Thrombospondin signaling of focal adhesion disassembly requires activation of phosphoinositide 3-kinase

Jeffrey A. Greenwood, Manuel A. Pallero, Anne B. Theibert, Joanne E. Murphy-Ullrich

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

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 α-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 32P(i)- 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.

Original languageEnglish (US)
Pages (from-to)1755-1763
Number of pages9
JournalJournal of Biological Chemistry
Volume273
Issue number3
DOIs
StatePublished - Jan 16 1998
Externally publishedYes

Fingerprint

Thrombospondins
Focal Adhesions
1-Phosphatidylinositol 4-Kinase
Phosphatidylinositols
Phosphotransferases
Adhesion
Chemical activation
Actins
Phosphotyrosine
Actin Cytoskeleton
Actinin
Amino Acids
Phosphorylation
Extracellular Matrix Proteins
Cell adhesion
Endothelial cells
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Cell Adhesion
Membranes
Endothelial Cells

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Thrombospondin signaling of focal adhesion disassembly requires activation of phosphoinositide 3-kinase. / Greenwood, Jeffrey A.; Pallero, Manuel A.; Theibert, Anne B.; Murphy-Ullrich, Joanne E.

In: Journal of Biological Chemistry, Vol. 273, No. 3, 16.01.1998, p. 1755-1763.

Research output: Contribution to journalArticle

Greenwood, Jeffrey A. ; Pallero, Manuel A. ; Theibert, Anne B. ; Murphy-Ullrich, Joanne E. / Thrombospondin signaling of focal adhesion disassembly requires activation of phosphoinositide 3-kinase. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 3. pp. 1755-1763.
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AB - 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 α-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 32P(i)- 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.

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