Tyrosine phosphorylation modifies protein kinase C delta-dependent phosphorylation of cardiac troponin I

Abstract

Our study identifies tyrosine phosphorylation as a novel protein kinase Cdelta (PKCdelta) activation mechanism that modifies PKCdelta-dependent phosphorylation of cardiac troponin I (cTnI), a myofilament regulatory protein. PKCdelta phosphorylates cTnI at Ser23/Ser24 when activated by lipid cofactors; Src phosphorylates PKCdelta at Tyr311 and Tyr332 leading to enhanced PKCdelta autophosphorylation at Thr505 (its activation loop) and PKCdelta-dependent cTnI phosphorylation at both Ser23/Ser24 and Thr144. The Src-dependent acquisition of cTnI-Thr144 kinase activity is abrogated by Y311F or T505A substitutions. Treatment of detergent-extracted single cardiomyocytes with lipid-activated PKCdelta induces depressed tension at submaximum but not maximum [Ca2+] as expected for cTnI-Ser23/Ser24 phosphorylation. Treatment of myocytes with Src-activated PKCdelta leads to depressed maximum tension and cross-bridge kinetics, attributable to a dominant effect of cTnI-Thr144 phosphorylation. Our data implicate PKCdelta-Tyr311/Thr505 phosphorylation as dynamically regulated modifications that alter PKCdelta enzymology and allow for stimulus-specific control of cardiac mechanics during growth factor stimulation and oxidative stress.

FIGURE 1.

Stimulus-dependent cTnI phosphorylation in cardiomyocytes. Adult ventricular myocytes were treated with vehicle (dimethyl sulfoxide (DMSO)), GF109203X (5 μm), or PP1 (10 μm) followed by H₂O₂ (5 mm) or PMA (100 nm) for 15 min. The gels were stained with Pro-Q Diamond (phosphoproteins stain) followed by Sypro Ruby (total protein stain), and the phospho/total protein ratios were determined (n = 4, means ± S.E.; ^*, p < 0.05). The results are normalized by setting the mean value for vehicle-treated samples to 100.

FIGURE 2.

PKCδ is recovered as a lipid-independent kinase from H₂O₂-treated cardiomyocytes. A, PKCδ immunoprecipitated from cardiomyocytes treated with vehicle (Me₂SO), PMA (100 nm), or H₂O₂ (5 mm), each for 15 min, was used in immune complex kinase assays with cTn complex as substrate (with 100 nm PMA and 5 μm GF109203X also included in kinase assays as indicated). Immunoblotting (IB) was used to validate equal PKCδ recovery from control and PMA- and H₂O₂-treated cultures and to show that PKCδ is tyrosine-phosphorylated in H₂O₂-treated cultures (and to a much lesser extent in PMA-treated cultures) but not in control cultures. The autoradiogram showing cTnI phosphorylation and quantification is from a representative experiment, with similar results obtained in separate experiments on four separate culture preparations. B, cardiomyocytes were treated with vehicle, 5 mm H₂O₂, or 5 mm H₂O₂ + 10 μm PP1 (each for 15 min); cell extracts were subjected to immunoblotting for PKCδ-Tyr(P)³¹¹ and Src-Tyr(P)⁴¹⁶ (with Src protein as a loading control, top right), and PKCδ pull-downs were immunoblotted for PKCδ-Tyr(P)³³² and Tyr(P) (with PKCδ as a loading control; the anti-PKCδ-Tyr(P)³³² antibody displays too much nonspecific immunoreactivity to be used directly on cell extracts, middle right). PKCδ in immune complexes also was used in kinase assays with cTn complex as substrate; a representative autoradiogram of cTnI phosphorylation is illustrated (bottom right). IP, immunoprecipitation.

FIGURE 3.

Src-dependent PKCδ phosphorylation maps to Tyr³¹¹ and Tyr³³² and leads to increased PKCδ-dependent cTnI phosphorylation. PKCδ was incubated in a kinase buffer containing [³²P]ATP without and with active Src and PKCδ phosphorylation was tracked by immunoblotting (A) and MALDI-TOF mass spectrometry (B) according to “Experimental Procedures.” C, in vitro kinase assays were performed with PKCδ in the absence or presence of Src, 100 nm PMA + 112 μm PS, and 5 μm GF109203X as indicated. Autoradiograms showing ³²P incorporation into PKCδ, Src, and cTnI are depicted (top left) with results for cTnI phosphorylation quantified (right). Immunoblotting (IB) with anti-Tyr(P) antibody shows Src autophosphorylation and Src-dependent tyrosine phosphorylation of PKCδ, but no cTnI tyrosine phosphorylation.

