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Activation of m-calpain (calpain II) by epidermal growth factor is limited by protein kinase A phosphorylation of m-calapin

Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Molecular and Cellular Biology (Impact Factor: 5.04). 05/2002; 22(8):2716-27. DOI: 10.1128/MCB.22.8.2716-2727.2002
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ABSTRACT 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.

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    • "The A-isomer has been shown to induce migration and proliferation, and the B-isomer to inhibit migration and proliferation (Lasagni et al., 2003). In fibroblasts, this receptor actuates the cAMP-PKA pathway, which results in negative phosphorylation of m-calpain to prevent growth-factorinduced motility (Shiraha et al., 2002). In keratinocytes, CXCR3 is linked to activation of PLCβ to induce a calcium-flux-mediated activation of μ-calpain enabling greater motility (Satish et al., 2005). "
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