v-Src-Induced Modulation of the Calpain-Calpastatin Proteolytic System Regulates Transformation

Abstract

v-Src-induced oncogenic transformation is characterized by alterations in cell morphology, adhesion, motility, survival, and proliferation. To further elucidate some of the signaling pathways downstream of v-Src that are responsible for the transformed cell phenotype, we have investigated the role that the calpain-calpastatin proteolytic system plays during oncogenic transformation induced by v-Src. We recently reported that v-Src-induced transformation of chicken embryo fibroblasts is accompanied by calpain-mediated proteolytic cleavage of the focal adhesion kinase (FAK) and disassembly of the focal adhesion complex. In this study we have characterized a positive feedback loop whereby activation of v-Src increases protein synthesis of calpain II, resulting in degradation of its endogenous inhibitor calpastatin. Reconstitution of calpastatin levels by overexpression of exogenous calpastatin suppresses proteolytic cleavage of FAK, morphological transformation, and anchorage-independent growth. Furthermore, calpastatin overexpression represses progression of v-Src-transformed cells through the G₁ stage of the cell cycle, which correlates with decreased pRb phosphorylation and decreased levels of cyclins A and D and cyclin-dependent kinase 2. Calpain 4 knockout fibroblasts also exhibit impaired v-Src-induced morphological transformation and anchorage-independent growth. Thus, modulation of the calpain-calpastatin proteolytic system plays an important role in focal adhesion disassembly, morphological transformation, and cell cycle progression during v-Src-induced cell transformation.

We thank W. Clark (Beatson Institute for Cancer Research) for providing polyclonal antibody against chicken cyclin A; Masatoshi Maki (Nagoya University) for providing calpastatin cDNA; Val Fincham for providing v-Src constructs; Joe Winnie and Brad Ozanne for providing v-Fos-transformed cells; Clare Pollock and Walter Kolch for providing K-Ras-transformed cells; and Ann Mclaren, Carolyn Wiltshire, and David Gillespie for providing v-Jun- and v-Myc transformed cells. We are also grateful to John Wyke for critical review of the manuscript.

This work was supported by the Cancer Research Campaign, United Kingdom. D. Riley received support from the Association for International Cancer Research and The Sylvia Aitkin Trust. D. A. Potter received support from the Walther Oncology Institute, (Indianapolis, Ind.), the Walther Oncology Center at Indiana University, p30 DK34928 (GRASP Digestive Disease Research Center Grant), and a Life Span New Initiatives Grant. P. A. Greer received support from the Canadian Institutes of Health Research. J. S. Elce received support from the Canadian Heart and Stroke Institute.