The survival of endothelial cells is dependent on interactions between the matrix and integrins mediated through focal adhesions. Focal adhesion kinase (FAK) is thought to play a key role in maintaining focal adhesion function and cell survival, whereas caspase-mediated FAK proteolysis is implicated in focal adhesion disassembly during apoptosis. We examined the relationship between changes in FAK phosphorylation and proteolysis during apoptosis of primary porcine aortic endothelial cells (PAEC) induced by staurosporine, a widely used apoptogenic agent in diverse cell types. Staurosporine-induced PAEC apoptosis was detected after 1h and was preceded by disruption and loss of FAK localization to focal adhesions within a few minutes, whereas staurosporine-induced cleavage of FAK occurred only after 8—24h. Staurosporine induced a very rapid dephosphorylation of FAK at Tyr861 and Tyr397 and caused dissociation of phosphorylated FAK from focal adhesions as early as 30s. The effect of staurosporine was very potent with striking inhibition of Tyr861 and Tyr397 phosphorylation and focal adhesion disruption occurring in the range 10—100nM. Selective inhibition of a known target of staurosporine, protein kinase C, using GF109203X, and of phosphoinositide 3′-kinase using wortmannin, did not reduce FAK tyrosine phosphorylation at Tyr861 and Tyr397, or cause disruption of focal adhesions. Cycloheximide, the protein synthesis inhibitor, induced PAEC apoptosis more slowly than staurosporine, but did not induce FAK dephosphorylation or rapid focal adhesion disruption, and instead caused a slower loss of focal adhesions and a marked increase in FAK proteolysis. These studies show that FAK dephosphorylation and focal adhesion disassembly are very early events mediating the onset of staurosporine-induced endothelial cell apoptosis and are dissociated from FAK proteolysis. Cycloheximide induces apoptosis through a pathway involving FAK proteolysis without dephosphorylation.
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October 2002
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Research Article|
October 01 2002
Staurosporine induces endothelial cell apoptosis via focal adhesion kinase dephosphorylation and focal adhesion disassembly independent of focal adhesion kinase proteolysis Available to Purchase
Jahangir KABIR;
Jahangir KABIR
Department of Medicine, BHF Laboratories, University College London, 5 University Street, London WC1E 6JJ, U.K.
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Melvin LOBO;
Melvin LOBO
Department of Medicine, BHF Laboratories, University College London, 5 University Street, London WC1E 6JJ, U.K.
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Ian ZACHARY
Ian ZACHARY
1
Department of Medicine, BHF Laboratories, University College London, 5 University Street, London WC1E 6JJ, U.K.
1To whom correspondence should be addressed (e-mail [email protected]).
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Publisher: Portland Press Ltd
Received:
April 25 2002
Revision Received:
June 25 2002
Accepted:
June 25 2002
Accepted Manuscript online:
June 25 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 367 (1): 145–155.
Article history
Received:
April 25 2002
Revision Received:
June 25 2002
Accepted:
June 25 2002
Accepted Manuscript online:
June 25 2002
Citation
Jahangir KABIR, Melvin LOBO, Ian ZACHARY; Staurosporine induces endothelial cell apoptosis via focal adhesion kinase dephosphorylation and focal adhesion disassembly independent of focal adhesion kinase proteolysis. Biochem J 1 October 2002; 367 (1): 145–155. doi: https://doi.org/10.1042/bj20020665
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