Calcium-dependent Tyrosine Kinase Research Articles

High expression of PYK2 is associated with poor prognosis and cancer progression in early-stage cervical carcinoma.

Proline-rich tyrosine kinase-2 (PYK2), also known as calcium dependent tyrosine kinase, regulates different signal transduction cascades that control cell proliferation, migration, and invasion. However, the role of PYK2 in cervical cancer remains to be elucidated. The current study retrospectively included 134 patients with cervical cancer from December 2007 to September 2014. PYK2 expression was detected in tissue microarray and cervical cancer cell lines. Statistical analysis was performed to evaluate its clinicopathological significance. Small interfering RNA (siRNA) was employed to suppress endogenous PYK2 expression in cervical cancer cells to observe the biological function. PYK2 expression was up-regulated in cervical cancer specimens compared with paired adjacent normal cervical tissue samples. Statistical analyses indicated that PYK2 expression might be an independent prognostic indicator for patients with early-stage cervical cancer. A nomogram model was constructed based on PYK2 expression and other clinicopathological risk factors, and it performed well in predicting patients survival. In cellular studies, down-regulation of PYK2 remarkably inhibited cellular proliferation, migration and invasion. PYK2 expression possessed the potential to serve as a novel prognostic marker in cervical cancer patients.

HBx targeting to mitochondria and ROS generation are necessary but insufficient for HBV-induced cyclooxygenase-2 expression

Chronic inflammation can be a major risk factor for cancer development and may contribute to the high worldwide incidence of hepatocellular carcinoma (HCC). Cyclooxygenase-2 (COX-2) is known to be an important mediator of inflammatory responses; however, its link to hepatitis B virus (HBV)-mediated inflammatory responses has not been established. Here, we demonstrate that the expression of COX-2 mRNA and protein was significantly elevated in cells transfected by HBV replicon but not in cells transfected by HBV genome lacking the HBx gene. Notably, COX-2 induction was correlated with HBx's ability to increase reactive oxygen species (ROS) levels. Consistently with this, antioxidant treatment and ectopic expression of manganese superoxide dismutase or catalase completely abolished COX-2 induction. Interestingly, a mitochondria localization-defective mutant of HBx (HBx(Delta 68-117)) neither increased intracellular ROS levels nor induced COX-2 expression. HBx(68-117), which encodes only amino acids 68-117 and is sufficient for mitochondria localization, increased ROS levels but did not induce COX-2 expression. Similarly, HBx targeting to the outer membrane of mitochondria (Mito-HBx) increased ROS but also failed to increase COX-2 expression, suggesting that other cytoplasmic signaling pathways are involved in HBx-mediated COX-2 induction. Indeed, inhibition of cytoplasmic calcium signaling by cyclosporine A, blocking mitochondrial permeability transition pore, and herbimycin, and inhibition of calcium-dependent tyrosine kinase suppressed HBV-mediated COX-2 induction. Thus, the data indicate that both mitochondrial ROS and cytoplasmic calcium signaling are necessary for the COX-2 induction. Our studies revealed a pathophysiological link between HBV infection and hepatic inflammation, and this chain of events might contribute to early steps in HBV-associated liver carcinogenesis.

