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• W2000263416 abstract "Protein kinase Cδ (PKCδ) activation is generally attributed to lipid cofactor-dependent allosteric activation mechanisms at membranes. However, recent studies indicate that PKCδ also is dynamically regulated through tyrosine phosphorylation in H2O2- and phorbol 12-myristate 13-acetate (PMA)-treated cardiomyocytes. H2O2 activates Src and related Src-family kinases (SFKs), which function as dual PKCδ-Tyr311 and -Tyr332 kinases in vitro and contribute to H2O2-dependent PKCδ-Tyr311/Tyr332 phosphorylation in cardiomyocytes and in mouse embryo fibroblasts. H2O2-dependent PKCδ-Tyr311/Tyr332 phosphorylation is defective in SYF cells (deficient in SFKs) and restored by Src re-expression. PMA also promotes PKCδ-Tyr311 phosphorylation, but this is not associated with SFK activation or PKCδ-Tyr332 phosphorylation. Rather, PMA increases PKCδ-Tyr311 phosphorylation by delivering PKCδ to SFK-enriched caveolae. Cyclodextrin treatment disrupts caveolae and blocks PMA-dependent PKCδ-Tyr311 phosphorylation, without blocking H2O2-dependent PKCδ-Tyr311 phosphorylation. The enzyme that acts as a PKCδ-Tyr311 kinase without increasing PKCδ phosphorylation at Tyr332 in PMA-treated cardiomyocytes is uncertain. Although in vitro kinase assays implicate c-Abl as a selective PKCδ-Tyr311 kinase, PMA-dependent PKCδ-Tyr311 phosphorylation persists in cardiomyocytes treated with the c-Abl inhibitor ST1571 and c-Abl is not detected in caveolae; these results effectively exclude a c-Abl-dependent process. Finally, we show that 1,2-dioleoyl-sn-glycerol mimics the effect of PMA to drive PKCδ to caveolae and increase PKCδ-Tyr311 phosphorylation, whereas G protein-coupled receptor agonists such as norepinephrine and endothelin-1 do not. These results suggest that norepinephrine and endothelin-1 increase 1,2-dioleoyl-sn-glycerol accumulation and activate PKCδ exclusively in non-caveolae membranes. Collectively, these results identify stimulus-specific PKCδ localization and tyrosine phosphorylation mechanisms that could be targeted for therapeutic advantage. Protein kinase Cδ (PKCδ) activation is generally attributed to lipid cofactor-dependent allosteric activation mechanisms at membranes. However, recent studies indicate that PKCδ also is dynamically regulated through tyrosine phosphorylation in H2O2- and phorbol 12-myristate 13-acetate (PMA)-treated cardiomyocytes. H2O2 activates Src and related Src-family kinases (SFKs), which function as dual PKCδ-Tyr311 and -Tyr332 kinases in vitro and contribute to H2O2-dependent PKCδ-Tyr311/Tyr332 phosphorylation in cardiomyocytes and in mouse embryo fibroblasts. H2O2-dependent PKCδ-Tyr311/Tyr332 phosphorylation is defective in SYF cells (deficient in SFKs) and restored by Src re-expression. PMA also promotes PKCδ-Tyr311 phosphorylation, but this is not associated with SFK activation or PKCδ-Tyr332 phosphorylation. Rather, PMA increases PKCδ-Tyr311 phosphorylation by delivering PKCδ to SFK-enriched caveolae. Cyclodextrin treatment disrupts caveolae and blocks PMA-dependent PKCδ-Tyr311 phosphorylation, without blocking H2O2-dependent PKCδ-Tyr311 phosphorylation. The enzyme that acts as a PKCδ-Tyr311 kinase without increasing PKCδ phosphorylation at Tyr332 in PMA-treated cardiomyocytes is uncertain. Although in vitro kinase assays implicate c-Abl as a selective PKCδ-Tyr311 kinase, PMA-dependent PKCδ-Tyr311 phosphorylation persists in cardiomyocytes treated with the c-Abl inhibitor ST1571 and c-Abl is not detected in caveolae; these results effectively exclude a c-Abl-dependent process. Finally, we show that 1,2-dioleoyl-sn-glycerol mimics the effect of PMA to drive PKCδ to caveolae