FIGURE 4.

Lipid-activated PKCδ is a cTnI-Ser²³/Ser²⁴ kinase; tyrosine-phosphorylated-PKCδ is a cTnI-Ser²³/Ser²⁴ kinase that also phosphorylates cTnI at Thr¹⁴⁴. A, schematic of WT-cTnI and cTnI mutants used in this study. B and C, in vitro kinase assays with PKCδ alone or PKCδ + Src performed with 112 μm PS + 175 nm PMA and cTnI complexes containing WT or mutant cTnIs as substrates. cTnI phosphorylation was quantified by PhosphorImager analysis (mean ± S.E., n = 3 top) and immunoblotting (IB) with anti-cTnI-Ser²³/Ser²⁴ and anti-pTXR (that recognizes cTnI-Thr¹⁴⁴ phosphorylation; representative experiment is depicted at the bottom). D, in vitro kinase assays with PKCδ alone or PKCδ + Src with complexes containing WT-cTnI and either 175 nm PMA or 7.2 μm 1,2-DAG (each with 112 μm PS) as lipid cofactor. PKCδ tyrosine (Tyr³¹¹ and Tyr³³²) was tracked by immunoblotting; cTnI phosphorylation was tracked by PhosphorImager analysis (top) and immunoblotting (bottom). Similar results were obtained in two additional separate experiments.

FIGURE 5.

cTnI phosphorylation by PKC isoforms, PKA, and PKD. In vitro kinase assays performed with PKCδ, PKCα, PKCβII, PKCε, or PKA alone or with Src as indicated are depicted in A; assays with PKD (alone or with Src or c-Abl) and control assays with Src and c-Abl alone are depicted in B. All of the reactions contained PS/PMA. Autoradiograms and anti-Tyr(P) blots showing the positions of the various enzymes is on top; cTnI phosphorylation quantified by PhosphorImager analysis (with a representative autoradiogram depicted) and immunoblotting for cTnI-Ser²³/Ser²⁴ and Thr¹⁴⁴ (detected as anti-pTXR) from a single experiments are illustrated at the bottom; the results were replicated in separate experiments.

FIGURE 6.

Tyr³¹¹ and Thr⁵⁰⁵ substitutions prevent Src-dependent changes in PKCδ substrate specificity. COS cells were transfected with plasmids that drive expression of WT and Y311F, Y332F, T505A, and T505E substituted forms of PKCδ fused to GFP. PKCδ was immunoprecipitated with anti-GFP, subjected to immunoblotting with anti-GFP to validate equal protein recovery (C), and subjected to immunocomplex kinase assays without and with lipid cofactor, Src, or c-Abl as indicated. cTnI phosphorylation was quantified by PhosphorImager (and is expressed relative to the basal level of phosphorylation for each PKCδ construct (A). PKCδ-Tyr³¹¹, -Tyr³³², and -Thr⁵⁰⁵ phosphorylation and cTnI-Ser²³/Ser²⁴ and Thr¹⁴⁴ phosphorylation were detected by immunoblot analysis according to the legend for B. The results were replicated in two separate experiments. D, a schematic that marks the various phosphorylation sites of PKCδ and the main kinases implicated in phosphorylating each site.

FIGURE 7.

Mechanical properties of skinned cardiomyocyte preparations before and after PKCδ treatments at maximal and submaximal [Ca²⁺]. The traces show the recovery of force following a mechanical detachment of cross-bridges, to drop force to near-zero values. A, top panel, representative records used for the determination of k_tr rates before (black) and after (red) PKCδ treatment demonstrate no change in rate of force recovery. Histograms (inset) summarize lack of effect of PKCδ on maximum Ca²⁺-activated parameters. A, bottom panel, force tracings demonstrate that Src (tyrosine-phosphorylated) PKCδ (blue) decreases the rate of recovery of force (i.e. return to equilibrium more slowly) and therefore k_tr. Histograms of maximum Ca²⁺-activated tension and k_tr values demonstrate significant decreases in these parameters in myocytes treated with Src (tyrosine-phosphorylated) PKCδ. B, top panel, tracings show no difference between control (black) and PKCδ (red) on the rate of force recovery at submaximal Ca²⁺, although submaximal tension production was significantly low (histogram). B, bottom panel, force tracings reflect a minor (although nearly significant p = 0.07) shift in force recovery with PKCδ pretreated with Src kinase (blue). In contrast to PKCδ alone, no significant change in submaximal tension production was observed (histogram, bottom). All of the measurements were performed in the presence of lipid activators PS/PMA. The results are reported as the means ± S.E. (^* indicates p < 0.05).