Protein kinase C-{delta} regulates the subcellular localization of Shc in H2O2-treated cardiomyocytes

Abstract

Protein kinase C-δ (PKCδ) exerts important cardiac actions as a lipid-regulated kinase. There is limited evidence that PKCδ also might exert an additional kinase-independent action as a regulator of the subcellular compartmentalization of binding partners such as Shc (Src homologous and collagen), a family of adapter proteins that play key roles in growth regulation and oxidative stress responses. This study shows that native PKCδ forms complexes with endogenous Shc proteins in H(2)O(2)-treated cardiomyocytes; H(2)O(2) treatment also leads to the accumulation of PKCδ and Shc in a detergent-insoluble cytoskeletal fraction and in mitochondria. H(2)O(2)-dependent recruitment of Shc isoforms to cytoskeletal and mitochondrial fractions is amplified by wild-type-PKCδ overexpression, consistent with the notion that PKCδ acts as a signal-regulated scaffold to anchor Shc in specific subcellular compartments. However, overexpression studies with kinase-dead (KD)-PKCδ-K376R (an ATP-binding mutant of PKCδ that lacks catalytic activity) are less informative, since KD-PKCδ-K376R aberrantly localizes as a constitutively tyrosine-phosphorylated enzyme to detergent-insoluble and mitochondrial fractions of resting cardiomyocytes; relatively little KD-PKCδ-K376R remains in the cytosolic fraction. The aberrant localization and tyrosine phosphorylation patterns for KD-PKCδ-K376R do not phenocopy the properties of native PKCδ, even in cells chronically treated with GF109203X to inhibit PKCδ activity. Hence, while KD-PKCδ-K376R overexpression increases Shc localization to the detergent-insoluble and mitochondrial fractions, the significance of these results is uncertain. Our studies suggest that experiments using KD-PKCδ-K376R overexpression as a strategy to competitively inhibit the kinase-dependent actions of native PKCδ or to expose the kinase-independent scaffolding functions of PKCδ should be interpreted with caution.

Fig. 1.

Protein kinase C-δ (PKCδ) coprecipitates with Shc (Src homologous and collagen) in H₂O₂-treated cardiomyocytes. Cardiomyocytes were treated for 15 min with vehicle, H₂O₂ (5 mM, unless indicated otherwise), or PMA (300 nM). Stimulation followed a 45-min pretreatment with vehicle, GF109203X (GFX; 5 μM), or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1; 10 μM) in B and C. Extracts were immunoprecipitated with anti-Shc (A and B) or anti-PKCδ (C), and equal amounts of protein were subjected to immunoblotting for PKCδ and Shc (to verify equal protein recovery/loading and to track PKCδ-Shc complex formation) as well as other antibodies indicated in individual panels. A white space in B denotes where data from two regions of a single gel were merged for purposes of presentation. In C, NS indicates a nonspecific immnunoreactive band that migrates between p52Shc and p66Shc. All results were replicated in at least 4 independent experiments. Results for H₂O₂-dependent PKCδ-Shc complex formation are quantified in A (n = 4; *P < 0.05). SOS1, Son of Sevenless 1.

Fig. 2.

PKCδ and Shc accumulate in a detergent-insoluble cytoskeletal fraction that is enriched in intermediate filament proteins in H₂O₂-treated cardiomyocytes. Cardiomyocytes were treated for 15 min with vehicle, the indicated concentration of H₂O₂, 1 μM norepinephrine (NE), or 100 nM EGF. Stimulation followed a 45-min pretreatment with vehicle or PP1 (10 μM) in C. Extracts were partitioned into detergent-soluble and detergent-insoluble fractions and subjected to immunoblotting with the indicated antibodies according to protocols described in materials and methods. Vehicle-treated samples were used for immunoblotting experiments in B. The white spaces in A and C show where data from two regions of a single gel were merged for purposes of presentation. Similar results were obtained in 3 separate experiments.

Fig. 3.

Wild-type (WT)-PKCδ and kinase-dead (KD)-PKCδ localize in, and recruit Shc to, the detergent-insoluble fraction. Adenoviral-mediated gene transfer was used to overexpress WT-PKCδ, KD-PKCδ, or β-galactosidase (β-Gal) as control (multiplicities of infection: 100 plaque-forming units/cell). A: forty-eight hours postinfection, cells were challenged for 15 min with vehicle, 300 nM PMA, or 5 mM H₂O₂ and then partitioned into detergent-soluble and detergent-insoluble fractions. WT-PKCδ and KD-PKCδ overexpression does not lead to any gross change in protein partitioning between detergent-soluble and detergent-insoluble fractions. B: immunoblotting on whole cell lysates or subcellular fractions was performed as described in materials and methods. The dashed lines in B denote where data from different regions of a single gel were merged for purposes of presentation. Similar results were obtained in 5 separate experiments.

Fig. 4.

The properties of KD-PKCδ (a constitutively tyrosine-phosphorylated enzyme that localizes to the detergent-insoluble fraction) do not phenocopy native PKCδ lacking catalytic activity. A: adenoviral-mediated gene transfer was used to overexpress WT-PKCδ or β-galactosidase as control. Cultures were treated with vehicle, GF109203X (GFX; 5 μM for 24 or 48 h), or H₂O₂ (5 mM for 15 min); stimulations were staggered, so that all stimulations were terminated 96 h postinfection. B: cultures were treated with vehicle, 300 nM PMA, or 300 nM PMA plus 5 μM GF109203X for 24 h and were then subjected to an acute 15-min challenge with vehicle or 5 mM H₂O₂. Immunoblotting was performed on whole cell lysates or subcellular fractions with antibodies that recognize PKCδ protein and PKCδ-Y³¹¹ phosphorylation according to protocols described in materials and methods. The results were replicated in 2 separate experiments.

Fig. 5.

PKCδ and Shc accumulate in the mitochondrial fractions of H₂O₂-treated cardiomyocytes. Cardiomyocytes were treated for 15 min with vehicle or 5 mM H₂O₂, partitioned into cytosolic, plasma membrane, and mitochondrial fractions, and then subjected to immunoblot analysis with the indicated antibodies according to protocols described in materials and methods. The white space in one panel (depicting immunoblotting for GM130) denotes merging of two regions of a single gel for presentation purposes. Similar results were obtained in 4 separate experiments.

Fig. 6.

WT-PKCδ and KD-PKCδ overexpression enhances Shc localization to the mitochondrial fraction. Adenoviral-mediated gene transfer was used to overexpress WT-PKCδ, KD-PKCδ, or β-galactosidase. Forty-eight hours postinfection, cells were challenged for 15 min with vehicle or 5 mM H₂O₂ and were then subjected to extraction and differential centrifugation to separate cytosolic and mitochondrial fractions. Immunoblotting with the indicated antibodies was performed as described in materials and methods. The data are representative of results in 4 separate experiments.