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J. Biol. Chem., Vol. 283, Issue 14, 9217-9223, April 4, 2008

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Phosphoinositide Binding to the Substrate Regulates Susceptibility to Proteolysis by Calpain^*

From the Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331

Calpain-mediated proteolysis regulates cytoskeletal dynamics and is altered during aging and the progression of numerous diseases or pathological conditions. Although several cytoskeletal proteins have been identified as substrates, how localized calpain activity is regulated and the mechanisms controlling substrate recognition are not clear. In this study, we report that phosphoinositide binding regulates the susceptibility of the cytoskeletal adhesion protein -actinin to proteolysis by calpains 1 and 2. At first, -actinin did not appear to be a substrate for calpain 2; however, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃) binding to -actinin resulted in nearly complete proteolysis of the full-length protein, producing stable breakdown products. Calpain 1 was able to cleave -actinin in the absence of phosphoinositide binding; however, PtdIns(3,4,5)P₃ binding increased the rate of proteolysis, and phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P₂) binding significantly inhibited cleavage. Phosphoinositide binding appeared to regulate calpain proteolysis of -actinin by modulating the exposure of a highly sensitive cleavage site within the calponin homology 2 domain. In U87MG glioblastoma cells, which contain elevated levels of PtdIns(3,4,5)P₃, -actinin colocalized with calpain within dynamic actin cytoskeletal structures. Furthermore, proteolysis of -actinin producing stable breakdown products was observed in U87MG cells treated with calcium ionophore to activate the calcium-dependent calpains. Additional evidence of PtdIns(3,4,5)P₃-mediated calpain proteolysis of -actinin was observed in rat embryonic fibroblasts. These results suggest that PtdIns(3,4,5)P₃ binding is a critical determinant for -actinin proteolysis by calpain. In conclusion, phosphoinositide binding to the substrate is a potential mechanism for regulating susceptibility to proteolysis by calpain.

Received for publication, September 5, 2007 , and in revised form, January 9, 2008.

^* This work was supported in part by NIGMS Grant GM 63711 (to J. A. G.) and by NIEHS Grant P30 ES00210 (to the Cell Imaging and Analysis Facility and Services Core of the Environmental Health Sciences Center at Oregon State University) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by a summer undergraduate research fellowship funded by Howard Hughes Medical Institute Grant 52003741 to Oregon State University.

² To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, ALS 2011, Oregon State University, Corvallis, OR 97331. Tel.: 541-737-4997; Fax: 541-737-0481; E-mail: jeffrey.greenwood{at}orst.edu.

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