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1.
Proline-rich tyrosine kinase 2 (PYK2), structurally related to focal adhesion kinase, has been shown to play a role in signaling cascades. Endothelial cells (ECs) under hemodynamic forces increase reactive oxygen species (ROS) that modulate signaling pathways and gene expression. In the present study, we found that bovine ECs subjected to cyclic strain rapidly induced phosphorylation of PYK2 and Src kinase. This strain-induced PYK2 and Src phosphorylation was inhibited by pretreating ECs with an antioxidant N-acetylcysteine. Similarly, ECs exposed to H(2)O(2) increased both PYK2 and Src phosphorylation. An increased association of Src to PYK2 was observed in ECs after cyclic strain or H(2)O(2) exposure. ECs treated with an inhibitor to Src (PPI) greatly reduced Src and PYK2 phosphorylation, indicating that Src mediated PYK2 activation. Whereas the protein kinase C (PKC) inhibitor (calphostin C) pretreatment was shown to inhibit strain-induced NADPH oxidase activity, ECs treated with either calphostin C or the inhibitor to NADPH oxidase (DPI) reduced strain-induced ROS levels and then greatly inhibited the Src and PYK2 activation. In contrast to the activation of PYK2 and Src with calcium ionophore (ionomycin), ECs treated with a Ca(2+) chelator inhibited both phosphorylation, indicating that PYK2 and Src activation requires Ca(2+). ECs transfected with antisense to PKCalpha, but not antisense to PKCepsilon(,) reduced cyclic strain-induced PYK2 activation. These data suggest that cyclic strain-induced PYK2 activity is mediated via Ca(2+)-dependent PKCalpha that increases NADPH oxidase activity to produce ROS crucial for Src and PYK2 activation. ECs under cyclic strain thus activate redox-sensitive PYK2 via Src and PKC, and this PYK2 activation may play a key role in the signaling responses in ECs under hemodynamic influence. 相似文献
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Walker DP Bi FC Kalgutkar AS Bauman JN Zhao SX Soglia JR Aspnes GE Kung DW Klug-McLeod J Zawistoski MP McGlynn MA Oliver R Dunn M Li JC Richter DT Cooper BA Kath JC Hulford CA Autry CL Luzzio MJ Ung EJ Roberts WG Bonnette PC Buckbinder L Mistry A Griffor MC Han S Guzman-Perez A 《Bioorganic & medicinal chemistry letters》2008,18(23):6071-6077
The synthesis and SAR for a series of diaminopyrimidines as PYK2 inhibitors are described. Using a combination of library and traditional medicinal chemistry techniques, a FAK-selective chemical series was transformed into compounds possessing good PYK2 potency and 10- to 20-fold selectivity against FAK. Subsequent studies found that the majority of the compounds were positive in a reactive metabolite assay, an indicator for potential toxicological liabilities. Based on the proposed mechanism for bioactivation, as well as a combination of structure-based drug design and traditional medicinal chemistry techniques, a follow-up series of PYK2 inhibitors was identified that maintained PYK2 potency, FAK selectivity and HLM stability, yet were negative in the RM assay. 相似文献
3.
