Phorbol 12-myristate 13-acetate-dependent protein kinase C delta-Tyr311 phosphorylation in cardiomyocyte caveolae

Abstract

Protein kinase Cdelta (PKCdelta) activation is generally attributed to lipid cofactor-dependent allosteric activation mechanisms at membranes. However, recent studies indicate that PKCdelta also is dynamically regulated through tyrosine phosphorylation in H(2)O(2)- and phorbol 12-myristate 13-acetate (PMA)-treated cardiomyocytes. H(2)O(2) activates Src and related Src-family kinases (SFKs), which function as dual PKCdelta-Tyr(311) and -Tyr(332) kinases in vitro and contribute to H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation in cardiomyocytes and in mouse embryo fibroblasts. H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation is defective in SYF cells (deficient in SFKs) and restored by Src re-expression. PMA also promotes PKCdelta-Tyr(311) phosphorylation, but this is not associated with SFK activation or PKCdelta-Tyr(332) phosphorylation. Rather, PMA increases PKCdelta-Tyr(311) phosphorylation by delivering PKCdelta to SFK-enriched caveolae. Cyclodextrin treatment disrupts caveolae and blocks PMA-dependent PKCdelta-Tyr(311) phosphorylation, without blocking H(2)O(2)-dependent PKCdelta-Tyr(311) phosphorylation. The enzyme that acts as a PKCdelta-Tyr(311) kinase without increasing PKCdelta phosphorylation at Tyr(332) in PMA-treated cardiomyocytes is uncertain. Although in vitro kinase assays implicate c-Abl as a selective PKCdelta-Tyr(311) kinase, PMA-dependent PKCdelta-Tyr(311) 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 PKCdelta to caveolae and increase PKCdelta-Tyr(311) 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 PKCdelta exclusively in non-caveolae membranes. Collectively, these results identify stimulus-specific PKCdelta localization and tyrosine phosphorylation mechanisms that could be targeted for therapeutic advantage.

FIGURE 1.

Agonist-specific differences in PKCδ localization and phosphorylation mechanisms in cardiomyocytes. Immunoblot analysis of cell extracts (panels A and B) or soluble and particulate fractions (panel D) from cells treated with NE (1 μm), ET-1 (100 nm), PDGF (50 ng/ml), H₂O₂ (5 mm), or PMA (300 nm). Stimulations were for 5 min unless indicated otherwise. All samples in panel B are from a single experiment. Agonist-dependent increases in PKCδ-Thr⁵⁰⁵ and -Tyr³¹¹ phosphorylation at 5 (n = 7) and 30 min (n = 3) are quantified in panel C (mean ± S.E., *, p < 0.05 compared with basal; ‡, p < 0.05 compared with H₂O₂).

FIGURE 2.

The role of Src family kinases in PKCδ-Tyr³¹¹ and -Tyr³³² phosphorylation mechanisms in cardiomyocytes. Cardiomyocytes were treated with NE (1 μm), H₂O₂ (5 mm, unless indicated otherwise), PMA (300 nm), or EGF (100 nm), with agonist stimulations preceded by a 45-min pretreatment interval with vehicle, GF109203X (GFX, 5 μm, in panel A), PP1 (10 μm, in panels A and C), or AG1478 (10 μm, in panel C) as indicated. Immunoblot analysis of Src-Tyr(P)⁴¹⁶ in panel A and pERK or ERK in panels B and C was on cell extracts. An anti-PKCδ protein antibody (Santa Cruz Biotechnology) was used for immunoprecipitation (IP) experiments in panels A and C. Antibodies that selectively recognize Src (Oncogene), Lyn (Santa Cruz Biotechnology), or Fyn (Santa Cruz Biotechnology) were used to immunoprecipitate individual SFKs (according to conditions that essentially clear SFK proteins from the cellular extracts) followed by immunoblotting (IB) with anti-Src-Tyr(P)⁴¹⁶ PSSA (as a surrogate measure of enzyme activity) and antibodies that detect the individual SFK proteins to ensure equivalent protein recovery and loading on the gel in panel B. Effects of 5 mm H₂O₂ or PMA on Src, Lyn, or Fyn activity are quantified in panel B (mean ± S.E., n = 6, *, p < 0.05). All other results were replicated in at least three separate experiments on separate culture preparations.

FIGURE 3.

H₂O₂- and PMA increase PKCδ tyrosine phosphorylation in Src⁺ cells, but not in SYF cells that lack Src, Yes, and Fyn expression. Panel A, SYF and Src⁺ cultures were treated with vehicle or H₂O₂ (alone or following a pretreatment with 10 μm PP1) followed by immunoprecipitation (IP) of PKCδ and immunoblotting (IB) for PKCδ-Tyr(P)³³² and overall tyrosine phosphorylation. Panel B, SYF and Src⁺ cultures were treated with vehicle, and the indicated concentrations of H₂O₂ or PMA (300 nm). Immunoblotting was on cell extracts with the indicated antibodies. Panel C, immunoblotting to compare Lyn protein and SFK activity (Src-Tyr(P)⁴¹⁶ immunoreactivity) in soluble and detergent-insoluble fractions from SYF cells, Src⁺ cells, and cardiomyocytes cultures (CM). Results were replicated in three separate experiments on separate culture preparations.