and increase PKCδ-Tyr311 phosphorylation, whereas G protein-coupled receptor agonists such as norepinephrine and endothelin-1 do not. These results suggest that norepinephrine and endothelin-1 increase 1,2-dioleoyl-sn-glycerol accumulation and activate PKCδ exclusively in non-caveolae membranes. Collectively, these results identify stimulus-specific PKCδ localization and tyrosine phosphorylation mechanisms that could be targeted for therapeutic advantage. Protein kinase C-δ (PKCδ) 2The abbreviations used are: PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SFK, Src-family kinases; DAG, 1,2-dioleoyl-sn-glycerol; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; diC8, 1,2-dioctanoyl-sn-glycerol; MES, 4-morpholineethane-sulfonic acid; PSSA, phospho-site specific antibodies; PS, phosphatidylserine; RP, reverse phase; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; AR, adrenergic receptor; NE, norepinephrine; ET-1, endothelin-1. 2The abbreviations used are: PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SFK, Src-family kinases; DAG, 1,2-dioleoyl-sn-glycerol; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; diC8, 1,2-dioctanoyl-sn-glycerol; MES, 4-morpholineethane-sulfonic acid; PSSA, phospho-site specific antibodies; PS, phosphatidylserine; RP, reverse phase; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; AR, adrenergic receptor; NE, norepinephrine; ET-1, endothelin-1. is a serine/threonine kinase that plays a vital role in signaling pathways that regulate cardiac contraction, ischemic preconditioning, and the pathogenesis of cardiac hypertrophy and failure (1Steinberg S.F. Biochem. J. 2004; 384: 449-459Crossref PubMed Scopus (323) Google Scholar). Traditional models of PKCδ activation have focused on lipid cofactor-dependent mechanisms that localize PKCδ in its active conformation to membranes. However, recent studies identify regulatory phosphorylation at Thr505 in the “activation loop” segment (a region flanked by the highly conserved DFG and APE sequences) as an additional mechanism that contributes to the dynamic control of PKCδ activity in some cellular contexts. Although activation loop phosphorylation is a stable modification that plays a critical role to structure other PKC isoforms in a favorable conformation for catalysis, PKCδ is catalytically active even without activation loop Thr505 phosphorylation. Rather, PKCδ-Thr505 phosphorylation plays a unique role as a dynamically regulated process that is increased by PMA and certain agonist-activated receptors and contributes to the control of PKCδ activity and/or substrate specificity (1Steinberg S.F. Biochem. J. 2004; 384: 449-459Crossref PubMed Scopus (323) Google Scholar, 2Rybin V.O. Sabri A. Short J. Braz J.C. Molkentin J.D. Steinberg S.F. J. Biol. Chem. 2003; 278: 14555-14564Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 3Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (967) Google Scholar, 4Stempka L. Schnolzer M. Radke S. Rincke G. Marks F. Gschwendt M. J. Biol. Chem. 1999; 274: 8886-8892Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 5Liu Y. Belkina N.V. Graham C. Shaw S. J. Biol. Chem. 2006; 281: 12102-12111Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 6Cheng N. He R. Tian J. Dinauer M.C. Ye R.D. J. Immunol. 