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Vol. 11, Issue 3, 929-939, March 2000

Caspase-mediated Cleavage of p130cas in Etoposide-induced Apoptotic Rat-1 Cells

Seunghyi Kook,^* Sang Ryeol Shim,^* Soo Jeon Choi,^* Joohong Ahnn,^* Jae Il Kim,^* Soo Hyun Eom,^* Yong Keun Jung,^* Sang Gi Paik, and Woo Keun Song^*

 ^*Department of Life Science, Kwangju Institute of Science and Technology, Kwangju 500-712, Korea; and  Department of Biology, Chungnam National University, Taejon 305-764, Korea

Apoptosis causes characteristic morphological changes in cells, including membrane blebbing, cell detachment from the extracellular matrix, and loss of cell-cell contacts. We investigated the changes in focal adhesion proteins during etoposide-induced apoptosis in Rat-1 cells and found that during apoptosis, p130cas (Crk-associated substrate [Cas]) is cleaved by caspase-3. Sequence analysis showed that Cas contains 10 DXXD consensus sites preferred by caspase-3. We identified two of these sites (DVPD⁴¹⁶G and DSPD⁷⁴⁸G) in vitro, and point mutations substituting the Asp of DVPD⁴¹⁶G and DSPD⁷⁴⁸G with Glu blocked caspase-3-mediated cleavage. Cleavage at DVPD⁴¹⁶G generated a 74-kDa fragment, which was in turn cleaved at DSPD⁷⁴⁸G, yielding 47- and 31-kDa fragments. Immunofluorescence microscopy revealed well-developed focal adhesion sites in control cells that dramatically declined in number in etoposide-treated cells. Cas cleavage correlated temporally with the onset of apoptosis and coincided with the loss of p125FAK (focal adhesion kinase [FAK]) from focal adhesion sites and the attenuation of Cas-paxillin interactions. Considering that Cas associates with FAK, paxillin, and other molecules involved in the integrin signaling pathway, these results suggest that caspase-mediated cleavage of Cas contributes to the disassembly of focal adhesion complexes and interrupts survival signals from the extracellular matrix.

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Corresponding author. E-mail address: wksong{at}pia.kjist.ac.kr.

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Molecular Biology of the Cell
Vol. 11, 929-939, March 2000
Copyright © 2000 by The American Society for Cell Biology

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