Degraded collagen fragments promote rapid disassembly of smooth muscle focal adhesions that correlates with cleavage of pp125(FAK), paxillin, and talin.
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 (pp125(FAK)), paxillin, and talin. Cleavage is suppressed by inhibitors of the proteolytic enzyme, calpain I. In vitro translated pp125(FAK) is a substrate for both calpain I- and II-mediated processing. Mapping of the proteolytic cleavage fragments of pp125(FAK) 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 pp125(FAK) 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 pp125(FAK) with the cytoskeletal fraction, while pp125(FAK) 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 pp125(FAK) cleavage. Thus, collagen fragments induce distinct integrin signals that lead to initiation of calpain-mediated cleavage of pp125(FAK), paxillin, and talin and dissolution of the focal adhesion complex.
Carragher
N O
NO
Department of Pathology, University of Washington School of Medicine, Seattle, Washington 98195-7470, USA.
Levkau
B
B
Ross
R
R
Raines
E W
EW
D000185:Actinin / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D001158:Arteries; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism*; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D017364:Collagenases / Q000378:metabolism; D050719:Crk-Associated Substrate Protein; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D007231:Infant, Newborn; D016023:Integrins / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000378:metabolism; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D051419:Paxillin; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins; D038981:Receptors, Collagen; D050717:Retinoblastoma-Like Protein p130; D016608:Talin / Q000378:metabolism*; D016596:Vinculin / Q000378:metabolism
D000199:Actins; C493841:BCAR1 protein, human; D015815:Cell Adhesion Molecules; D050719:Crk-Associated Substrate Protein; D003598:Cytoskeletal Proteins; D016023:Integrins; D061965:Matrix Metalloproteinase Inhibitors; C496061:PXN protein, human; D051419:Paxillin; D010446:Peptide Fragments; D010750:Phosphoproteins; D011506:Proteins; D038981:Receptors, Collagen; D050717:Retinoblastoma-Like Protein p130; D016608:Talin; D000185:Actinin; D016596:Vinculin; D003094:Collagen; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D002154:Calpain; D017364:Collagenases; D020782:Matrix Metalloproteinases