Depolarization activates ERK and proline-rich tyrosine kinase 2 (PYK2) independently in different cellular compartments in hippocampal slices 总被引:1,自引:0,他引:1
Corvol JC Valjent E Toutant M Enslen H Irinopoulou T Lev S Hervé D Girault JA 《The Journal of biological chemistry》2005,280(1):660-668
In the hippocampus, extracellular signal-regulated kinase (ERK) and the non-receptor protein proline-rich tyrosine kinase 2 (PYK2) are activated by depolarization and involved in synaptic plasticity. Both are also activated under pathological conditions following ischemia, convulsions, or electroconvulsive shock. Although in non-neuronal cells PYK2 activates ERK through the recruitment of Src-family kinases (SFKs), the link between these pathways in the hippocampus is not known. We addressed this question using K(+)-depolarized rat hippocampal slices. Depolarization increased the phosphorylation of PYK2, SFKs, and ERK. These effects resulted from Ca(2+) influx through voltage-gated Ca(2+) channels and were diminished by GF109203X, a protein kinase C inhibitor. Inhibition of SFKs with PP2 decreased PYK2 tyrosine phosphorylation dramatically, but not its autophosphorylation on Tyr-402. Moreover, PYK2 autophosphorylation and total tyrosine phosphorylation were profoundly altered in fyn-/- mice, revealing an important functional relationship between Fyn and PYK2 in the hippocampus. In contrast, ERK activation was unaltered by PP2, Fyn knock-out, or LY294002, a phosphatidyl-inositol-3-kinase inhibitor. ERK activation was prevented by MEK inhibitors that had no effect on PYK2. Immunofluorescence of hippocampal slices showed that PYK2 and ERK were activated in distinct cellular compartments in somatodendritic regions and nerve terminals, respectively, with virtually no overlap. Activation of ERK was critical for the rephosphorylation of a synaptic vesicle protein, synapsin I, following depolarization, underlining its functional importance in nerve terminals. Thus, in hippocampal slices, in contrast to cell lines, depolarization-induced activation of non-receptor tyrosine kinases and ERK occurs independently in distinct cellular compartments in which they appear to have different functional roles. 相似文献
4.
John G. Allen Matthew R. Lee Chun-Ya E. Han Jon Scherrer Shaun Flynn Christy Boucher Huilin Zhao Anne B. O’Connor Philip Roveto David Bauer Russell Graceffa William G. Richards Philip Babij 《Bioorganic & medicinal chemistry letters》2009,19(17):4924-4928
A screening campaign of a diverse collection of ~250,000 small molecule compounds was performed to identify inhibitors of proline-rich tyrosine kinase 2 (Pyk2) with potential osteogenic activity in osteoblast cells. Compounds were prioritized based on selectivity following a counter-screen against focal adhesion kinase (FAK), a closely related kinase. 4-Amino and 5-aryl substituted pyridinone series were identified that showed strong biochemical potency against Pyk2 and up to 3700-fold selectivity over FAK. Modeling analysis suggested that structural differences in the substrate binding cleft could explain the high selectivity of these chemical series against FAK. Representative compounds from each series showed inhibition of Pyk2 autophosphorylation in 293T cells (IC50 ~0.11 μM), complete inhibition of endogenous Pyk2 in A7r5 cells and increased levels of osteogenic markers in MC3T3 osteoblast cells (EC50’s ~0.01 μM). These results revealed a new class of compounds with osteogenic-inducing activity in osteoblast cells and a starting point for the development of more potent and selective Pyk2 inhibitors. 相似文献
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Chieffi P Barchi M Di Agostino S Rossi P Tramontano D Geremia R 《Molecular reproduction and development》2003,65(3):330-335
Prolin-rich kinase 2 (PYK2) is a nonreceptor tyrosine kinase related to the focal adhesion kinase (FAK) p125(FAK). PYK2 is rapidly phosphorylated on tyrosine residues in response to various stimuli, such as tumor necrosis factor-alpha (TNF-alpha), changes in osmolarity, elevation in intracellular calcium concentration, angiotensin, and UV irradiation. PYK2 has ligand sequences for Src homology 2 and 3 (SH-2 and SH-3), and has binding sites for paxillin and p130(cas). Activation of PYK2 leads to modulation of ion channel function, phosphorylation of tyrosine residues, and activation of the MAP kinase signaling pathways. Immunocytochemistry shows that PYK2 is present in mouse germinal and Sertoli cells (ser). Northern blot and immunoprecipitation analysis demonstrate that, among germinal cells, PYK2 is more abundant in spermatocytes (spc) and spermatids (spt); in addition, immunofluorescence analysis clearly shows that the diffuse cytoplasmic localization of PYK2 changes in a specific cellular compartment in spt and spermatozoa. 相似文献