2007; 179: 7720-7728Crossref PubMed Scopus (50) Google Scholar). PKCδ also is phosphorylated at Tyr311 and Tyr332, two tyrosine residues that are unique to the hinge region of PKCδ (and not conserved in other PKC isoforms). Our recent studies focused on mechanisms that regulate PKCδ-Tyr311 phosphorylation, showing that oxidative stress resulting from H2O2 treatment leads to the release of PKCδ from membranes and a global increase in PKCδ-Tyr311 phosphorylation in both soluble and particulate subcellular compartments. H2O2-dependent PKCδ-Tyr311 phosphorylation is via a PP1-sensitive mechanism; this is presumed to reflect a role for Src or a related PP1-sensitive Src family kinase (SFK), because these enzymes are activated in H2O2-treated cardiomyocytes. PMA also increases PKCδ-Tyr311 phosphorylation via a PP1-sensitive pathway. However, PMA-dependent PKCδ-Tyr311 phosphorylation is via a different signaling pathway that is confined to the membrane fraction and is not associated with a global increase in SFK activity. In vitro studies suggest that PMA increases PKCδ-Tyr311 phosphorylation by inducing a conformational change that renders PKCδ a better substrate for phosphorylation by Src (7Rybin V.O. Guo J. Gertsberg Z. Elouardighi H. Steinberg S.F. J. Biol. Chem. 2007; 282: 23631-23638Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 8Rybin V.O. Guo J. Sabri A. Elouardighi H. Schaefer E. Steinberg S.F. J. Biol. Chem. 2004; 279: 19350-19361Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). According to this formulation, PMA promotes PKCδ-Tyr311 phosphorylation by delivering the enzyme in an active conformation to a Src-enriched membrane fraction. Because caveolae have been identified as signaling domains for SFKs in other cell types, and our previous studies demonstrated that PMA delivers PKCδ to the caveolae compartment (9Rybin V.O. Xu X. Steinberg S.F. Circ. Res. 1999; 84: 980-988Crossref PubMed Scopus (139) Google Scholar), this study examines the role of caveolae as platforms for cross-talk between PKCδ and SFKs in cardiomyocytes. Insofar as PKCδ-Tyr311 phosphorylation is predicted to be associated with a coordinate increase in PKCδ phosphorylation at Tyr332 (a site that also is a target for in vitro Src-dependent phosphorylation (7Rybin V.O. Guo J. Gertsberg Z. Elouardighi H. Steinberg S.F. J. Biol. Chem. 2007; 282: 23631-23638Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar)), and PKCδ-Tyr332 phosphorylation may impart functionally important properties (as a consensus binding sequence for the SH2 domain of the adapter protein Shc (10Leitges M. Gimborn K. Elis W. Kalesnikoff J. Hughes M.R. Krystal G. Huber M. Mol. Cell. Biol. 2002; 22: 3970-3980Crossref PubMed Scopus (115) Google Scholar) or to influence PKCδ proteolytic cleavage, which contributes to the induction of apoptosis (11Lu W. Lee H.K. Xiang C. Finniss S. Brodie C. Cell. Signal. 2007; 19: 2165-2173Crossref PubMed Scopus (30) Google Scholar)), the mechanisms that regulate PKCδ-Tyr332 phosphorylation also were examined. Materials—Antibodies were from the following sources: PKCδ-Thr(P)505, PKCδ-Tyr(P)311, Src-Tyr(P)416, ERK-Thr(P)202/Tyr(P)204, Abl, and FAK, Cell Signaling Technology; PKCϵ and Caveolin-3 (BD Transduction Laboratories); PKCδ and PKCδ-Tyr(P)332, Santa Cruz Biotechnology; Src, Oncogene;, Lyn, Fyn, and Yes, Santa Cruz Biotechnology; anti-Tyr(P), Clone 4G10, BD Transduction Laboratories; and FAK-Tyr(P)397, BIO-SOURCE. Recombinant human PKCδ (rPKCδ) was from Sigma; active Src kinase was from Panvera; Lyn, Fyn, Yes, PDGFR β, FAK, JAK2, and c-Abl were from Upstate Biotechnologies. PMA and platelet-derived growth factor (PDGF) were from Sigma. 