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• W2012382163 abstract "The signal transduction pathway linking physiological concentrations of [Arg8]vasopressin (AVP) to an increase in frequency of Ca2+ spiking was examined in confluent cultures of A7r5 vascular smooth muscle cells. Immunoprecipitation/Western blot studies revealed a robust increase in tyrosine phosphorylation of the non-receptor tyrosine kinase, PYK2, in A7r5 cells treated with 4β-phorbol 12-myristate 13-acetate or ionomycin. 100 pm AVP also induced PYK2 tyrosine phosphorylation, and this effect was inhibited by protein kinase C inhibitors Ro-31-8220 (1–10 μm) or chelerythrine chloride (1–20 μm). In fura-2-loaded A7r5 cells, the stimulation of Ca2+ spiking by 100 pm AVP or 1 nm 4β-phorbol 12-myristate 13-acetate was completely blocked by PP2 (10 μm, a Src family kinase inhibitor). Salicylate (20 mm, recently identified as a PYK2 inhibitor) and the tyrosine kinase inhibitor, tyrphostin A47 (50 μm), but not its inactive analog, tyrphostin A63, also blocked AVP-stimulated Ca2+ spiking. PYK2 phosphorylation was inhibited by both PP2 and salicylate, whereas tyrphostin A47 failed to inhibit PYK2 tyrosine phosphorylation. ERK1/2 kinases did not appear to be involved because 1) 100 pm AVP did not appreciably increase ERK1/2 phosphorylation and U-0126 (2.5 μm) did not inhibit AVP-stimulated Ca2+ spiking; and 2) epidermal growth factor (10 nm) robustly stimulated ERK1/2 phosphorylation but did not induce Ca2+ spiking. Delayed rectifier K+ channels may mediate the PYK2 activity because Kv1.2 channel protein co-immunoprecipitated with PYK2 and tyrosine phosphorylation of Kv1.2 was stimulated by AVP and inhibited by Ro-31-8220, PP2, and salicylate but not tyrphostin A47. Our findings are consistent with a role for PYK2 and phosphorylation of K+ channels in the stimulation of Ca2+ spiking by physiological concentrations of AVP. The signal transduction pathway linking physiological concentrations of [Arg8]vasopressin (AVP) to an increase in frequency of Ca2+ spiking was examined in confluent cultures of A7r5 vascular smooth muscle cells. Immunoprecipitation/Western blot studies revealed a robust increase in tyrosine phosphorylation of the non-receptor tyrosine kinase, PYK2, in A7r5 cells treated with 4β-phorbol 12-myristate 13-acetate or ionomycin. 100 pm AVP also induced PYK2 tyrosine phosphorylation, and this effect was inhibited by protein kinase C inhibitors Ro-31-8220 (1–10 μm) or chelerythrine chloride (1–20 μm). In fura-2-loaded A7r5 cells, the stimulation of Ca2+ spiking by 100 pm AVP or 1 nm 4β-phorbol 12-myristate 13-acetate was completely blocked by PP2 (10 μm, a Src family kinase inhibitor). Salicylate (20 mm, recently identified as a PYK2 inhibitor) and the tyrosine kinase inhibitor, tyrphostin A47 (50 μm), but not its inactive analog, tyrphostin A63, also blocked AVP-stimulated Ca2+ spiking. PYK2 phosphorylation was inhibited by both PP2 and salicylate, whereas tyrphostin A47 failed to inhibit PYK2 tyrosine phosphorylation. ERK1/2 kinases did not appear to be involved because 1) 100 pm AVP did not appreciably increase ERK1/2 phosphorylation and U-0126 (2.5 μm) did not inhibit AVP-stimulated Ca2+ spiking; and 2) epidermal growth factor (10 nm) robustly stimulated ERK1/2 phosphorylation but did not induce Ca2+ spiking. Delayed rectifier K+ channels may mediate the PYK2 activity because Kv1.2 channel protein co-immunoprecipitated with PYK2 and tyrosine phosphorylation of Kv1.2 was stimulated by AVP and inhibited by Ro-31-8220, PP2, and salicylate but not tyrphostin A47. Our findings are