Calcium Signals Prime Macrophages

Interferon-α (IFN-α) stimulates macrophages to produce an inflammatory response. Wang et al . show that the activation of signal transducer and activator of transcription 1 (STAT1), but not STAT2, downstream of the IFN-α receptor (IFNAR) is regulated by crosstalk from signals generated by immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as integrins (mediating cell adhesion) and CCR5 (a G protein-coupled receptor for the chemokines CCL3, CCL4, and CCL5). STAT activation requires tyrosine phosphorylation, which then allows STAT proteins to dimerize and translocate to the nucleus to regulate gene expression. Wang et al . found basal phosphorylation of STAT1 on Ser 727 in unstimulated primary human macrophages. Ser 727 phosphorylation, as well as tyrosine phosphorylation (Tyr 701 ) stimulated by IFN-α, was inhibited by the calcium/calmodulin kinase (CaMK) inhibitor KN-93, the calmodulin inhibitor W-7, or the calcium chelator BAPTA. Thus, a basal calcium signal appears to be involved in this process of sensitizing cells to respond to IFN-α stimulation with STAT1 tyrosine phosphorylation. Although CaMK activity was likely to regulate STAT1 Ser 727 phosphorylation, it was not directly responsible for the tyrosine phosphorylation. Instead the calcium-dependent tyrosine kinase Pyk2 was found to interact with the tyrosine kinases associated with the IFNAR, Jak1, and Tyk2, and a role for Pyk2 in enhancing IFN-α-stimulated STAT1 tyrosine phosphorylation was confirmed by experiments with pharmacological inhibitors, cells expressing dominant-negative mutants, cells in which Pyk2 was depleted, and Pyk2-knockout cells. STAT2 activation was not affected by the treatments that altered STAT1 activation, suggesting that these two STATs may have different sensitivities to IFN-α. Dose-response experiments with a Jak1 inhibitor indicated that phosphorylation of STAT2 required low Jak1 activity, whereas STAT1 tyrosine phosphorylation required higher Jak1 activity. Thus, the basal calcium signal may help boost the STAT1 activation in response to low IFN-α signaling. The IFNAR does not produce a calcium signal; however, receptors that couple to the ITAM adaptor DAP12, which is basally active in macrophages, do stimulate calcium signals. At least two receptors that couple to DAP12 appear to contribute to the basal calcium signal and priming of the STAT1 response: integrins and CCR5. In experiments with mouse bone marrow macrophages, which are more adherent than the primary human macrophages from blood, detachment of the cells from the substrate decreased Pyk2 phosphorylation and decreased STAT1 activation in response to IFN-α. Primary human macrophages were found to produce ligand for the CCR5 receptor, and blocking CCR5 activity with a function-blocking antibody decreased CaMK activation and decreased STAT1 activation in response to IFN-α. Finally, in a mouse model of systemic lupus erythematosus, STAT1 was basally phosphorylated at Ser 727 in cells from the kidney (an affected organ in the mouse model). When the mice were treated with IFN-α, the kidney cells exhibited increased STAT1 activation compared with that in cells from control mice. The enhanced STAT1 activation and the basal Ser 727 phosphorylation were reduced in mice treated with the CaMK inhibitor KN-93. Thus, crosstalk through receptors coupled to DAP12 contributes a signal that tunes the IFN-α response and may be amenable to therapeutic intervention for treatment of autoimmune disease. L. Wang, I. Tassiulas, K.-H. Park-Min, A. C. Reid, H. Gil-Henn, J. Schlessinger, R. Baron, J. J. Zhang, L. B. Ivashkiv, 'Tuning' of type I interferon-induced Jak-STAT1 signaling by calcium-dependent kinases in macrophages. Nat. Immunol. 9 , 186-193 (2008). [PubMed]

Abstract 1446: Central Role of Calcium-Dependent Tyrosine Kinase PYK2 in eNOS-Mediated Vascular Function and Angiogenic Response -Unmasked by PYK2 Knockout Mice-

Background: Endothelial nitric oxide synthase (eNOS) is activated by PI3-kinase/Akt or Ca 2+ -dependent CaM kinases, whereas the involvement of Ca 2+ -dependent tyrosine kinase PYK2 remains to be determined. Methods and Results: We newly generated the PYK2-deficient (−/−) mice and investigated the effects of PYK2 on NO-mediated vascular functions and angiogenic response. The PYK2−/− mice exhibited a significant reduction in the basal eNOS phosphorylation and VEGF- or Acetylcholin-mediated response in the aorta and aortic endothelial cells (ECs) compared with the wild-type mice, and blood flow recovery and neovessel formation after hindlimb ischemia were reduced with affecting mobilization of endothelial progenitors. Acetylcholin-mediated aortic vasorelaxation and cGMP production were decreased (54% and 44%, P<0.01, respectively) and blood pressure in the PYK2−/− mice is higher than the wild-type mice (mean 97 vs. 86 mmHg, P<0.05). In the PYK2−/− mice, VEGF-mediated Akt phosphorylation, NO production and cytoplasmic Ca 2+ mobilization were markedly decreased. And also the phosphorylation of c-Src and PLCγ1, interaction of c-Src/PLCγ1 were clearly reduced. VEGF-dependent migration, tube formation and actin cytoskeletal reorganization were markedly inhibited in PYK2−/− ECs, while addition of L-arginine or cGMP-dependent kinase activator reversed the attenuated response. Conclusion: These findings demonstrate that PYK2 is involved in the receptor-activated signaling events leading to the phosphorylation of eNOS and thus regulates eNOS-mediated vasoactive function and angiogenesis.