1,2-Dioleoyl-sn-glycerol (DAG) and 1,2-dioctanoyl-sn-glycerol (diC8) were from Avanti Polar Lipids, Inc. All other chemicals were reagent grade. Cell Culture—Cardiomyocytes were isolated from the hearts of 2-day-old Wistar rats by a trypsin dispersion procedure using a differential attachment procedure to enrich for cardiomyocytes followed by irradiation as described previously (2Rybin V.O. Sabri A. Short J. Braz J.C. Molkentin J.D. Steinberg S.F. J. Biol. Chem. 2003; 278: 14555-14564Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 12Lau Y.H. Robinson R.B. Rosen M.R. Bilezikian J.P. Circ. Res. 1980; 47: 41-48Crossref PubMed Scopus (66) Google Scholar, 13Steinberg S.F. Robinson R.B. Lieberman H.B. Stern D.M. Rosen M.R. Circ. Res. 1991; 68: 1216-1229Crossref PubMed Scopus (80) Google Scholar). The yield of cardiomyocytes typically is 2.5–3 × 106 cells per neonatal ventricle. Cells were plated on protamine sulfate-coated culture dishes at a density of 5 × 106 cells/100-mm dish. Experiments were performed on cultures grown for 5 days in minimal essential medium (Invitrogen) supplemented with 10% fetal calf serum and then serum deprived for the subsequent 24 h. SYF and Src+ cells obtained from ATCC were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 100 μg/ml hygromycin at 37 °C, in a 5% CO2 atmosphere. Preparation of Soluble and Particulate or Detergent-insoluble Fractions—Soluble and particulate fractions were prepared according to methods published previously (7Rybin V.O. Guo J. Gertsberg Z. Elouardighi H. Steinberg S.F. J. Biol. Chem. 2007; 282: 23631-23638Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In brief, cells were washed with phosphate-buffered saline and then transferred to ice-cold homogenization buffer (20 mm Tris-HCl, pH 7.5, 2 mm EDTA, 2 mm EGTA, 6 mm β-mercaptoethanol, 50 μg/ml aprotinin, 48 μg/ml leupeptin, 5 μm pepstatin A, 1 mm phenylmethylsulfonyl fluoride, 0.1 mm sodium vanadate, and 50 mm sodium fluoride), lysed by sonication, and centrifuged at 100,000 × g for 1 h. The supernatant was saved as the soluble fraction and the particulate fraction was solubilized in SDS-PAGE sample buffer. Some studies used a different biochemical fractionation method to prepare soluble and detergent-insoluble fractions. Briefly, after washing with phosphate-buffered saline, cells were solubilized in detergent-containing lysis buffer (50 mm HEPES, pH 7.4, 1 mm EGTA, 150 mm NaCl, 1.5 mm MgCl2, 10% glycerol, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 0.5 mm phenylmethylsulfonyl fluoride, 1 mm sodium vanadate, 100 mm sodium fluoride, 10 mm sodium pyrophosphate, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS), sonicated, and centrifuged at 14,000 × g for 15 min. The supernatant was saved as the soluble fraction and the detergent-insoluble fraction was solubilized in SDS-PAGE sample buffer. Preparation of Caveolae Membranes—Fractions enriched in the muscle-specific caveolin-3 isoform were prepared according to a detergent-free purification scheme described previously (9Rybin V.O. Xu X. Steinberg S.F. Circ. Res. 1999; 84: 980-988Crossref PubMed Scopus (139) Google Scholar, 14Rybin V.O. Xu X. Lisanti M.P. Steinberg S.F. J. Biol. Chem. 2000; 275: 41447-41457Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). All steps were carried out at 4 °C. Briefly, cells from five 100-mm diameter dishes were washed twice with ice-cold phosphate-buffered saline and then scraped into 0.5 m sodium carbonate, pH 11.0 (0.5 ml/dish). Cells from five dishes were combined (total volume, 2.5 ml) for each preparation. The extract was sequentially disrupted by homogenization with a loose-fitting Dounce homogenizer (10 strokes), a Polytron tissue grinder (three 10-s bursts), and a tip sonicator (three 20-s bursts). The homogenate was then adjusted to 40% sucrose by adding an equal volume of 80% sucrose prepared in MES-buffered saline (25 mm MES, pH 6.5, and 0.15 m NaCl), placed on the bottom of an ultracentrifuge tube, overlaid with a 5–30% discontinuous sucrose gradient (3 ml of 5% sucrose and 4 ml of 35% sucrose, both in MES-buffered saline containing 0.25 m sodium carbonate), and centrifuged at 260,000 × g for 16 