consistent with a role for PYK2 and phosphorylation of K+ channels in the stimulation of Ca2+ spiking by physiological concentrations of AVP. Periodic or oscillatory increases in cytosolic free [Ca2+] ([Ca2+] i) 1[Ca2+] icytosolic free Ca2+concentrationAVP[Arg8]vasopressinfura-2-AMfura-2 acetoxymethyl esterEGFepidermal growth factorEGFRepidermal growth factor receptorMAPKsmitogen-activated protein kinasesPKCprotein kinase CPLCphospholipase CPLDphospholipase DPMA4β-phorbol 12-myristate 13-acetatePYK2proline-rich tyrosine kinase 2SFKsSrc family kinasesTyrA47tyrphostin A47TyrA63tyrphostin A63ERKextracellular signal-regulated kinaseANOVAanalysis of variance in vascular smooth muscle cells are believed to underlie arterial vasomotion. These rhythmic contractions of resistance arteries and arterioles are important for local perfusion of tissues (1Nicoll P.A. Webb R.L. Angiology. 1955; 6: 291-310Crossref PubMed Scopus (80) Google Scholar) as well as a determinant of blood pressure and peripheral resistance (2Gratton R.J. Gandley R.E. McCarthy J.F. Michaluk W.K. Slinker B.K. McLaughlin M.K. J. Appl. Physiol. 1998; 85: 2255-2260Crossref PubMed Scopus (28) Google Scholar). Vasomotion correlates with action potentials in the smooth muscle cells of the artery wall (3Steedman W.M. J. Physiol. (Lond.). 1966; 180: 382-400Crossref Scopus (34) Google Scholar, 4Nicoll P.A. Immunochemistry. 1975; 12: 511-515Crossref PubMed Scopus (7) Google Scholar, 5Droogmans G. Raeymaekers L. Casteels R. J. Gen. Physiol. 1977; 70: 129-148Crossref PubMed Scopus (157) Google Scholar, 6Von Der Weid P.-Y Bény J.-L. J. Physiol. (Lond.). 1993; 471: 13-24Crossref Scopus (75) Google Scholar, 7Gokina N.I. Bevan R.D. Walters C.L. Bevan J.A. Circ. Res. 1996; 78: 148-151Crossref PubMed Scopus (52) Google Scholar). This activity depends on activation of L-type voltage-sensitive Ca2+ channels (5Droogmans G. Raeymaekers L. Casteels R. J. Gen. Physiol. 1977; 70: 129-148Crossref PubMed Scopus (157) Google Scholar, 7Gokina N.I. Bevan R.D. Walters C.L. Bevan J.A. Circ. Res. 1996; 78: 148-151Crossref PubMed Scopus (52) Google Scholar) and may be triggered or enhanced by vasoconstrictor hormones (3Steedman W.M. J. Physiol. (Lond.). 1966; 180: 382-400Crossref Scopus (34) Google Scholar, 8Stein P.G. Driska S.P. Circ. Res. 1984; 55: 480-485Crossref PubMed Scopus (19) Google Scholar, 9Gerstberger R. Meyer J.-U. Rettig R. Printz M. Intaglietta M. Int. J. Microcirc. Clin. Exp. 1987; 7: 3-14Google Scholar, 10Fujii K. Heistad D.D. Faraci F.M. Am. J. Physiol. 1990; 258: H1829-H1834PubMed Google Scholar, 11Achakri H. Stergiopulos N. Hoogerwerf N. Hayoz D. Brunner H.R. Meister J.J. J. Vasc. Res. 1995; 32: 237-246Crossref PubMed Scopus (31) Google Scholar, 12Wesselman J.P.M. Van Bavel E. Pfaffendorf M. Spaan J.A.E. J. Vasc. Res. 1996; 33: 32-41Crossref PubMed Scopus (76) Google Scholar). The mechanisms whereby vasoconstrictor hormones stimulate Ca2+-dependent action potentials in vascular smooth muscle cells have not been elucidated. cytosolic free Ca2+concentration [Arg8]vasopressin fura-2 acetoxymethyl ester epidermal growth factor epidermal growth factor receptor mitogen-activated protein kinases protein kinase C phospholipase C phospholipase D 4β-phorbol 12-myristate 13-acetate proline-rich tyrosine kinase 2 Src family kinases tyrphostin A47 tyrphostin A63 extracellular signal-regulated kinase analysis of variance AVP is a potent vasoconstrictor peptide. It binds to heptahelical V1a vasopressin receptors on vascular smooth muscle cells, leading to G protein-dependent activation of phospholipase C (PLC) and the consequent release of Ca2+ from intracellular stores. This signal transduction pathway is activated independently of L-type voltage-sensitive Ca2+ channels (13Byron K.L. Taylor C.W. J. Biol. Chem. 1993; 268: 6945-6952Abstract Full Text PDF PubMed Google Scholar) and requires nanomolar concentrations of AVP for half-maximal activation (14Doyle V.M. Rüegg U.T. Biochem. Biophys. Res. Commun. 