Central Role of Calcium-Dependent Tyrosine Kinase PYK2 in Endothelial Nitric Oxide Synthase–Mediated Angiogenic Response and Vascular Function

The involvement of Ca2+-dependent tyrosine kinase PYK2 in the Akt/endothelial NO synthase pathway remains to be determined. Blood flow recovery and neovessel formation after hind-limb ischemia were impaired in PYK2-/- mice with reduced mobilization of endothelial progenitors. Vascular endothelial growth factor (VEGF)-mediated cytoplasmic Ca2+ mobilization and Ca2+-independent Akt activation were markedly decreased in the PYK2-deficient aortic endothelial cells, whereas the Ca2+-independent AMP-activated protein kinase/protein kinase-A pathway that phosphorylates endothelial NO synthase was not impaired. Acetylcholine-mediated aortic vasorelaxation and cGMP production were significantly decreased. Vascular endothelial growth factor-dependent migration, tube formation, and actin cytoskeletal reorganization associated with Rac1 activation were inhibited in PYK2-deficient endothelial cells. PI3-kinase is associated with vascular endothelial growth factor-induced PYK2/Src complex, and inhibition of Src blocked Akt activation. The vascular endothelial growth factor-mediated Src association with PLCgamma1 and phosphorylation of 783Tyr-PLCgamma1 also were abolished by PYK2 deficiency. These findings demonstrate that PYK2 is closely involved in receptor- or ischemia-activated signaling events via Src/PLCgamma1 and Src/PI3-kinase/Akt pathways, leading to endothelial NO synthase phosphorylation, and thus modulates endothelial NO synthase-mediated vasoactive function and angiogenic response.

7‐Ketocholesterol‐induced apoptosis

Oxysterols, and particularly 7-ketocholesterol, appear to be strongly involved in the physiopathology of atherosclerosis. These molecules are suspected to be cytotoxic to the cells of the vascular wall and monocytes/macrophages, particularly by inducing apoptosis. Previous studies have demonstrated that 7-ketocholesterol-induced apoptosis is triggered by a sustained increase of cytosolic-free Ca2+, which elicits the mitochondrial pathway of apoptosis by activation of the calcium-dependent phosphatase calcineurin, leading to dephosphorylation of the 'BH3 only' protein BAD. However, thorough study of the results suggests that other pathways are implicated in 7-ketocholesterol-induced cytotoxicity. In this study, we demonstrate the involvement of two other calcium-dependent pathways during 7-ketocholesterol-induced apoptosis. The activation of the MEK-->ERK pathway by the calcium-dependent tyrosine kinase PYK 2, a survival pathway which delays apoptosis as shown by the use of the MEK inhibitor U0126, and a pathway involving another pro-apoptotic BH3 only protein, Bim. Indeed, 7-ketocholesterol treatment of human monocytic THP-1 cells induces the release of Bim-LC8 from the microtubule-associated dynein motor complex, and its association with Bcl-2. Therefore, it appears that 7-ketocholesterol-induced apoptosis is a complex phenomenon resulting from calcium-dependent activation of several pro-apoptotic pathways and also one survival pathway.

Endothelin signalling and regulation of protein kinases in glomerular mesangial cells.

The molecular mechanisms of endothelin (ET)-dependent activation of extracellular signal-regulated kinase (ERK)and p38 mitogen-activated protein (MAP) kinase were studied in rat and human renal glomerular mesangial cells. ET-1 induced a rapid and transient activation of Ras in renal mesangial cells, which was dependent upon the formation of the Shc/Grb2/Sos1 signalling complex and resulted in transient ERK activation. We have observed that Pyk2, a calcium-dependent cytoplasmic tyrosine kinase, was expressed in human renal mesangial cells and was tyrosine phosphorylated after ET-1 treatment. ET-1-induced activation of p38 MAPK pathway (but not ERK pathway) was inhibited in human and in rat glomerular mesangial cells expressing dominant-negative form of Pyk2, suggesting the engagement of Pyk2 in ET-1-mediated activation of p38 MAP kinase cascade. Contractive responsiveness of renal mesangial cells was shown to depend on activation of the p38 MAP kinases. Thus, p38 MAP kinase stimulation could perhaps partially account for ET-1 contractive properties, whereas ET-1-induced cell proliferation occurs primarily via Ras-dependent activation of the ERK.

