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Focal adhesion kinase N-terminus in breast carcinoma cells induces rounding, detachment and apoptosis.

ABSTRACT: Focal adhesion kinase (FAK) has a central role in adhesion-mediated cell signalling. The N-terminus of FAK is thought to function as a docking site for a number of proteins, including the Src-family tyrosine kinases. In the present study, we disrupted FAK signalling by expressing the N-terminal domain of FAK (FAK-NT) in human breast carcinoma cells, BT474 and MCF-7 lines, and non-malignant epithelial cells, MCF-10A line. Expression of FAK-NT led to rounding, detachment and apoptosis in human breast cancer cells. Apoptosis was accompanied by dephosphorylation of FAK Tyr(397), degradation of the endogenous FAK protein and activation of caspase-3. Over-expression of FAK rescued FAK-NT-mediated cellular rounding. Expression of FAK-NT in non-malignant breast epithelial cells did not lead to rounding, loss of FAK phosphorylation or apoptosis. Thus FAK-NT contributes to cellular adhesion and survival pathways in breast cancer cells which are not required for survival in non-malignant breast epithelial cells.

SUBMITTER: Beviglia L

PROVIDER: S-EPMC1223465 | biostudies-other | 2003 Jul

REPOSITORIES: biostudies-other

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Project description: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 1 h 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 8-24 h. Staurosporine induced a very rapid dephosphorylation of FAK at Tyr(861) and Tyr(397) and caused dissociation of phosphorylated FAK from focal adhesions as early as 30 s. The effect of staurosporine was very potent with striking inhibition of Tyr(861) and Tyr(397) phosphorylation and focal adhesion disruption occurring in the range 10-100 nM. 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 Tyr(861) and Tyr(397), 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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Focal adhesion kinase N-terminus in breast carcinoma cells induces rounding, detachment and apoptosis.

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