FAK is phosphorylated in response to 2β1 ligation and mediates 2β1-induced inhibition of AICD and Fas-L expression.

(A) The cells were stimulated or not with collagen I at 20 and 50 μg/mL either alone or in combination with 20 μg/mL of soluble anti-CD3 for 10 minutes. The cells were washed and lysed and subjected to immunoprecipitation with anti-FAK antibodies. The immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine antibodies (top panel) and with antibodies against FAK (lower panel). (B) Jurkat cells were cotransfected with a plasmid encoding the dominant-negative form of FAK (FRNK), wild-type form of FAK (FAK), combination of FRNK- and FAK-encoding plasmids, or a control plasmid, together with a plasmid encoding GFP as described in “Materials and methods.” Viable cells were recovered 48 hours later by Ficoll gradient and stimulated with or without PMA and ionomycin in the presence or absence of 50 μg/mL collagen I for 24 hours. The cells were then washed and propidium iodide was added for 15 minutes on ice, and apoptosis was analyzed by FACScan®. The analysis was carried out on the double positive cell population for propidium iodide and fluorescent GFP. (C) Jurkat cells were cotransfected with plasmids encoding FRNK, FAK, Fas-L promoter reporter construct, and β-galactosidase. After 48 hours, the cells were washed and treated with the various stimuli as in (B) and luciferase activity (RLU) was determined after 18 hours.