Glycogen Synthase Kinase 3β Is Tyrosine Phosphorylated by PYK2

Glycogen synthase kinase 3β (GSK3β) is a Ser/Thr kinase that is involved in numerous cellular activities. GSK3β is activated by tyrosine phosphorylation. However, very little is known about the tyrosine kinases that are responsible for phosphorylating GSK3β. In this report, we investigated the ability of the calcium-dependent tyrosine kinase, proline-rich tyrosine kinase 2 (PYK2) to tyrosine phosphorylate GSK3β. In transfected CHO cells, it was demonstrated that PYK2 tyrosine phosphorylates GSK3β in situ. The two kinases also coimmunoprecipitated. Furthermore, GSK3β was tyrosine phosphorylated in vitro by an active, wild type PYK2, but not by the inactive, kinase dead form of PYK2. Therefore, this study is the first to demonstrate that GSK3β is a substrate of PYK2 both in vitro and in situ.

Inhibition of the Calcium-dependent Tyrosine Kinase (CADTK) Blocks Monocyte Spreading and Motility

Freshly isolated peripheral blood monocytes lack focal adhesion kinase (p125(FAK)) but activate a second member of this kinase family, calcium-dependent tyrosine kinase (CADTK; also known as Pyk2/CAKbeta/RAFTK/FAK2), upon adhesion or stimulation with chemokines. To study the role of CADTK in monocyte adherence and motility, we performed immunocytochemical localization that showed CADTK at the leading edge and ruffling lamellipodial structures in freshly isolated, adhered human monocytes. We next introduced CADTK/CAKbeta-related non-kinase (CRNK), the C-terminal noncatalytic domain of CADTK, into monocytes by electroporation and showed that it inhibited CADTK autophosphorylation. Introduction of the fusion protein glutathione S-transferase (GST)-CRNK also reduced (i) cell spreading, as reflected in a reduced cell area 30 min after adhesion, (ii) adhesion-induced phosphotyrosine increases and redistribution into lamellipodia, and (iii) adhesion-induced extracellular signal-regulated protein kinase (ERK) activation. In control experiments, introduction of GST or GST-C3 transferase (an inhibitor of RhoA GTPase activity) by electroporation did not affect these parameters. Monocytes adhered in the presence of autologous serum were highly motile even after introduction of GST (83% motile cells). However, only 26% of monocytes with introduced GST-CRNK were motile. In contrast, GST-CRNK-treated monocytes were fully capable of phagocytosis and adhesion-induced cytokine gene induction, suggesting that CADTK is not involved in these cellular activities and that GST-CRNK introduction does not inhibit global monocyte functions. These results suggest that CADTK is crucial for the in vitro monocyte cytoskeletal reorganization necessary for cell motility and is likely to be required in vivo for recruitment to sites of inflammation.

Outside-in Signaling Pathway Linked to CD146 Engagement in Human Endothelial Cells

CD146 (S-Endo 1 Ag or MUC18) is a transmembrane glycoprotein expressed on endothelial cells on the whole vascular tree. CD146 is located at the intercellular junction where it plays a role in the cohesion of the endothelial monolayer. CD146 engagement initiates an outside-in signaling pathway involving the protein tyrosine kinases FYN and FAK as well as paxillin. Here we report that CD146 engagement by its specific monoclonal antibody in human umbilical vein endothelial cells induces a Ca(2+) influx that is sensitive to thapsigargin and EGTA treatment, indicating that CD146 engagement initiates a store-operated calcium mobilization. In addition, biochemical and pharmacological analysis revealed that CD146 engagement initiates the tyrosine phosphorylation of phospholipase C-gamma, Pyk2, and p130(Cas). Pharmacological inhibition of Ca(2+) flux with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethyl ester and EGTA indicated that an increase in Ca(2+) is required for Pyk2 and p130(Cas) tyrosine phosphorylation. Moreover, a complex association was observed between Pyk2, p130(Cas), and paxillin. These results indicate that CD146 is coupled to a FYN-dependent pathway that triggers Ca(2+) flux via phospholipase C-gamma activation leading subsequently to the tyrosine phosphorylation of downstream targets such as Pyk2, p130(Cas), FAK, and paxillin. In addition to its role in cell-cell adhesion, CD146 is a signaling molecule involved in the dynamics of actin cytoskeleton rearrangement.