FIGURE 4.

PMA-dependent PKCδ-Tyr³¹¹ phosphorylation is confined to caveolae (and is disrupted by treatment with cyclodextrin); H₂O₂ does not drive PKCδ to caveolae; H₂O₂ increases PKCδ-Tyr³¹¹ phosphorylation in both caveolae and F8–13 fractions. Panels A–D, cardiomyocyte cultures were treated for 20 min with 300 nm PMA without or with a 45-min pretreatment with GF109203X (GFX, 5 μm), PP1 (10 μm), or Gö6976 (5 μm) or with 5 mm H₂O₂ as indicated. Caveolae membranes (Cav) were separated from heavy gradient fractions (F8-13) and the insoluble pellet (P) and samples were subjected to immunoblotting with the indicated antibodies as described under “Experimental Procedures.” Some variability in the detection of PKCδ-Tyr(P)³¹¹ immunoreactivity in the F8–13 fractions between experiments (compare panels A and B) is attributable to differences in protein loading and gel exposure time. Panel E, immunoblotting on cell extracts from cardiomyocyte cultures incubated for 1 h at 37 °C in SFM containing vehicle, 2% cyclodextrin, or 2% cyclodextrin complexed with 1.3 mm cholesterol and then challenged with PMA or H₂O₂ as indicated. For each panel, the results were replicated in three to six separate experiments on separate culture preparations.

FIGURE 5.

In vitro kinase assays showing PKCδ phosphorylation by Src family kinases, PDGFR, FAK, and JAK2. Panels A and C, in vitro kinase assays were performed with PKCδ and active Src, Lyn, Fyn, Yes, PDGFR, FAK, or JAK2 according “Experimental Procedures.” Proteins were separated by SDS-PAGE and subjected to autoradiography and immunoblotting for PKCδ protein and phosphorylation. Panel B, cardiomyocyte cultures were treated for 10 min with H₂O₂ (5 mm) without or with a 45-min pretreatment with PP1 (10 μm). Caveolae membranes were prepared and subjected to immunoblotting with the indicated antibodies according “Experimental Procedures.” All results were replicated in two or three separate experiments.

FIGURE 6.

c-Abl selectively phosphorylates PKCδ at Tyr³¹¹, but c-Abl inhibition does not prevent PMA-dependent PKCδ-Tyr³¹¹ phosphorylation. Panel A, in vitro kinase assays were performed with PKCδ and active Src or c-Abl without and with PS alone (112 μm), PS plus PMA (175 nm), PS plus DAG (7.2 μm), or PS plus diC8 (7.2 μm). Proteins were separated by SDS-PAGE and subjected to immunoblotting for PKCδ protein and phosphorylation. Panel B, PKCδ was incubated in a kinase buffer containing [³²P]ATP without and with active Src or c-Abl and PKCδ phosphorylation was tracked by MALDI-TOF mass spectroscopy according “Experimental Procedures.” Panel C, immunoblotting for c-Abl and Src protein partitioning in caveolae membranes and the F8–13 heavy gradient fractions; immunoblotting for caveolin-3 is included to validate the preparation. Panel D, cardiomyocyte cultures were pretreated for 45 min with PP1 (10 μm) or AG1295 (25 μm) or for 2 h with ST1571 (5 mm) prior to stimulation for 10 min with PDGF (50 ng/ml), PMA (300 nm), or H₂O₂ (5 mm) as indicated. Immunoblotting was on cell lysates with the indicated antibodies according “Experimental Procedures.”

FIGURE 7.

Norepinephrine and endothelin-1 do not drive PKCδ to caveolae or increase PKCδ-Tyr³¹¹ phosphorylation. Agonist treatments were according to protocols described in the legend to Fig. 1. Stimulations were preceded by a 30-min pretreatment with vehicle or U73122 (5 μm) in panel B. Immunoblotting was on caveolae and other gradient fractions (panel A) or cell extracts (panel B). Results were replicated in three separate experiments on different culture preparations.

FIGURE 8.

DAG analogues mimic the effect of PMA to drive PKCδ to caveolae and increase PKCδ-Tyr³¹¹ phosphorylation. Incubations were for 20 min with 300 nm PMA or 58 μm diC8 and immunoblotting was on soluble and particulate fractions (panel A) or caveolae and other gradient fractions (panel B) with the indicated antibodies. Results were replicated in three separate experiments on different culture preparations.