to 18 h in a SW40 rotor (Beckman Coulter, Palo Alto, CA). After centrifugation, 13 1-ml fractions were collected. A pooled caveolae fraction (fractions 4–5, containing all of the buoyant caveolin-3 immunoreactivity and 0.5–1% total starting cell protein), a pooled fraction 8–13 (F8–13, which contains the bulk of the cellular material including the cytosol and most of the particulate membrane fraction), and the insoluble pellet (P, which is solubilized in SDS-PAGE sample buffer) were subjected to SDS-PAGE and immunoblotting. The caveolin-3-enriched membrane fraction isolated according to this method is biochemically distinct from the surrounding phospholipid bilayer and is operationally defined as caveolae in this study. However, it is important to note that this buoyant membrane fraction undoubtedly contains both true caveolae (specialized lipid raft membranes that contain caveolin and form invaginations at, or vesicles close to, the surface membrane) and morphologically featureless lipid rafts (that coexist and may even associate with caveolae (15Oh P. Schnitzer J.E. Mol. Biol. Cell. 2001; 12: 685-698Crossref PubMed Scopus (346) Google Scholar)). Biochemical methods to separate these distinct membrane subdomains and experiments to resolve their discrete cellular functions are beyond the scope of this study. Immunoprecipitation and Immunoblot Analysis—Immunoblotting on lysates or immunoprecipitated PKCδ was according to methods described previously or the manufacturer's instructions (2Rybin V.O. Sabri A. Short J. Braz J.C. Molkentin J.D. Steinberg S.F. J. Biol. Chem. 2003; 278: 14555-14564Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 7Rybin V.O. Guo J. Gertsberg Z. Elouardighi H. Steinberg S.F. J. Biol. Chem. 2007; 282: 23631-23638Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 9Rybin V.O. Xu X. Steinberg S.F. Circ. Res. 1999; 84: 980-988Crossref PubMed Scopus (139) Google Scholar). All anti-PKC antibodies (including the phosphosite specific antibodies (PSSAs) that specifically recognize PKCδ phosphorylation at Thr505, Tyr311, and Tyr332) have been validated (2Rybin V.O. Sabri A. Short J. Braz J.C. Molkentin J.D. Steinberg S.F. J. Biol. Chem. 2003; 278: 14555-14564Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Of note, the PKCδ-Tyr(P)311 antibody is a highly specific reagent that can be used to track PKCδ phosphorylation (at endogenous levels of enzyme expression) in experiments on cell lysates. The anti-PKCδ-Tyr(P)332 antibody is less specific and requires immunoprecipitation for studies of endogenous PKCδ phosphorylation. In each figure, each panel represents the results from a single gel (exposed for a uniform duration); detection was with enhanced chemiluminescence. In Vitro PKCδ Phosphorylation by Src and Other Tyrosine Kinases—0.03 μg of recombinant human PKCδ (rPKCδ) was preincubated in the absence or presence of Src kinase (0.18 units) in 110 μl of a reaction buffer containing 43 mm Tris-Cl, pH 7.5, 5.45 mm MgCl2, 0.75 mm EDTA, 0.77 mm EGTA, 0.3 mm dithiothreitol, 125 mm NaCl, 5% glycerol, 0.006% Brij-35, 0.04 mm phenylmethylsulfonyl fluoride, 0.2 mm benzamidine, and [γ-32P]ATP (13 μCi, 83 μm). Incubations were carried out for 30 min at 30 °C in the absence or presence of 91 μg/ml phosphatidylserine (PS) alone or with PMA (175 nm), DAG (7.2 μm), or diC8 (7.2 μm) and were stopped by adding 37 μl of 4× SDS-PAGE sample buffer. Samples were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted for PKCδ protein and phosphorylation. For peptide mapping studies, proteins were separated by SDS-PAGE, transferred to nitrocellulose, and the band corresponding to PKCδ was excised from the membrane, cut into small pieces, and treated for 30 