Angiotensin II Induces Human Monocyte Migration and Pyk2/ Paxillin Phosphorylation

12 Angiotensin II (AII) has been shown to enhance the development of atherosclerotic lesions. Transendothelial migration of monocytes is an early critical step in the atherosclerotic process. To elucidate mechanisms by which AII promotes atherogenesis, we investigated its effects on human monocyte migration. AII induced migration of human peripheral blood monocytes (HPBM) and human THP-1 monocytes at concentrations between 0.01-1μmol/L, with 3.3-fold induction in HPBM and 4.8-fold in THP-1cells at 1μmol/L AII (both p< 0.01 vs. unstimulated cells). To identify cell signalling pathways involved in AII-directed migration, we blocked extracellular regulated protein kinase (ERK1/2) - MAPK activation with PD98059 (PD) and p38-MAPK activity with SB203580 (SB). Pretreatment of HPBM with PD (30μmol/L) and SB (10μmol/L) blocked AII (1μmol/L) stimulated migration by 75.6 +/- 3.1% (p< 0.01) and 52.8 +/- 2.6% (p< 0.01), respectively. AII (1μmol/L) directed migration of THP-1 monocytes was inhibited by 67.7 +/- 15.9% (PD 30μmol/L, p< 0.01) and 47.2 +/- 12.3% (SB 10μmol/L, p<0.05). Addition of the AII receptor type 1 (AT1R) antagonist Losartan (LOS) completely supressed AII induced monocyte migration, demonstrating an AT1R mediated mechanism (HPBM: LOS 100μmol/L 83.1 +/- 6.5% inhibition (p< 0.05)/ THP-1: LOS 100μmol/L 91.3+/- 21.5% inhibition (p<0.01)). Cell movement requires the phosphorylation of cytoskeleton-associated tyrosine kinases and formation of focal adhesion complexes. Western immunblotting with phosphospecific antibodies showed that AII (1μmol/L) induced the rapid tyrosine phosphorylation of PYK2 , a calcium-dependent tyrosine kinase, in THP-1 cells by 3.6-fold peaking after 10min. Phosphorylation of the focal adhesion related protein paxillin was enhanced in THP-1 cells by AII (1μmol/L) with a 4.3-fold induction after 2 min. This study demonstrates a potential proatherogenic mechanism of AII by stimulating human monocyte migration through an AT1R-ERK1/2-p38-dependent process, which might be, in part, mediated by AII induced PYK2/Paxillin phosphorylation.

β2 Integrin-dependent tyrosine phosphorylation of proline-rich tyrosine kinase 2 in platelet-activating factor-activated eosinophils

Platelet-activating factor (PAF) can activate various cellular functions, including degranulation in eosinophils. In the present study, we examined tyrosine phosphorylation of intracellular proteins induced by PAF in eosinophils derived from cord blood mononuclear cells. Platelet-activating factor induced tyrosine phosphorylation of many intracellular proteins, including 42, 123 and 150 kDa proteins. Immunoprecipitation studies showed that the 123 kDa phosphorylated protein was proline-rich tyrosine kinase 2 (PYK-2; also known as related adhesion focal tyrosine kinase (RAFTK), cell adhesion kinase (β (CAK(β) and calcium-dependent tyrosine kinase (CADTK)). Furthermore, blocking of cellular adhesion through β2 integrin by anti-CD18 monoclonal antibody inhibited tyrosine phosphorylation of PYK-2 as well as the degranulation response. These findings suggest that tyrosine phosphorylation of PYK-2 is involved in a signaling pathway mediated by cellular adhesion through β2 integrin in PAF-activated eosinophils.

