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Molecular and Cellular Biology, April 2002, p. 2716-2727, Vol. 22, No. 8
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.8.2716-2727.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain

Hidenori Shiraha,1 Angela Glading,1 Jeffrey Chou,1 Zongchao Jia,2 and Alan Wells1*

Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261,1 Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N62

Received 10 July 2001/ Returned for modification 20 August 2001/ Accepted 4 January 2002

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.

* Corresponding author. Mailing address: Department of Pathology, University of Pittsburgh, S713 Scaife, Terrace and Lothrop Street, Pittsburgh, PA 15261. Phone: (412) 624-0973. Fax: (412) 647-8567. E-mail: wells{at}msx.upmc.edu.

Molecular and Cellular Biology, April 2002, p. 2716-2727, Vol. 22, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.8.2716-2727.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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