min at 37 °C with polyvinylpyrrolidone (0.5%, w/v) in acetic acid (100 mm), followed by 5 water washes (to remove the acid) and a 10-min incubation at room temperature in the dark with 100 mm iodoacetate to carboxymethylate PKCδ. Membrane pieces were then washed three times with water and twice with 50 mm ammonium bicarbonate and incubated overnight at 37 °C in 60 μl of a buffer containing 42 mm ammonium bicarbonate, 17 μm HCl, and 10 μg of sequencing grade trypsin. Digested peptides were eluted from the membrane by sonication, lyophilized, and the residue was reconstituted in 0.1% trifluoroacetic acid and fractionated by RP-HPLC on a Vydac semimicro C18 column (2.1 × 250 mm). Peptides were eluted with a linear gradient from 0.1% trifluoroacetic acid in water to 0.1% trifluoroacetic acid in acetonitrile over 140 min at a flow rate of 1 ml/min. The eluant was monitored at 220 nm and fractions were collected every 30 s for Cherenkov counting. Radioactive peptides of interest were submitted to the Howard Hughes Medical Institute/Columbia University Protein Chemistry Core Facility for sequencing by MALDI-MS. Distinct Agonist-dependent PKCδ Phosphorylation Patterns in Cardiomyocytes—We previously reported that treatment with PMA, the α1-adrenergic receptor (α1-AR) agonist norepinephrine (NE), and H2O2 results in distinct PKCδ phosphorylation patterns in cardiomyocytes. Fig. 1 extends these studies to examine PKCδ regulation by endothelin-1 (ET-1) and PDGF. Fig. 1A shows that PKCδ is recovered from resting cardiomyocytes with little-to-no Thr505 or Tyr311 phosphorylation. NE and ET-1 increase PKCδ phosphorylation at Thr505. In each case, PKCδ-Thr505 phosphorylation is maximal at 5 min (the first time point sampled in the experiments) and sustained for at least another 10 min of continuous stimulation (Fig. 1B). NE- and ET-1-dependent increases in PKCδ-Thr505 phosphorylation are similar in magnitude, and comparable with the stimulatory effect of PMA (Fig. 1, A and C). We previously reported that PMA induces a rapid and sustained increase in PKCδ-Thr505 phosphorylation that falls only as PKCδ protein abundance decreases due to down-regulation during chronic stimulation (2Rybin V.O. Sabri A. Short J. Braz J.C. Molkentin J.D. Steinberg S.F. J. Biol. Chem. 2003; 278: 14555-14564Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Fig. 1B shows that the NE-dependent increase in PKCδ-Thr505 phosphorylation also is sustained for at least 60 min, whereas the ET-1 response wanes with stimulation intervals greater than 30 min. PDGF also increases PKCδ-Thr505 phosphorylation, but this response is quite modest in magnitude when compared with the considerably more robust increases in PKCδ-Thr505 phosphorylation induced by NE, ET-1, or PMA. PDGF-dependent PKCδ-Thr505 phosphorylation is detected at 5 min and wanes as the stimulation interval is prolonged to >30 min (Fig. 1, B and C). PDGF increases PKCδ phosphorylation at Tyr311, whereas NE and ET-1 treatments for 5 min (Fig. 1A) or selected time points up to 60 min of incubation (Ref. 8Rybin V.O. Guo J. Sabri A. Elouardighi H. Schaefer E. Steinberg S.F. J. Biol. Chem. 2004; 279: 19350-19361Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar and data not shown) do not lead to a detectable increase in PKCδ-Tyr311 phosphorylation. The magnitude of the PDGF-dependent increase in PKCδ-Tyr311 phosphorylation is comparable with the stimulatory effect of PMA (Fig. 1, A and C). However, PDGF and PMA responses are not necessarily mediated by the identical signaling mechanism. Using an anti-Src-Tyr(P)416 PSSA that selectively recognizes the activation loop phosphorylated/activated forms of Src and related SFKs, Fig. 1B shows that PDGF