Annexin VII and Annexin XI Are Tyrosine Phosphorylated in Peroxovanadate-treated Dogs and in Platelet-derived Growth Factor-treated Rat Vascular Smooth Muscle Cells

The intraperitoneal administration of peroxovanadate results in the rapid accumulation of many tyrosine-phosphorylated proteins in the liver and kidney of treated animals. The availability of large pools of tyrosine-phosphorylated proteins derived from normal tissues facilitates the purification and identification of previously unknown targets for cellular tyrosine kinases. Using this procedure, we have thus far identified four proteins in the liver and kidney of peroxovanadate-treated dogs. Two of these, annexin VII and annexin XI, were novel and had not been previously reported to be substrates of tyrosine kinases while the remaining two, ezrin and clathrin, have been reported to be tyrosine phosphorylated in some cell culture systems. In the present study, isolated proteins were identified both by sequence analysis and immunological methods. Annexin VII and annexin XI are present in cultured rat vascular smooth muscle cells and both were tyrosine phosphorylated in response to a physiological ligand, platelet-derived growth factor-BB (PDGF-BB). Furthermore, the extent of tyrosine phosphorylation in response to PDGF-BB was augmented by the co-addition of peroxovanadate to cell cultures. In vitro phosphorylation assays showed that PDGF receptor, calcium-dependent tyrosine kinase (CADTK/Pyk-2), Src kinase, and epidermal growth factor receptor all were able to phosphorylate purified annexin VII and XI on tyrosine residues. These findings confirm the usefulness of phosphatase inhibition by peroxovanadate as a tool for identifying previously unknown physiological targets for cellular protein tyrosine kinases.

Interactions between Two Cytoskeleton-associated Tyrosine Kinases: Calcium-dependent Tyrosine Kinase and Focal Adhesion Tyrosine Kinase

The calcium-dependent tyrosine kinase (CADTK), also known as Pyk2/RAFTK/CAKbeta/FAK2, is a cytoskeleton-associated tyrosine kinase. We compared CADTK regulation with that of the highly homologous focal adhesion tyrosine kinase (FAK). First, we generated site-specific CADTK mutants. Mutation of Tyr402 eliminated autophosphorylation and significantly decreased kinase activity. Mutation of Tyr881, a putative Src kinase phosphorylation site predicted to bind Grb2, had little effect on CADTK regulation. Src family tyrosine kinases resulted in CADTK tyrosine phosphorylation even when co-expressed with the Tyr402/Tyr881 double mutant, suggesting that Src/Fyn etc. phosphorylate additional tyrosine residues. Interestingly, CADTK tyrosine-phosphorylated FAK when both were transiently expressed, but FAK did not phosphorylate CADTK. Biochemical experiments confirmed direct CADTK phosphorylation of FAK. This phosphorylation utilized tyrosine residues other than Tyr397, Tyr925, or Tyr576/Tyr577, suggesting that new SH2-binding sites might be created by CADTK-dependent FAK phosphorylation. Last, expression of the CADTK carboxyl terminus (CRNK) abolished CADTK but not FAK autophosphorylation. In contrast, FAK carboxyl terminus overexpression inhibited both FAK and CADTK autophosphorylation, suggesting that a FAK-dependent cytoskeletal function may be necessary for CADTK activation. Thus, CADTK and FAK, which both bind to some, but not necessarily the same, cytoskeletal elements, may be involved in coordinate regulation of cytoskeletal structure and signaling.

Endothelin-1 Stimulates Hydrolysis of Phosphatidylcholine by Phospholipases C and D in Intact Rat Mesenteric Arteries