1985; 131: 469-476Crossref PubMed Scopus (74) Google Scholar, 15Byron K.L. Circ. Res. 1996; 78: 813-820Crossref PubMed Scopus (42) Google Scholar). We have identified previously a novel signal transduction pathway in A7r5 vascular smooth muscle cells that is activated by physiological concentrations of AVP (between 10 and 100 pm) and leads to oscillations of [Ca2+] i (Ca2+ spiking) that increase in frequency with [AVP] (15Byron K.L. Circ. Res. 1996; 78: 813-820Crossref PubMed Scopus (42) Google Scholar, 16Shiels A. Lucchesi P.A. Moran C. Byron K.L. J. Mol. Cell. Cardiol. 1998; 30: 190Google Scholar). This effect of low [AVP] is dependent on L-type voltage-sensitive Ca2+ channels (15Byron K.L. Circ. Res. 1996; 78: 813-820Crossref PubMed Scopus (42) Google Scholar) and correlates with action potential generation (16Shiels A. Lucchesi P.A. Moran C. Byron K.L. J. Mol. Cell. Cardiol. 1998; 30: 190Google Scholar), suggesting that it may represent an effect equivalent to stimulation of arterial vasomotion in vivo. We have recently shown that AVP-stimulated Ca2+ spiking in A7r5 cells involves phospholipase D (17Li Y. Shiels A.J. Maszak G. Byron K.L. Am. J. Physiol. 2001; 280: H2658-H2664Crossref PubMed Google Scholar) and activation of one or more protein kinase C (PKC) isoforms (18Fan J. Byron K.L. J. Physiol. (Lond.). 2000; 524: 821-831Crossref Scopus (34) Google Scholar). It remains to be elucidated how activation of PKC ultimately produces Ca2+ spiking. One possibility is that PKC activation leads to membrane depolarization and consequently to activation of L-type voltage-sensitive Ca2+ channels. We have preliminary data that suggest that inhibition of delayed rectifier K+channels (K v channels) may provide the trigger for L-type Ca2+ channel activation (16Shiels A. Lucchesi P.A. Moran C. Byron K.L. J. Mol. Cell. Cardiol. 1998; 30: 190Google Scholar). The present study examines the possibility that one or more tyrosine kinases may serve as intermediary links in this signaling cascade. In particular, we focus on the non-receptor tyrosine kinase PYK2 (proline-rich tyrosine kinase 2, also known as RAFTK or CADTK), a member of the focal adhesion kinase (p125FAK) family, which is activated by stimuli that increase [Ca2+] i or activate PKC in cultured rat aortic smooth muscle cells (19Sabri A. Govindarajan G. Griffin T.M. Byron K.L. Samarel A.M. Lucchesi P.A. Circ. Res. 1998; 83: 841-851Crossref PubMed Scopus (137) Google Scholar, 20Rocic P. Govindarajan G. Sabri A. Lucchesi P.A. Am. J. Physiol. 2001; 280: C90-C99Crossref PubMed Google Scholar, 21Eguchi S. Iwasaki H. Inagami T. Numaguchi K. Yamakawa T. Motley E.D. Owada K.M. Marumo F. Hirata Y. Hypertension. 1999; 33: 201-206Crossref PubMed Google Scholar). PYK2 has also been linked with inhibition of delayed rectifier K+ channels in non-muscle cells (22Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1253) Google Scholar, 23Felsch J.S. Cachero T.G. Peralta E.