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Fibronectin fragments (FN-f), including the 110-kDa fragment that binds the alpha5beta1 integrin, stimulate collagenase-3 (MMP-13) production and cartilage destruction. In the present study, treatment of chondrocytes with the 110-kDa FN-f or an activating antibody to the alpha5beta1 integrin was found to increase tyrosine autophosphorylation (Tyr-402) of the proline-rich tyrosine kinase-2 (PYK2) without significant change in autophosphorylation (Tyr-397) of focal adhesion kinase (FAK). The tyrosine kinase inhibitor tyrphostin A9, shown previously to block a PYK2-dependent pathway, blocked the FN-f-stimulated increase in MMP-13, whereas tyrphostin A25 did not. FN-f-stimulated PYK2 phosphorylation and MMP-13 production was also blocked by reducing intracellular calcium levels. Adenovirally mediated overexpression of wild type but not mutant PYK2 resulted in increased MMP-13 production. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate stimulated PYK2 phosphorylation and MMP-13 production. MMP-13 expression stimulated by either phorbol 12-myristate 13-acetate or FN-f was blocked by PKC inhibitors including the PKCdelta inhibitor rottlerin. Furthermore, PKCdelta translocation from cytosol to membrane was noted within 5 min of stimulation with FN-f. Immortalized human chondrocytes, transiently transfected with MMP-13 promoter-luciferase reporter constructs, showed increased promoter activity after FN-f treatment that was inhibited by co-transfection with either of two dominant negative mutants of PYK2 (Y402F and K457A). No inhibition was seen after cotransfection with wild type PYK2, a dominant negative of FAK (FRNK) or empty vector plasmid. FN-f-stimulated MMP-13 promoter activity was also inhibited by chemical inhibitors of ERK, JNK, and p38 mitogen-activated protein (MAP) kinases or by co-transfection of dominant negative MAP kinase mutant constructs. These studies have identified a novel pathway for the MAP kinase regulation of MMP-13 production which involves FN-f stimulation of the alpha5beta1 integrin and activation of the nonreceptor tyrosine kinase PYK2 by PKC, most likely PKCdelta 相似文献
8.
Hartigan JA Xiong WC Johnson GV 《Biochemical and biophysical research communications》2001,284(2):485-489
Glycogen synthase kinase 3beta (GSK3beta) is a Ser/Thr kinase that is involved in numerous cellular activities. GSK3beta is activated by tyrosine phosphorylation. However, very little is known about the tyrosine kinases that are responsible for phosphorylating GSK3beta. In this report, we investigated the ability of the calcium-dependent tyrosine kinase, proline-rich tyrosine kinase 2 (PYK2) to tyrosine phosphorylate GSK3beta. In transfected CHO cells, it was demonstrated that PYK2 tyrosine phosphorylates GSK3beta in situ. The two kinases also coimmunoprecipitated. Furthermore, GSK3beta 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 GSK3beta is a substrate of PYK2 both in vitro and in situ. 相似文献
9.
Abnormal vascular smooth muscle cell (VSMC) growth plays a key role in the pathogenesis of hypertension and atherosclerosis. Angiotensin II (Ang II) elicits a hypertrophic growth response characterized by an increase in protein synthesis in the absence of DNA synthesis and cell proliferation. Intracellular signaling mechanisms linking angiotensin type I receptor activation to protein synthesis in VSMC have not been fully characterized. The present study investigates the role of the nonreceptor proline-rich tyrosine kinase 2 (PYK2) in Ang II-induced VSMC protein synthesis and in the regulation of two signaling pathways that have been implicated in the control of protein synthesis, the extracellular signal-regulated kinase (ERK1/2) and the phosphatidylinositol 3-kinase/Akt pathways. PYK2 antisense oligonucleotides were used to down-regulate PYK2 expression in cultured VSMC. An 80% down-regulation in PYK2 expression resulted in an approximately 80% inhibition of ERK1/2 (3.8 +/- 1.3 versus 16.6 +/- 1.8), p70S6 kinase (1.03 +/- 0.03 versus 3.8 +/- 0.5), and Akt activation (3.0 +/- 0.8 versus 16.0 +/- 1.0) by Ang II. Furthermore, PYK2 down-regulation resulted in a complete inhibition of Ang II-induced VSMC protein synthesis. These data conclusively identify PYK2 as an upstream regulator of both the ERK1/2 and the phosphatidylinositol 3-kinase/Akt pathways that are involved in Ang II-induced VSMC protein synthesis. 相似文献
