Degraded Collagen Fragments Promote Rapid Disassembly of Smooth Muscle Focal Adhesions That Correlates with Cleavage of Pp125FAK, Paxillin, and Talin

doi:10.1083/jcb.147.3.619

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© The Rockefeller University Press, 0021-9525/1999//619 $5.00
The Journal of Cell Biology, Volume 147, Number 3, , 1999 619-630

Original Article

Degraded Collagen Fragments Promote Rapid Disassembly of Smooth Muscle Focal Adhesions That Correlates with Cleavage of Pp125^FAK, Paxillin, and Talin

Neil O. Carragher^a, Bodo Levkau^a, Russell Ross^a, and Elaine W. Raines^a

^a Department of Pathology, University of Washington School of Medicine, Seattle, Washington 98195-7470
University of Washington School of Medicine, Department of Pathology, Box 357470, Seattle, WA 98195-7470.(206) 685-3018(206) 685-7441

ewraines{at}u.washington.edu

Active matrix metalloproteinases and degraded collagen are observedin disease states, such as atherosclerosis. To examine whetherdegraded collagen fragments have distinct effects on vascularsmooth muscle cells (SMC), collagenase-digested type I collagenwas added to cultured human arterial SMC. After addition ofcollagen fragments, adherent SMC lose their focal adhesion structuresand round up. Analysis of components of the focal adhesion complexdemonstrates rapid cleavage of the focal adhesion kinase (pp125^FAK),paxillin, and talin. Cleavage is suppressed by inhibitors ofthe proteolytic enzyme, calpain I. In vitro translated pp125^FAKis a substrate for both calpain I– and II–mediatedprocessing. Mapping of the proteolytic cleavage fragments ofpp125^FAK predicts a dissociation of the focal adhesion targeting(FAT) sequence and second proline-rich domain from the tyrosinekinase domain and integrin-binding sequence. Coimmunoprecipitationstudies confirm that the ability of pp125^FAK to associate withpaxillin, vinculin, and p130cas is significantly reduced inSMC treated with degraded collagen fragments. Further, thereis a significant reduction in the association of intact pp125^FAKwith the cytoskeletal fraction, while pp125^FAK cleavage fragmentsappear in the cytoplasm in SMC treated with degraded collagenfragments. Integrin-blocking studies indicate that integrin-mediatedsignals are involved in degraded collagen induction of pp125^FAKcleavage. Thus, collagen fragments induce distinct integrinsignals that lead to initiation of calpain-mediated cleavageof pp125^FAK, paxillin, and talin and dissolution of the focaladhesion complex.

Key Words: extracellular matrix • matrix metalloproteinase • calpain • focal adhesion kinase • pp125^FAK

1.used in this paper: ALLN, calpain inhibitor 1; ECL, enhancedchemiluminescence; ECM, extracellular matrix; FAK, focal adhesionkinase; FAT, focal adhesion targeting (sequence); FRNK, pp125FAK-relatednon-kinase; MMP, matrix metalloproteinase; pp125FAK, focal adhesionkinase; RIPA, radioimmunoprecipitation assay; SMC, smooth musclecells; TIMP, tissue inhibitors of metalloproteinase; ZVAD-fmk,benzyloxycarbonyl Val-Ala-Asp fluoromethyl ketone (caspase inhibitor)

Dr. Levkau's present address is Institute for ArteriosclerosisResearch, Münster, Germany.

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