induces a modest increase in Src activity. In contrast, PMA promotes PKCδ-Tyr311 phosphorylation without increasing Src-Tyr416 phosphorylation (Fig. 1B and Ref. 8Rybin V.O. Guo J. Sabri A. Elouardighi H. Schaefer E. Steinberg S.F. J. Biol. Chem. 2004; 279: 19350-19361Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Finally, Fig. 1B shows that H2O2 induces a robust increase in PKCδ-Tyr311 phosphorylation (to a level that exceeds PKCδ-Tyr311 phosphorylation in PDGF- or PMA-treated cardiomyocytes) and that the H2O2 response is associated with a massive increase in Src-Tyr416 phosphorylation. PKCδ partitions between the soluble and particulate fractions of resting cardiomyocytes (Fig. 1D). PMA, NE, and ET-1 translocate PKCδ protein from the soluble to the particulate fraction and increase PKCδ-Thr505 phosphorylation exclusively in the particulate fraction (Fig. 1D and data not shown). The PMA-dependent increase in PKCδ-Tyr311 phosphorylation also is detected exclusively in the particulate fraction. In contrast, treatment with H2O2 (which does not translocate PKCδ to the particulate fraction, but rather releases PKCδ from the particulate fraction) leads to an increase in PKCδ-Tyr311 phosphorylation in both the soluble and particulate fractions. Fig. 1D also shows that H2O2 does not increase PKCδ-Thr505 phosphorylation and NE does not increase PKCδ-Tyr311 phosphorylation. Tyr332 has been identified as another major phosphorylation site on heterologously overexpressed PKCδ in H2O2-treated COS-7 cells (16Konishi H. Yamauchi E. Taniguchi H. Yamamoto T. Matsuzaki H. Takemura Y. Ohmae K. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6587-6592Crossref PubMed Scopus (215) Google Scholar). Because preliminary studies demonstrated that the anti-PKCδ-Tyr(P)332 antibody used in our studies is not sufficiently sensitive or specific to be used directly in immunoblotting studies on cell lysates, PKCδ was immunoprecipitated from resting and agonist-treated cardiomyocyte cultures followed by immunoblot analysis with anti-PKCδ-Tyr(P)332, anti-PKCδ-Tyr(P)311, a general anti-Tyr(P) antibody (to track total PKCδ tyrosine phosphorylation), and an anti-PKCδ protein antibody (to validate equal protein recovery and loading). Fig. 2A shows that high concentrations of H2O2 promote PKCδ-Tyr311 and -Tyr332 phosphorylation in association with an increase in Src-Tyr(P)416 SFKs or increasing PKCδ-Tyr332 phosphorylation; NE does not increase Src phosphorylation at Tyr416 or PKCδ phosphorylation at either tyrosine residue. We previously used an immunoprecipitation strategy with antibodies that discriminate individual SFKs (Src, Lyn, and Fyn) followed by immunoblotting with the anti-Src-Tyr(P)416 PSSA to compare the time course for Src activation and PKCδ-Tyr311 phosphorylation. These previous studies established that Src activation precedes PKCδ-Tyr311 phosphorylation; H2O2 induces a similar rapid and robust increase in Src, Lyn, and Fyn activity that is maximal by 2 min, whereas the H2O2-dependent increase in PKCδ-Tyr311 phosphorylation is detectable at 2 min and increases further as the incubation interval is prolonged to 5–30 min (8Rybin V.O. Guo J. Sabri A. Elouardighi H. Schaefer E. Steinberg S.F. J. Biol. Chem. 2004; 279: 19350-19361Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Fig. 2B uses a similar strategy to compare the H2O2 requirements for SFK activation and PKCδ-Tyr311/Tyr332 phosphorylation. These studies show that high H2O2 concentrations (5 mm, a level of oxidative stress that typically leads to cellular necrosis) activate Src, Lyn, and Fyn, whereas lower H2O2 concentrations (0.1–1 mm) do not detectably increase Src, Lyn, or Fyn activity (although control experiments show that 0.1–1 mm H2O2 activates ERK and previous literature links low H2O2 concentrations to changes in gene expression, cardioprotection, and at least some features of the cardiac hypertrophic response (17Kemp T.J. Causton H.C. Clerk A. Biochem. Biophys. Res. Commun. 