A characteristic of endothelin-1 (ET-1)-induced contraction is the prolonged duration of the response. Phosphatidylcholine (PC) hydrolysis has been implicated in sustained agonist-induced effects through the formation of the second messengers phosphatidic acid (PA) and diacylglycerol (DAG). Therefore, we have investigated the activation of PC-phospholipase D (PLD) and PC-phospholipase C (PLC) in intact rat mesenteric small arteries stimulated with ET-1 and determined whether PC-derived DAG is necessary for ET-1-induced contraction. PLD activity, measured as phosphatidylethanol (PEt) production in vessels labelled with [³H] or [¹⁴C]myristate, was increased in a concentration- and time-dependent manner following ET-1 stimulation, as were [³H]PA levels, peaking at 10 min (ET-1 100 nM) and remaining above control levels for up to 20 min. Inclusion of 0.5% ethanol during ET-1 stimulation reduced [³H]PA levels, but did not alter the time course of formation. In addition, [¹⁴C]choline release was increased, confirming PLD-mediated PC hydrolysis. In contrast, DAG levels, measured by [³H]myristate labelling and mass assay, increased transiently at 5 min of ET-1 stimulation only. Analysis of the subclasses of DAG demonstrated an increase in all types of DAG without any enrichment with arachidonate-containing species, indicating PC, not inositol lipid hydrolysis was the source of DAG. The PC-PLC inhibitor D609, 2.5 µg/ml, completely abolished the ET-1-induced increase in [¹⁴C]DAG without affecting the increase in [¹⁴C]PA, PLD activity or the contractile response. In [¹⁴C]choline-labelled vessels, [¹⁴C]phosphocholine levels were increased by ET-1 with a similar time course to DAG production. Removal of extracellular Ca²⁺ and addition of 0.1 or 2 mM EGTA completely inhibited ET-1-stimulated PLD activity. The tyrosine kinase inhibitor tyrphostin A23 (100 µM) abolished ET-1-induced [³H]PA, [³H]PEt and [¹⁴C]DAG increases, whilst the negative analogue A1 was without effect. These data suggest that ET-1 couples via a calcium-dependent tyrosine kinase mechanism to PLD and PC-PLC in vascular smooth muscle. PC-derived DAG did not appear to be necessary for the contractile response, whereas sustained formation of PA, generated by PLD activity, implies that this lipid second messenger could be involved in the prolonged contractile response to this peptide.

Interactions between Two Cytoskeleton-associated Tyrosine Kinases: Calcium-dependent Tyrosine Kinase and Focal Adhesion Tyrosine Kinase

Interactions between Two Cytoskeleton-associated Tyrosine Kinases: Calcium-dependent Tyrosine Kinase and Focal Adhesion Tyrosine Kinase

Angiotensin II stimulates ERK via two pathways in epithelial cells: protein kinase C suppresses a G-protein coupled receptor-EGF receptor transactivation pathway.

In GN4 rat liver epithelial cells, angiotensin II (Ang II) produces intracellular calcium and protein kinase C (PKC) signals and stimulates ERK and JNK activity. JNK activation appears to be mediated by a calcium-dependent tyrosine kinase (CADTK). To define the ERK pathway, we established GN4 cells expressing an inhibitory Ras(N17). Induction of Ras(N17) blocked EGF- but not Ang II- or phorbol ester (TPA)-dependent ERK activation. In control cells, Ang II and TPA produced minimal increases in Ras-GTP level and Raf kinase activity. PKC depletion by chronic TPA exposure abolished TPA-dependent ERK activation but failed to diminish the effect of Ang II. In PKC-depleted cells, Ang II increased Ras-GTP level and activated Raf and ERK in a Ras-dependent manner. In PKC depleted cells, Ang II stimulated Shc and Cbl tyrosine phosphorylation, suggesting that without PKC, Ang II activates another tyrosine kinase. PKC-depletion did not alter Ang II-dependent tyrosine phosphorylation or activity of p125(FAK), CADTK, Fyn or Src, but PKC depletion or incubation with GF109203X resulted in Ang II-dependent EGF receptor tyrosine phosphorylation. In PKC-depleted cells, EGF receptor-specific tyrosine kinase inhibitors blocked Ang II-dependent EGF receptor and Cbl tyrosine phosphorylation, and ERK activation. In summary, Ang II can activate ERK via two pathways; the latent EGF receptor, Ras-dependent pathway is equipotent to the Ras-independent pathway, but is masked by PKC action. The prominence of this G-protein coupled receptor to EGF receptor pathway may vary between cell types depending upon modifiers such as PKC.

