SR-48,968. We conclude that (1) endogenous tachykinins play an important role in producing changes in lung mechanics and airway microvascular leakage during the early phase of endotoxemia and (2) activation of tachykinin NK(2) receptors is responsible for the former response, while activation of both tachykinin NK(1) and NK(2) receptors is involved in the latter response.

Inhibition of mitochondrial respiration by endogenous nitric oxide: A critical step in Fas signaling

We have found that activation of human adult T cell leukemia (Jurkat) cells with anti-Fas Ab leads, in a concentration-dependent manner, to an early burst of production of nitric oxide (NO), which inhibits cell respiration. This results in mitochondrial hyperpolarization, dependent on the hydrolysis of glycolytic ATP by the F1F(o)-ATPase acting in reverse mode. During this early phase of activation, there is a transient release of superoxide anion. All these processes can be prevented by an inhibitor of NO synthase. Approximately 2 h after stimulation with anti-Fas Ab, a distinct second phase can be detected. This comprises a concentration-dependent collapse in mitochondrial membrane potential, a second wave of free radical production, and activation of caspase-8 leading to apoptosis. This second phase is abolished by an inhibitor of caspase activation. In contrast, inhibition of NO synthesis leads to an enhancement and acceleration of these latter processes, suggesting that the early NO-dependent phase represents a protective mechanism. The significance of the two phases in relation to cell survival and death remains to be studied.

Regulation of G Protein-linked Guanine Nucleotide Exchange Factors for Rho, PDZ-RhoGEF, and LARG by Tyrosine Phosphorylation: EVIDENCE OF A ROLE FOR FOCAL ADHESION KINASE

A recently identified family of guanine nucleotide exchange factors for Rho that includes PDZ-RhoGEF, LARG, and p115RhoGEF exhibits a unique structural feature consisting in the presence of area of similarity to regulators of G protein signaling (RGS). This RGS-like (RGL) domain provides a structural motif by which heterotrimeric G protein alpha subunits of the Galpha(12) family can bind and regulate the activity of RhoGEFs. Hence, these newly discovered RGL domain-containing RhoGEFs provide a direct link from Galpha(12) and Galpha(13) to Rho. Recently available data suggest, however, that tyrosine kinases can regulate the ability of G protein-coupled receptors (GPCRs) to stimulate Rho, although the underlying molecular mechanisms are still unknown. Here, we found that the activation of thrombin receptors endogenously expressed in HEK-293T cells leads to a remarkable increase in the levels of GTP-bound Rho within 1 min (11-fold) and a more limited but sustained activation (4-fold) thereafter, which lasts even for several hours. Interestingly, tyrosine kinase inhibitors did not affect the early phase of Rho activation, immediately after thrombin addition, but diminished the levels of GTP-bound Rho during the delayed phase. As thrombin receptors stimulate focal adhesion kinase (FAK) potently, we explored whether this non-receptor tyrosine kinase participates in the activation of Rho by GPCRs. We obtained evidence that FAK can be activated by thrombin, Galpha(12), Galpha(13), and Galpha(q) through both Rho-dependent and Rho-independent mechanisms and that PDZ-RhoGEF and LARG can in turn be tyrosine-phosphorylated through FAK in response to thrombin, thereby enhancing the activation of Rho in vivo. These data indicate that FAK may act as a component of a positive feedback loop that results in the sustained activation of Rho by GPCRs, thus providing evidence of the existence of a novel biochemical route by which tyrosine kinases may regulate the activity of Rho through the tyrosine phosphorylation of RGL-containing RhoGEFs.

A novel role for Gqα in α-thrombin-mediated mitogenic signalling pathways

α-Thrombin activates several G-proteins including members of the Gq, Gi, and G12/13 families, although the physiological importance of these proteins is still not completely understood. We specifically investigated the role of Gqα in modulating α-thrombin-induced mitogenesis. In Gqa1 cells, a stable cell line expressing reduced amounts of Gqα, concentrations of α-thrombin (1 NIH unit/ml), which induce cell cycle reentry and progression into S phase in wild-type IIC9 cells, do not stimulate phosphatidylinositol (PI) hydrolysis, the rapid early phase of ERK activity, and transit through G1 into S phase as quantified by cyclin-dependent kinase (CDK)4–cyclin D activity and [ 3H]thymidine incorporation. Interestingly, high concentrations of α-thrombin restore these activities and cell cycle progression into S phase. While, it is well documented that α-thrombin-induced sustained ERK activity mediates important responses for transit through G1 into S phase, the importance of the rapid, Gq-dependent phase as a prerequisite for α-thrombin-mediated mitogenesis has not been appreciated.