2003; 307: 416-421Crossref PubMed Scopus (57) Google Scholar, 18Kwon S.H. Pimentel D.R. Remondino A. Sawyer D.B. Colucci W.S. J. Mol. Cell Cardiol. 2003; 35: 615-621Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar)). The steep concentration-response curves for H2O2-dependent SFK activation identified in these experiments suggest an amplification mechanism. The results could reflect oxidative-inactivation of an antioxidant enzyme or an H2O2 response localized to mitochondria (where oxidative stress responses are amplified as a result of ROS-induced ROS release once H2O2 exceeds a threshold concentration (19Georgiou G. Masip L. Science. 2003; 300: 592-594Crossref PubMed Scopus (102) Google Scholar, 20Zorov D.B. Juhaszova M. Sollott S.J. Biochim. Biophys. Acta. 2006; 1757: 509-517Crossref PubMed Scopus (810) Google Scholar)). Finally, we used a pharmacologic approach as an initial strategy to identify the kinase pathway(s) that promote PKCδ-Tyr311 and -Tyr332 phosphorylation. Fig. 2, A and C, show that agonist-dependent PKCδ-Tyr311 and -Tyr332 (and Src-Tyr416) phosphorylation are fully abrogated by PP1. Although a recent study attributed H2O2-dependent PKCδ-Tyr332 phosphorylation to the epidermal growth factor receptor (which is recovered in a multiprotein complex with PKCδ from H2O2-treated COS-7 cells (21Morita M. Matsuzaki H. Yamamoto T. Fukami Y. Kikkawa U. J. Biochem. (Tokyo). 2008; 143: 31-38Crossref PubMed Scopus (23) Google Scholar)), agonist-dependent PKCδ-Tyr311 and -Tyr332 phosphorylation is preserved in cardiomyocytes treated with AG1478 (an epidermal growth factor receptor inhibitor, Fig. 2C); control experiments validate the efficacy of AG1478 treatment showing that AG1478 abrogates epidermal growth factor-dependent activation of ERK (Fig. 2C). Agonist-dependent PKCδ-Tyr311 and -Tyr332 phosphorylation is also preserved in cardiomyocytes treated with GF109203X (a general PKC inhibitor) or PP3 (a structurally similar, but functionally inactive, PP1 analogue that serves as a negative control; Fig. 2A and data not shown). These results implicate Src or a related PP1-sensitive SFK in the PKCδ-Tyr311 and -Tyr332 phosphorylation pathway in cardiomyocytes. This conclusion gains further support from studies in SYF cells (that lack the major SFKs, Src, Yes, and Fyn) and Src+ cells (SYF cells engineered to re-express Src). In SYF cells, a high concentration of H2O2 leads to a robust increase in PKCδ tyrosine phosphorylation (identified with anti-PKCδ-Tyr(P)332 antibodies in PKCδ pull-downs (Fig. 3A) and with anti-PKCδ-Tyr(P)311 in cell lysates (Fig. 3B)); PMA also induces a modest increase in PKCδ-Tyr311 phosphorylation in Src+ cells (Fig. 3B). In contrast, H2O2-dependent PKCδ-Tyr311 phosphorylation is detected only at very low levels, and PMA-dependent PKCδ-Tyr311 phosphorylation is not detected in SYF cells. Control experiments establish that the defect in PKCδ tyrosine phosphorylation in H2O2-treated SYF cells is not due to a generalized defect in H2O2-dependent responses, because H2O2-dependent activation of ERK is similar in SYF and Src+ cells. The observation that SYF cells support a low level of H2O2-dependent PKCδ-Tyr311 phosphory" @default.
• W2000263416 created "2016-06-24" @default.
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• W2000263416 date "2008-06-01" @default.
• W2000263416 modified "2023-10-14" @default.
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