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Bodo Levkau^*, Barbara Herren, Hidenori Koyama, Russell Ross^*, and Elaine W. Raines^*

From the ^* Department of Pathology, University of Washington School of Medicine, Seattle, Washington 98195-7470; The Cruciform Project, The Wolfson Institute for Biomedical Research, University College London, London, United Kingdom WC1E 6JJ; and Second Department of Internal Medicine, Osaka City University Medical School, Osaka, Japan 545

Normal endothelial and epithelial cells undergo apoptosis when cell adhesion and spreading are prevented, implying a requirement for antiapoptotic signals from the extracellular matrix for cell survival. We investigated some of the molecular changes occurring in focal adhesions during growth factor deprivation–induced apoptosis in confluent monolayers of human umbilical vein endothelial cells. Among the first morphologic changes after initiation of the apoptotic process are membrane blebbing, loss of focal adhesion sites, and retraction from the matrix followed by detachment. We observe a specific proteolytic cleavage of focal adhesion kinase (pp125^FAK), an important component of the focal adhesion complex, and identify pp125^FAK as a novel substrate for caspase-3 and caspase-3–like apoptotic caspases. The initial cleavage precedes detachment, and coincides with loss of pp125^FAK and paxillin from focal adhesion sites and their redistribution into the characteristic membrane blebs of apoptotically dying cells. Cleavage of pp125^FAK differentially affects its association with signaling and cytoskeletal components of the focal adhesion complex; binding of paxillin, but not pp130^Cas (Cas, Crk-associated substrate) and vinculin, to the COOH terminally truncated pp125^FAK is abolished. Therefore, caspase-mediated cleavage of pp125^FAK may be participating in the disassembly of the focal adhesion complex and actively interrupting survival signals from the extracellular matrix, thus propagating the cell death program.

This work was supported in part by National Institutes of Health grant HL18645 to R. Ross and E.W. Raines. B. Levkau is a recipient of a training research scholarship by the Deutsche Forschungsgemeinschaft of Germany.

Address correspondence to Elaine W. Raines, University of Washington School of Medicine, Department of Pathology, Box 357470, J507, Seattle WA 98195-7470. Phone: 206-685-7441; Fax: 206-685-3018; E-mail: ewraines{at}u.washington.edu

¹ Abbreviations used in this paper: Ac-DEVD-CHO, N-acetyl-Asp-Glu-Val-Asp-aldehyde; FAK, focal adhesion kinase; FRNK, pp125^FAK-related nonkinase; GF, growth factor; HUVEC, human umbilical vein endothelial cell; PARP, poly(ADP-ribose) polymerase; PCNA, proliferating cell nuclear antigen; SH, Src homology domain; ZVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone.

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