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Molecular and Cellular Biology, July 2006, p. 5481-5496, Vol. 26, No. 14
0270-7306/06/$08.00+0     doi:10.1128/MCB.02243-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Spatial Localization of m-Calpain to the Plasma Membrane by Phosphoinositide Biphosphate Binding during Epidermal Growth Factor Receptor-Mediated Activation{dagger}

Hanshuang Shao,1 Jeff Chou,1,{ddagger} Catherine J. Baty,2 Nancy A. Burke,2 Simon C. Watkins,2 Donna Beer Stolz,2 and Alan Wells1*

Departments of Pathology,1 Cell Biology & Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 152132

Received 21 November 2005/ Returned for modification 22 December 2005/ Accepted 26 April 2006

Calpain activity is required for de-adhesion of the cell body and rear to enable productive locomotion of adherent cells during wound repair and tumor invasion. Growth factors activate m-calpain (calpain 2, CAPN2) via ERK/mitogen-activated protein kinases, but only when these kinases are localized to the plasma membrane. We thus hypothesized that m-calpain is activated by epidermal growth factor (EGF) only when it is juxtaposed to the plasma membrane secondary to specific docking. Osmotic disruption of NR6 fibroblasts expressing the EGF receptor demonstrated m-calpain being complexed with the substratum-adherent membrane with this increasing in an EGF-dependent manner. m-Calpain colocalized with phosphoinositide biphosphate (PIP2) with exogenous phospholipase C removal of phosphoinositides, specifically, PI(4,5)P2 but not PI(4)P1 or PIP3, releasing the bound m-calpain. Downregulation of phosphoinositide production by 1-butanol resulted in diminished PIP2 in the plasma membrane and eliminated EGF-induced calpain activation. This PIP2-binding capacity resided in domain III of calpain, which presents a putative C2-like domain. This active conformation of this domain appears to be partially masked in the holoenzyme as both activation of m-calpain by phosphorylation at serine 50 and expression of constitutively active phosphorylation mimic glutamic acid-increased m-calpain binding to the membrane, consistent with blockade of this cascade diminishing membrane association. Importantly, we found that m-calpain was enriched toward the rear of locomoting cells, which was more pronounced in the plasma membrane footprints; EGF further enhanced this enrichment, in line with earlier reports of loss of PIP2 in lamellipodia of motile cells. These data support a model of m-calpain binding to PIP2 concurrent with and likely to enable ERK activation and provides a mechanism by which cell de-adhesion is directed to the cell body and tail as phospholipase C-{gamma} hydrolyzes PIP2 in the protruding lamellipodia.

* Corresponding author. Mailing address: Department of Pathology, University of Pittsburgh, Scaife Hall, S-711, 3550 Terrace St., Pittsburgh, PA 15261. Phone: (412) 624-0973. Fax: (412) 624-8946. E-mail: wellsa{at}upmc.edu.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

{ddagger} Present address: Fred Hutchinson Cancer Center, Seattle, Wash.

Molecular and Cellular Biology, July 2006, p. 5481-5496, Vol. 26, No. 14
0270-7306/06/$08.00+0     doi:10.1128/MCB.02243-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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