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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 Pp125FAK, Paxillin, and Talin
ewraines{at}u.washington.edu
Active matrix metalloproteinases and degraded collagen are observed in disease states, such as atherosclerosis. To examine whether degraded collagen fragments have distinct effects on vascular smooth muscle cells (SMC), collagenase-digested type I collagen was added to cultured human arterial SMC. After addition of collagen fragments, adherent SMC lose their focal adhesion structures and round up. Analysis of components of the focal adhesion complex demonstrates rapid cleavage of the focal adhesion kinase (pp125FAK), paxillin, and talin. Cleavage is suppressed by inhibitors of the proteolytic enzyme, calpain I. In vitro translated pp125FAK is a substrate for both calpain I– and II–mediated processing. Mapping of the proteolytic cleavage fragments of pp125FAK predicts a dissociation of the focal adhesion targeting (FAT) sequence and second proline-rich domain from the tyrosine kinase domain and integrin-binding sequence. Coimmunoprecipitation studies confirm that the ability of pp125FAK to associate with paxillin, vinculin, and p130cas is significantly reduced in SMC treated with degraded collagen fragments. Further, there is a significant reduction in the association of intact pp125FAK with the cytoskeletal fraction, while pp125FAK cleavage fragments appear in the cytoplasm in SMC treated with degraded collagen fragments. Integrin-blocking studies indicate that integrin-mediated signals are involved in degraded collagen induction of pp125FAK cleavage. Thus, collagen fragments induce distinct integrin signals that lead to initiation of calpain-mediated cleavage of pp125FAK, paxillin, and talin and dissolution of the focal adhesion complex.
Key Words: extracellular matrix • matrix metalloproteinase • calpain • focal adhesion kinase • pp125FAK
© 1999 The Rockefeller University Press
1.used in this paper: ALLN, calpain inhibitor 1; ECL, enhanced chemiluminescence; ECM, extracellular matrix; FAK, focal adhesion kinase; FAT, focal adhesion targeting (sequence); FRNK, pp125FAK-related non-kinase; MMP, matrix metalloproteinase; pp125FAK, focal adhesion kinase; RIPA, radioimmunoprecipitation assay; SMC, smooth muscle cells; TIMP, tissue inhibitors of metalloproteinase; ZVAD-fmk, benzyloxycarbonyl Val-Ala-Asp fluoromethyl ketone (caspase inhibitor)Dr. Levkau's present address is Institute for Arteriosclerosis Research, Münster, Germany.
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