Protein kinase C-{delta} regulates the subcellular localization of Shc in H2O2-treated cardiomyocytes.
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.
Guo
Jianfen
J
Department of Pharmacology, College of Physicians and Surgeons, Columbia Univ., 630 West 168 St., New York, NY 10032, USA.
Cong
Lin
L
Rybin
Vitalyi O
VO
Gertsberg
Zoya
Z
Steinberg
Susan F
SF
D000818:Animals; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress; D051745:Protein Kinase C-delta / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D016877:Oxidants; D017382:Reactive Oxygen Species; D055765:Shc Signaling Adaptor Proteins; D006861:Hydrogen Peroxide; D051745:Protein Kinase C-delta