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5051-5056Crossref PubMed Scopus (95) Google Scholar). Src family kinases (SFKs) and epidermal growth factor receptors (EGFR) are tyrosine kinases that have been implicated as activators and/or downstream mediators of PYK2 in other systems (19Sabri A. Govindarajan G. Griffin T.M. Byron K.L. Samarel A.M. Lucchesi P.A. Circ. Res. 1998; 83: 841-851Crossref PubMed Scopus (137) Google Scholar, 21Eguchi S. Iwasaki H. Inagami T. Numaguchi K. Yamakawa T. Motley E.D. Owada K.M. Marumo F. Hirata Y. Hypertension. 1999; 33: 201-206Crossref PubMed Google Scholar, 24Inagami T. Eguchi S. Numaguchi K. Motley E.D. Tang H. Matsumoto T. Yamakawa T. J. Am. Soc. Nephrol. 1999; 10: 57-61PubMed Google Scholar, 25Tang H. Nishishita T. Fitzgerald T. Landon E.J. Inagami T. J. Biol. Chem. 2000; 275: 13420-13426Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 26Dikic I Toliwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (879) Google Scholar). Activation of PYK2 is associated with its autophosphorylation on tyrosine 402. This phosphotyrosine moiety may then serve as a docking site for the SH2 domain of Src (22Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1253) Google Scholar, 26Dikic I Toliwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (879) Google Scholar). Another tyrosine residue in PYK2 (Tyr-881) may also be phosphorylated and serve as a docking site for Grb2, leading to activation of ERK1/2, members of the family of mitogen-activated protein kinases (MAPKs) (22Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1253) Google Scholar, 26Dikic I Toliwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (879) Google Scholar). The roles of SFKs and transactivation of EGFR or MAPKs in AVP-stimulated Ca2+ spiking have not been examined previously. The results of the present study are consistent with roles for SFKs and PYK2 activation leading to tyrosine phosphorylation of delayed rectifier K+ channels in this novel signal transduction pathway. However, activation of EGFR or ERK1/2 does not appear to be either necessary or sufficient to induce Ca2+ spiking in A7r5 cells. Cell culture media were from Invitrogen or MediaTech (Herndon, VA). Fura-2-AM, fura-2 pentapotassium salt, fluo3-AM, and Pluronic F127 were from Molecular Probes, Inc. (Eugene, OR). Monoclonal anti-PKC and anti-PYK2 antibodies and polyclonal anti-phosphotyrosine antibodies were from Transduction Laboratories (San Diego, CA). Monoclonal anti-Kv1.2 and anti-phosphotyrosine (clone 4G10) and polyclonal anti-PYK2 were from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal anti-Kv1.2 channel antibodies were from Chemicon (Temecula, CA). Monoclonal anti-Src antibodies were from Oncogene Research Products (San Diego, CA). Anti-phospho-ERK antibodies were from Promega (Madison, WI). AVP, epidermal growth factor, salicylate, and ionomycin were from Sigma. 4β-Phorbol 12-myristate 13-acetate, chelerythrine chloride, tyrphostins A47 and A63, and Ro-31-8220 were from Calbiochem. U-0126 was from Biomol (Plymouth Meeting, PA). Pefabloc SC“ was from Roche Molecular Biochemicals. A7r5 cells were cultured as described previously (13Byron K.L. Taylor C.W. J. Biol. Chem. 1993; 268: 6945-6952Abstract Full Text PDF PubMed Google Scholar). Cells were subcultured onto rectangular (9 × 22-mm, number 1½) glass coverslips or plastic tissue culture dishes (Corning Glass). Confluent cell monolayers were used 2–5 days after plating. Essentially as described previously (15Byron K.L. Circ. Res. 1996; 78: 813-820Crossref PubMed Scopus (42) Google Scholar, 18Fan J. Byron K.L. J. Physiol. (Lond.). 2000; 524: 821-831Crossref Scopus (34) Google Scholar), coverslips were washed twice with control medium (135 mm NaCl, 5.9 mm KCl, 1.5 mm CaCl2, 1.2 mm MgCl2, 11.5 mm glucose, 11.6 mm HEPES, pH 7.3) and then incubated in the same medium with 2 μm fura-2-AM, 0.1% bovine serum albumin, and 0.02% Pluronic F127 detergent (27Poenie M. Alderton J. Steinhardt R. Tsien R. Science. 