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Chapman TM Bouloc N Buxton RS Chugh J Lougheed KE Osborne SA Saxty B Smerdon SJ Taylor DL Whalley D 《Bioorganic & medicinal chemistry letters》2012,22(9):3349-3353
A high-throughput screen against PknB, an essential serine-threonine protein kinase present in Mycobacterium tuberculosis (M. tuberculosis), allowed the identification of an aminoquinazoline inhibitor which was used as a starting point for SAR investigations. Although a significant improvement in enzyme affinity was achieved, the aminoquinazolines showed little or no cellular activity against M. tuberculosis. However, switching to an aminopyrimidine core scaffold and the introduction of a basic amine side chain afforded compounds with nanomolar enzyme binding affinity and micromolar minimum inhibitory concentrations against M. tuberculosis. Replacement of the pyrazole head group with pyridine then allowed equipotent compounds with improved selectivity against a human kinase panel to be obtained. 相似文献
12.
Rodríguez-Fernández JL Sánchez-Martín L Rey M Vicente-Manzanares M Narumiya S Teixidó J Sánchez-Madrid F Cabañas C 《The Journal of biological chemistry》2001,276(44):40518-40527
We have examined the role of the small GTPase Rho and its downstream effector, the Rho-associated kinase (ROCK), in the control of the adhesive and signaling function of the lymphocyte function-associated antigen-1 (LFA-1) integrin in human T-lymphocytes. Inhibition of Rho (either by treatment with C3-exoenzyme or transfection with a dominant-negative form of Rho (N19Rho)) or ROCK (by treatment with Y-27632) results in the following: (a) partial disorganization and aggregation of cortical filamentous actin (F-actin); (b) induction of LFA-1-mediated cellular adhesion to the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1) through a mechanism involving clustering of LFA-1 molecules, rather than alterations in the level of expression or in the affinity state of this integrin; and (c) induction of cellular polarization and activation of the tyrosine kinase PYK2. Transfection of T-cells with a constitutively active form of Rho (V14Rho) blocks the clustering of LFA-1 on the membrane and the LFA-1-mediated activation of PYK2. Importantly, the activation of PYK2 caused by inhibition of Rho or ROCK takes place only when the T-cells are plated onto ICAM-1 but not when they are either prevented from interacting with ICAM-1 with anti-LFA-1 blocking antibodies or when they are plated on the nonspecific poly-l-lysine substrate. These results indicate that the small GTPase Rho regulates the tyrosine kinase PYK2 in T-cells through the F-actin-mediated control of the activity of the integrin LFA-1. These findings represent a novel paradigm for the regulation of the activity of a cytoplasmic tyrosine kinase by the small GTPase Rho. 相似文献
13.
Conformational searches on three closely related pp60(c-src) protein tyrosine kinase inhibitors of varying potencies were performed to determine a structural basis for their activity. The first was a linear peptide (PDNEYAFFQf), the second its 10-membered cyclic analogue, and the third a cyclic analogue with a para carboxyphenylalanine in place of one the F residues. A common backbone conformation with an antiparallel beta-sheet-like geometry capped by similar beta-turns was found for all three peptides, which may be a binding conformation and gives a candidate pharmacophore for further testing. The interaction between some polar side chains and between some of the aromatic rings may be important for maintaining the correct conformation. The differences in potencies of these inhibitors may be attributed to certain thermodynamic and chemical reasons. 相似文献
14.
