Fig. 5. 5-deficient cell spreading induced by activation of specific PKC isozyme. (A) 5-deficient cells were plated on FN for 30 minutes in the presence or absence of PMA (3 nM), , or peptide activators (1 µM, labeled by the up arrow) or all three peptide activators together. In the absence of any activators, the cells do not attach. PMA treatment promotes rapid attachment and spreading. The activation of , or PKC all promote cell attachment, and and PKC activation promote cell spreading. All three activators together are nearly as effective as PMA. (B) 5-deficient cells treated as in A were assessed for FAK phosphorylation after 30 minutes on FN using an anti-phosphotyrosine antibody. FAK phosphorylation increased in the presence of the PKC activators in parallel with the effect on cell spreading shown in A. A duplicate blot was probed with an anti-FAK antibody to confirm equal loading (lower panel). FAK phosphorylation was quantified to calculate the percentage of activation (shown below each lane), with control levels being defined as no activation and PMA treatment defined as maximal activation. (C) 5-deficient cell spreading induced by , and PKC activators is inhibited by the corresponding specific inhibitors. 5-deficient cells were treated with individual PKC isozyme activators in the presence or absence of isozyme-specific inhibitors. FAK phosphorylation was determined by western blot analysis, and the level of phosphorylation was quantified. The experiment was repeated three times with similar results, and the results of a representative experiment are shown. The data were calculated as percentages of maximum activation obtained after 3 nM PMA treatment.