1986; 233: 886-889Crossref PubMed Scopus (373) Google Scholar) for 90–120 min at room temperature (20–23 °C) in the dark. The cells were then washed twice and incubated in the dark in control medium (or pretreated with drugs) for 1–5 h prior to the start of the experiment. Fura-2 fluorescence (at 510 nm) was measured in cell populations with a PerkinElmer Life Sciences LS50B fluorescence spectrophotometer. Background fluorescence was determined at the end of the experiment by quenching the fura-2 fluorescence for 10–15 min in the presence of 5 μm ionomycin and 6 mm MnCl2 in Ca2+-free medium. After background fluorescence was subtracted, the ratio of fluorescence at 340 nm excitation to that at 380 nm was calculated and calibrated in terms of [Ca2+] i . We found that salicylate interfered with the measurement of fura-2 fluorescence, so fluo3 was used to measure [Ca2+] i responses in the presence of salicylate. A7r5 cells were incubated for 1 h in the presence of 10 μm fluo3-AM, 0.1% bovine serum albumin, and 0.02% Pluronic F127 detergent, then washed, and incubated in control medium in the absence of fluo3-AM for at least 30 min. For these experiments, a single excitation wavelength (505 nm) was used, and emitted fluorescence (at 535 nm) was collected at 0.5-s intervals. Calibration of fura-2 fluorescence in terms of [Ca2+] i was carried out as described previously (28Byron K.L. Villereal M.L. J. Biol. Chem. 1989; 264: 18234-18239Abstract Full Text PDF PubMed Google Scholar) using solutions of known Ca2+ concentration to construct a standard curve. The Ca2+ concentration of the standard solutions was calculated using software (MaxChelator, version 6.60) that accounts for binding of Ca2+ to each constituent of the solution. For analysis of fluorescence ratios recorded from cells, the equation [Ca2+] i =K D ·β·((r −R min)/(R max −r)) (29Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar) was fit to the standard curve (using SigmaPlot® software, SPSS Inc., Chicago, IL) and used to convert ratios (r) into [Ca2+] i . In situcalibration of fura-2 fluorescence by direct determination of minimum and maximum ratios (R min andR max, respectively (29Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar)) from within cells yields similar calibrated values. Traces shown are representative of at least three similar experiments. A7r5 cells were grown to confluence on 100-mm tissue culture dishes (Corning Glass). Cells were washed and incubated in control medium (see above) for 3 h at room temperature, followed by treatment for the indicated time in control medium ± agonist. The medium was aspirated, and 0.8 ml of ice-cold lysis buffer (1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 100 mm NaF, 10 mm sodium pyrophosphate, 1 mm EGTA, 1.5 mmMgCl2, 10% glycerol, 150 mm NaCl, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mmNa3VO4, 50 mm HEPES, pH 7.4) was added to the dish on ice for 10 min. Cell lysates were collected, sonicated for 15 s, and centrifuged at 16,000 × gfor 20 min at 4 °C. The protein concentration in the supernatant was determined using a bicinchoninic acid protein assay (Pierce), and 600 μg of protein from each sample was incubated with 3 μg of polyclonal anti-phosphotyrosine antibody overnight at 4 °C with rocking. 