Thrombospondin signaling of focal adhesion disassembly requires activation of phosphoinositide 3-kinase.
Thrombospondin is an extracellular matrix protein involved in modulating cell adhesion. Thrombospondin stimulates a rapid loss of focal adhesion plaques and reorganization of the actin cytoskeleton in cultured bovine aortic endothelial cells. The focal adhesion labilizing activity of thrombospondin is localized to the amino-terminal domain, specifically amino acids 17-35. Use of a synthetic peptide (hep I), containing amino acids 17-35 of thrombospondin, enables us to examine the signaling mechanisms specifically involved in thrombospondin-induced disassembly of focal adhesions. We tested the hypothesis that activation of phosphoinositide 3-kinase is a necessary step in the thrombospondin-induced signaling pathway regulating focal adhesion disassembly. Both wortmannin and LY294002, membrane permeable inhibitors of phosphoinositide 3-kinase activity, blocked hep I-induced disassembly of focal adhesions. Similarly, wortmannin inhibited hep I-mediated actin microfilament reorganization and the hep I-induced translocation of alpha-actinin from focal adhesion plaques. Hep I also stimulated phosphoinositide 3-kinase activity approximately 2-3-fold as measured in anti-phosphoinositide 3-kinase and anti-phosphotyrosine immunoprecipitates. Increased immunoreactivity for the 85-kDa regulatory subunit in anti-phosphotyrosine immunoprecipitates suggests that the p85/p110 form of phosphoinositide 3-kinase is involved in this pathway. In 32Pi-labeled cells, hep I increased levels of phosphatidylinositol (3,4,5)-trisphosphate, the major product of phosphoinositide 3-kinase phosphorylation. These results suggest that thrombospondin signals the disassembly of focal adhesions and reorganization of the actin cytoskeleton by a pathway involving stimulation of phosphoinositide 3-kinase activity.
Greenwood
J A
JA
Department of Pathology, Division of Molecular and Cellular Pathology, University of Alabama at Birmingham 35294, USA. jeffrey@vh.path.uab.edu
Pallero
M A
MA
Theibert
A B
AB
Murphy-Ullrich
J E
JE
D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002448:Cell Adhesion; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D019699:Thrombospondins / Q000502:physiology*; D014157:Transcription Factors; D000077191:Wortmannin
D000730:Androstadienes; D002867:Chromones; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D009025:Morpholines; D009842:Oligopeptides; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D019699:Thrombospondins; D014157:Transcription Factors; D000077191:Wortmannin; C110208:Hep I peptide; C101206:SUB1 protein, S cerevisiae; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one