Bandyopadhyay G Sajan MP Kanoh Y Standaert ML Burke TR Quon MJ Reed BC Dikic I Noel LE Newgard CB Farese R 《The Journal of biological chemistry》2000,275(52):40817-40826
Glucose serves as both a nutrient and regulator of physiological and pathological processes. Presently, we found that glucose and certain sugars rapidly activated extracellular signal-regulated kinase (ERK) by a mechanism that was: (a) independent of glucose uptake/metabolism and protein kinase C but nevertheless cytochalasin B-inhibitable; (b) dependent upon proline-rich tyrosine kinase-2 (PYK2), GRB2, SOS, RAS, RAF, and MEK1; and (c) amplified by overexpression of the Glut1, but not Glut2, Glut3, or Glut4, glucose transporter. This amplifying effect was independent of glucose uptake but dependent on residues 463-468, IASGFR, in the Glut1 C terminus. Accordingly, glucose effects on ERK were amplified by expression of Glut4/Glut1 or Glut2/Glut1 chimeras containing IASGFR but not by Glut1/Glut4 or Glut1/Glut2 chimeras lacking these residues. Also, deletion of Glut1 residues 469-492 was without effect, but mutations involving serine 465 or arginine 468 yielded dominant-negative forms that inhibited glucose-dependent ERK activation. Glucose stimulated the phosphorylation of tyrosine residues 402 and 881 in PYK2 and binding of PYK2 to Myc-Glut1. Our findings suggest that: (a) glucose activates the GRB2/SOS/RAS/RAF/MEK1/ERK pathway by a mechanism that requires PYK2 and residues 463-468, IASGFR, in the Glut1 C terminus and (b) Glut1 serves as a sensor, transducer, and amplifier for glucose signaling to PYK2 and ERK. 相似文献
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Young-Won Chin Jae Yang Kong Sun-Young Han 《Bioorganic & medicinal chemistry letters》2013,23(6):1768-1770
The Fms-like tyrosine kinase 3 (FLT3), a receptor tyrosine kinase, is involved in the proliferation, differentiation and apoptosis of hematopoietic cells. FLT3 is highly overexpressed in acute myeloid leukemia (AML) of the majority of patients. Screening for flavonoids including flavones, flavanones, flavonols, and flavanonols disclosed that luteolin was potent FLT3 enzyme inhibitor. Furthermore, luteolin suppressed cell proliferation in MV4;11 cells with constitutively activated FLT3. 相似文献
17.
Farmer LJ Bemis G Britt SD Cochran J Connors M Harrington EM Hoock T Markland W Nanthakumar S Taslimi P Ter Haar E Wang J Zhaveri D Salituro FG 《Bioorganic & medicinal chemistry letters》2008,18(23):6231-6235
A series of SYK inhibitors based on the phenylamino pyrimidine thiazole lead 4 were prepared and evaluated for biological activity. Lead optimization provided compounds with nanomolar K(i)'s against SYK and potent inhibition in mast cell degranulation assays. 相似文献
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《Bioorganic & medicinal chemistry letters》2014,24(17):4080-4083
Erythropoietin-producing hepatocellular receptor tyrosine kinase subtype A2 (EphA2) is an attractive therapeutic target for suppressing tumor progression. In our efforts to discover novel small molecules to inhibit EphA2, a class of compound based on 4-substituted quinazoline containing 7-(morpholin-2-ylmethoxy) group was identified as a novel hit by high throughput screening campaign. Structural modification of parent quinazoline scaffolds by introducing substituents on aniline displayed potent inhibitory activities toward EphA2. 相似文献
20.
Lai JY Cox PJ Patel R Sadiq S Aldous DJ Thurairatnam S Smith K Wheeler D Jagpal S Parveen S Fenton G Harrison TK McCarthy C Bamborough P 《Bioorganic & medicinal chemistry letters》2003,13(18):3111-3114
A series of oxindoles demonstrating inhibition of the phosphorylation of biotinylated substrates of Syk and IgE/Fc epsilon RI triggered basophil cell degranulation has been identified. A study of the SAR around sulfonamide 31 (IC(50)=5 nM, EC(50)=1400 nM) is discussed. The modest cellular activity representative of the sulfonamide series was overcome when the Polar Surface Area was lowered to <110 A(2), leading to the identification of amide 32 (IC(50)=145 nM, EC(50)=100 nM). 相似文献