40 μl of packed protein A-Sepharose beads (Sigma) were then added to each sample and incubated with rocking for 60 min at 4 °C. The beads were then pelleted by centrifugation at 14,000 ×g and washed three times in 500 μl of lysis buffer. The procedure for the Kv1.2 or PYK2 immunoprecipitation was similar except that a milder lysis buffer was used to preserve protein-protein interactions (100 mm NaCl, 1% Nonidet P-40 (IGEPAL CA-630), 0.25% sodium deoxycholate, 30 mm sodium pyrophosphate, 5 mm β-glycerophosphate, 10 μg/ml leupeptin, 0.5 mm Pefabloc, 10 μg/ml aprotinin, 500 μm Na3VO4, 20 mmHEPES, pH 7.4). 700 μg of cell lysates were incubated with 4 μg of monoclonal Kv1.2 antibodies or 5 μg of polyclonal anti-PYK2 antibodies overnight at 4 °C, and immune complexes were collected by incubation with 40 μl of packed protein G-agarose beads. For Western blotting, the immunoprecipitates were subjected to SDS-PAGE, electrophoretically transferred to a nitrocellulose membrane, and immunoblotted with the indicated antibody. After blotting, the membrane was washed and treated with horseradish peroxidase-conjugated secondary antibody (goat anti-mouse or anti-rabbit IgG). The immunoreactive bands were visualized using enhanced chemiluminescence reagents (Amersham Biosciences) exposed to Hyperfilm (Amersham Biosciences) in the linear range of the film density. The films were scanned, and densitometric analysis was performed with NIH image software. A variation of these methods was used to measure phosphorylation of ERK1/2. A7r5 cells grown on 100-mm plastic tissue culture dishes were equilibrated for 2 h in control medium at room temperature. The cells were then pretreated for 1 h with 2.5 μmU-0126 or vehicle or 30 min with 20 mm salicylate or vehicle, followed by treatment for up to 30 min with 100 pmAVP in the presence or absence of U-0126 or 20 mmsalicylate. The cells were then lysed in 50 mm sodium pyrophosphate, 50 mm NaF, 50 mm NaCl, 5 mm EDTA, 5 mm EGTA, 0.1 mmNaVO4, 0.01% Triton X-100, 10 μg ml−1aprotinin, 10 μg ml−1 leupeptin, 0.5 mmPefabloc, 10 mm HEPES, pH 7.4, scraped off the dish, and centrifuged at 12,000 × g at 4 °C for 10 min. The supernatant (a volume containing 40 μg of protein) was subjected to SDS-PAGE, electrophoretically transferred to a nitrocellulose membrane, and immunoblotted with polyclonal antibodies against phospho-ERK proteins (Promega, Madison, WI; 1:10,000 dilution). The membranes were re-probed for total ERK protein using polyclonal anti-ERK antibodies (Upstate Biotechnology, Inc.). Data are expressed as mean ± S.E. for at least n = 3 experiments and were analyzed using InStat (Graphpad) or SigmaStat (SPSS Scientific) statistical software. One-way repeated measures analysis of variance (ANOVA) followed by Bonferroni's test or a Dunnett's test was used for comparisons among multiple groups. Paired Student's t test was used to evaluate the effects of PP2 on AVP-stimulated Ca2+spiking. Stimuli that activate PKC or elevate [Ca2+] i have been found to activate the tyrosine kinase, PYK2, leading to its autophosphorylation on a tyrosine residue. The presence of PYK2 in A7r5 cells was confirmed by Western blot analysis that identified a band at ∼112 kDa (Fig.1) that did not cross-react with p125FAK antibodies (not shown). Immunoprecipitation using anti-phosphotyrosine antibodies revealed an increase in tyrosine-phosphorylated PYK2 in response to both PMA (1 nm) and ionomycin (1 μm), indicating that it can be activated by either PKC or increased [Ca2+] i in A7r5 cells (Fig. 2).Figure 2PYK2 activation by PMA or ionomycin.Tyrosine phosphorylation of PYK2 was assessed by immunoprecipitation (IP) with anti-phosphotyrosine (pTyr) antibodies followed by immunoblotting with anti-PYK2. Left panel shows a representative immunoblot from cells treated with PMA (1 nm, 10 min) or ionomycin (Iono, 1 μm, 10 min); right panel shows a quantitative densitometric analysis from five experiments (mean ± S.E.). Results are presented as a fold increase above control, which was set at 1. A one-way repeated measures ANOVA was performed. * indicates significant difference from control, p < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 100 pm AVP was also found to stimulate PYK2 phosphorylation. The time course for stimulation of tyrosine phosphorylation of PYK2 by 100 pm AVP is shown in Fig.3, A and B. A significant increase in tyrosine phosphorylation was detected after 2 min, followed by a further increase, which plateaued between 5 and 20 min and then declined at 30 min. The Ca2+-spiking response to 100 pm AVP was typically delayed by several minutes (on average 4.2 ± 0.6 min, as reported previously (18Fan J. Byron K.L. J. Physiol. (Lond.). 2000; 524: 821-831Crossref Scopus (34) Google Scholar)) but is sustained for as long as AVP is present, at least up to 1 h (18Fan J. Byron K.L. J. Physiol. (Lond.). 2000; 524: 821-831Crossref Scopus (34) Google Scholar). Treatment of A7r5 cells with PLD (2.5 units/ml, 15 min), which has been shown previously to stimulate Ca2+ spiking in A7r5 cells (17Li Y. Shiels A.J. Maszak G. Byron K.L. Am. J. Physiol. 2001; 280: H2658-H2664Crossref PubMed Google Scholar), also stimulated PYK2 tyrosine phosphorylation (not shown). The stimulation of PYK2 tyrosine phosphorylation by 100 pm AVP was inhibited in a concentration-dependent manner by the selective PKC inhibitor Ro-31-8220 (Fig. 4). This drug was shown previously to block AVP-stimulated Ca2+ spiking (18Fan J. Byron K.L. J. Physiol. (Lond.). 2000; 524: 821-831Crossref Scopus (34) Google Scholar). Similar results were obtained using another structurally unrelated PKC inhibitor, chelerythrine chloride (0.1–20 μm, not shown). Salicylate has been reported recently (30Wang Z. Brecher P. Hypertension. 2001; 37: 148-153Crossref PubMed Scopus (21) Google Scholar) to inhibit selectively PYK2 tyrosine phosphorylation in angiotensin II-stimulated cardiac fibroblasts. Salicylate (20 mm) inhibited AVP-stimulated PYK2 tyrosine phosphorylation by 82% (p< 0.01, n = 3; Fig.5 A) and completely abolished AVP-stimulated Ca2+ spiking (Fig. 5 B) in A7r5 cells. In three independent paired experiments, the mean frequency of Ca2+ spiking in cells treated with 100 pm AVP alone was 7.8 ± 1.1 min−1, whereas no spiking was observed in cells treated with 100 pm AVP in the presence of 20 mm salicylate. This concentration of salicylate did not prevent 100 nm AVP-stimulated release of Ca2+ from intracellular stores, the [Ca2+] i response to a high external [K+] solution (not shown), or EGF-stimulated ERK1/2 phosphorylation (see below). PP2 is a relatively selective inhibitor of SFKs (31Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1784) Google Scholar). 10 μm PP2 completely inhibited AVP-stimulated Ca2+ spiking, whereas its inactive analog, PP3, had no effect (Fig. 5 C). PP2 also abolished PMA-stimulated Ca2+ spiking (not shown). AVP-stimulated tyrosine phosphorylation of PYK2 was significantly inhibited (by 65 ± 3%,n = 3, p < 0.05) by PP2 (Fig.5 D). SFKs have been found to associate with active PYK2 by binding to its phosphorylated tyrosine (Tyr-402; see Refs. 22Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1253) Google Scholar and 26Dikic I Toliwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (879) Google Scholar). We examined the possibility that Src and PYK2 might become associated following AVP treatment. Co-immunoprecipitation results are shown