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Receptor-independent cardiac protein kinase Calpha activation by calpain-mediated truncation of regulatory domains.

ABSTRACT: Protein kinase (PK)Cs and calpain cysteine proteases are highly expressed in myocardium. Ischemia produces calcium overload that activates calpains and conventional PKCs. However, calpains can proteolytically process PKCs, and the potential in vivo consequences of this interaction are unknown.To determine the biochemical and pathophysiological consequences of calpain-mediated cardiac PKC? proteolysis.Isolated mouse hearts subjected to global ischemia/reperfusion demonstrated cleavage of PKC?. Calpain 1 overexpression was not sufficient to produce PKC? cleavage in normal hearts, but ischemia-induced myocardial PKC? cleavage and myocardial injury were greatly increased by cardiac-specific expression of calpain 1. In contrast, calpain 1 gene ablation or inhibition with calpastatin prevented ischemia/reperfusion induced PKC? cleavage; infarct size was decreased and ventricular function enhanced in infarcted calpain 1 knockout hearts. To determine consequences of PKC? fragmentation on myocardial protein phosphorylation, transgenic mice were created conditionally expressing full-length PKC? or its N-terminal and C-terminal calpain 1 cleavage fragments. Two-dimensional mapping of ventricular protein extracts showed a distinct PKC? phosphorylation profile that was exaggerated and distorted in hearts expressing the PKC? C-terminal fragment. MALDI mass spectroscopy revealed hyperphosphorylation of myosin-binding protein C and phosphorylation of atypical substrates by the PKC? C-terminal fragment. Expression of parent PKC? produced a mild cardiomyopathy, whereas myocardial expression of the C-terminal PKC? fragment induced a disproportionately severe, rapidly lethal cardiomyopathy.Proteolytic processing of PKC? by calcium-activated calpain activates pathological cardiac signaling through generation of an unregulated and/or mistargeted kinase. Production of the PKC? C-terminal fragment in ischemic hearts occurs via a receptor-independent mechanism.

SUBMITTER: Kang MY

PROVIDER: S-EPMC2948630 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Receptor-independent cardiac protein kinase Calpha activation by calpain-mediated truncation of regulatory domains.

Kang Min-Young MY Zhang Yan Y Matkovich Scot J SJ Diwan Abhinav A Chishti Athar H AH Dorn Gerald W GW

Circulation research 20100805 7

<h4>Rationale</h4>Protein kinase (PK)Cs and calpain cysteine proteases are highly expressed in myocardium. Ischemia produces calcium overload that activates calpains and conventional PKCs. However, calpains can proteolytically process PKCs, and the potential in vivo consequences of this interaction are unknown.<h4>Objective</h4>To determine the biochemical and pathophysiological consequences of calpain-mediated cardiac PKCα proteolysis.<h4>Methods and results</h4>Isolated mouse hearts subjected ...[more]

PMID: 20689063

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Project description:We have shown previously that the ELR-negative CXC chemokines interferon-inducible protein 10, monokine induced by gamma interferon, and platelet factor 4 inhibit epidermal growth factor (EGF)-induced m-calpain activation and thereby EGF-induced fibroblast cell motility (H. Shiraha, A. Glading, K. Gupta, and A. Wells, J. Cell Biol. 146:243-253, 1999). However, how this cross attenuation could be accomplished remained unknown since the molecular basis of physiological m-calpain regulation is unknown. As the initial operative attenuation signal from the CXCR3 receptor was cyclic AMP (cAMP), we verified that this second messenger blocked EGF-induced motility of fibroblasts (55% +/- 4.5% inhibition) by preventing rear release during active locomotion. EGF-induced calpain activation was inhibited by cAMP activation of protein kinase A (PKA), as the PKA inhibitors H-89 and Rp-8Br-cAMPS abrogated cAMP inhibition of both motility and calpain activation. We hypothesized that PKA might negatively modulate m-calpain in an unexpected manner by directly phosphorylating m-calpain. A mutant human large subunit of m-calpain was genetically engineered to negate a putative PKA consensus sequence in the regulatory domain III (ST369/370AA) and was expressed in NR6WT mouse fibroblasts to represent about 30% of total m-calpain in these cells. This construct was not phosphorylated by PKA in vitro while a wild-type construct was, providing proof of the principle that m-calpain can be directly phosphorylated by PKA at this site. cAMP suppressed EGF-induced calpain activity of cells overexpressing a control wild-type human m-calpain (83% +/- 3.7% inhibition) but only marginally suppressed that of cells expressing the PKA-resistant mutant human m-calpain (25% +/- 5.5% inhibition). The EGF-induced motility of the cells expressing the PKA-resistant mutant also was not inhibited by cAMP. Structural modeling revealed that new constraints resulting from phosphorylation at serine 369 would restrict domain movement and help "freeze" m-calpain in an inactive state. These data point to a novel mechanism of negative control of calpain activation, direct phosphorylation by PKA.

Dataset Information

Receptor-independent cardiac protein kinase Calpha activation by calpain-mediated truncation of regulatory domains.

Publications

Receptor-independent cardiac protein kinase Calpha activation by calpain-mediated truncation of regulatory domains.

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