A Calcium-dependent Tyrosine Kinase Splice Variant in Human Monocytes: ACTIVATION BY A TWO-STAGE PROCESS INVOLVING ADHERENCE AND A SUBSEQUENT INTRACELLULAR SIGNAL

Freshly isolated human monocytes do not express p125(FAK) but upon adherence to substrata activate the highly related calcium-dependent tyrosine kinase (CADTK), also known as Pyk2, CAKbeta, RAFTK, and FAK2. The monocyte CADTK was 5 kDa smaller than protein from epithelial cells; isolation and sequencing of the monocyte CADTK cDNA revealed a predicted 42-amino acid deletion between the two proline-rich domains of the enzyme. The nucleic acid sequence suggests that the deletion is caused by alternative RNA splicing. This species was also found in T and B lymphocytes and appears to be the predominant form of cytoskeletal associated tyrosine kinase in non-neoplastic, circulating, hematopoietic cells. CADTK was not activated when monocytes maintained in suspension were treated with agents that produce an intracellular calcium (thapsigargin) or protein kinase C (phorbol 12-myristate 13-acetate) signal including a chemokine, RANTES, that binds to the HIV co-receptor, CCK5. In contrast, monocyte adherence to tissue culture plastic-stimulated CADTK tyrosine phosphorylation, a process that was enhanced by thapsigargin, phorbol 12-myristate 13-acetate, and RANTES but that was completely blocked by preincubation with cytochalasin D. When compared with plastic, adherence to fibronectin- or collagen-coated surfaces produced only minimal CADTK activation but permitted significant stimulation by added thapsigargin. These data suggest that in a cell type that lacks p125(FAK), CADTK plays an early role in post-adherence signaling. Its activation involves two stages, cytoskeletal engagement, which is permissive, and co-stimulatory signals (calcium or protein kinase C) generated by extensive cell surface engagement, agonists, or inflammatory chemokines.

Regulation of a Calcium-dependent Tyrosine Kinase in Vascular Smooth Muscle Cells by Angiotensin II and Platelet-derived Growth Factor: DEPENDENCE ON CALCIUM AND THE ACTIN CYTOSKELETON

Regulation of a Calcium-dependent Tyrosine Kinase in Vascular Smooth Muscle Cells by Angiotensin II and Platelet-derived Growth Factor: DEPENDENCE ON CALCIUM AND THE ACTIN CYTOSKELETON

Regulation of a Calcium-dependent Tyrosine Kinase in Vascular Smooth Muscle Cells by Angiotensin II and Platelet-derived Growth Factor: DEPENDENCE ON CALCIUM AND THE ACTIN CYTOSKELETON

A novel, p125FAK homologue, CADTK, has been detected in neural, epithelial, or hematopoietic cells but not in fibroblasts. We now demonstrate CADTK expression in a mesenchymal cell, rat aortic smooth muscle cells (RSMC). Angiotensin II (Ang II) or platelet-derived growth factor (PDGF-BB and PDGF-AA) markedly stimulated CADTK tyrosine phosphorylation in RSMC but did not affect p125FAK phosphorylation. The PDGF-depedent CADTK tyrosine phosphorylation was slower and more prolonged than that of Ang II, correlating well with the differential effects of these agonists on cytosolic calcium ([Ca2+]i) signaling. An intracellular calcium chelator inhibited both the rapid and sustained activation of CADTK by Ang II and PDGF. Extracellular calcium chelation inhibited the PDGF-stimulated increase in CADTK tyrosine phosphorylation as well as the sustained (but not the early) activation by Ang II. In contrast, p125FAK tyrosine phosphorylation was maximal in quiescent, adherent RSMC and was not affected by incubation with EGTA. Depletion of protein kinase C activity partially inhibited both the Ang II- and PDGF-induced CADTK tyrosine phosphorylation. Additional results confirm a relation between CADTK and the cytoskeleton. First, the tyrosine phosphorylation of paxilin correlated with activation of CADTK; this increase was inhibited by EGTA. Second, cytochalasin D blocked the PDGF- or Ang II-stimulated tyrosine phosphorylation of CADTK, suggesting a role for the cytoskeleton in agonist-dependent CADTK activation. Third, immunofluorescence analysis of CADTK localization demonstrated actin-like cytoskeleton staining extending into focal contacts. These results suggest that in mesenchymal cells, CADTK is localized to and activated by an actin cytoskeleton-dependent mechanism; a mechanism that is regulated in a calcium and protein kinase C-dependent manner independently of p125FAK.