in Fig. 6. A7r5 cells were treated for varying times with 100 pm AVP followed by immunoprecipitation of PYK2. Although PYK2 was readily detected in the immunoprecipitates (and depleted from the supernatants), Src was not detectable in the immunoprecipitates at any time point (but was readily detected in the supernatants). Similar results were obtained by immunoprecipitating Src and probing for PYK2 (not shown). The effects of another tyrosine kinase inhibitor, tyrphostin A47 (TyrA47), on the Ca2+-spiking responses to 100 pm AVP or 1 nm PMA are shown in Fig. 7(A–F). TyrA47 (50 μm) completely abolished the Ca2+-spiking response to both agents, whereas the inactive analog, TyrA63 (50 μm), did not affect the responses. However, in contrast to salicylate or PP2, neither TyrA47 nor TyrA63 prevented AVP- or PMA-stimulated tyrosine phosphorylation of PYK2 (Fig. 7 G). Transactivation of EGF receptors (EGFR) and activation of ERK1 and ERK2 MAPKs have been implicated as downstream effectors in other PYK2-mediated cell responses (9–22, 24Inagami T. Eguchi S. Numaguchi K. Motley E.D. Tang H. Matsumoto T. Yamakawa T. J. Am. Soc. Nephrol. 1999; 10: 57-61PubMed Google Scholar, 25Tang H. Nishishita T. Fitzgerald T. Landon E.J. Inagami T. J. Biol. Chem. 2000; 275: 13420-13426Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 32Tang H. Zhao Z.J. Landon E.J. Inagami T. J. Biol. Chem. 2000; 275: 8389-8396Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Activation of ERK1/2 requires dual threonine and tyrosine phosphorylation, both catalyzed by another highly specific kinase, MEK. EGF at concentrations ranging from 1 pm to 100 nm failed to stimulate Ca2+ spiking in A7r5 cells, whereas 50 pm AVP elicited a robust Ca2+-spiking response in the same cells (not shown). Despite its inability to stimulate Ca2+spiking, EGF (10 nm) robustly activated ERK1/2 (Fig.8 A). Salicylate (20 mm) did not prevent EGF-stimulated ERK1/2 phosphorylation (Fig. 8 A), whereas U-0126 (2.5 μm, a selective MEK inhibitor) completely abolished this effect (Fig. 8 B). Phosphorylation of ERK1/2 in response to 100 pm AVP was undetectable in 6 of 9 experiments (Fig. 8 A) and U-0126 did not inhibit AVP-stimulated Ca2+ spiking (Fig.8 C; frequency of Ca2+ spiking in response to 100 pm AVP was 3.8 ± 0.5 min−1 in the absence of U-0126 and 5.3 ± 0.9 min−1 in the presence of U-0126, p > 0.1, n = 4). These results suggest that activation of EGF receptors or ERK1/2 MAPKs is neither necessary nor sufficient to elicit the Ca2+-spiking effect. We next determined whether Kv1.2-delayed rectifier K+channels might be a potential effector for PYK2 in the stimulation of Ca2+ spiking. Kv1.2 channels have been shown to be tyrosine-phosphorylated by PYK2, leading to an inhibition of outward K+ currents in Xenopus oocytes (22Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1253) Google Scholar). We found that, in A7r5 cells, treatment of the cells with 100 pm AVP significantly increased tyrosine phosphorylation of the Kv1.2 channel protein (Fig. 9 A) and that Kv1.2 channel protein co-immunoprecipitated with PYK2 (Fig.9 B). The amounts of Kv1.2 detected in the PYK2 immunoprecipitates from untreated cells were similar to those from cells treated with 100 pm AVP in five independent experiments (Fig. 9 B and results not shown). AVP-stimulated tyrosine phosphorylation of Kv1.2 was significantly inhibited by PP2, Ro-31-8220, and salicylate, but not by tyrphostin A47 (% inhibition = 100, 36.9, 31.3, and 18.1, respectively; Fig.9 C). We have identified previously (15Byron K.L. 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