FIGURE 5.

The phosphorylation of Cool-1 is correlated with EGF-stimulated focal complex disassembly. NIH 3T3 cells transfected with A, an empty vector (control), B, Myc-tagged wild-type Cool-1, or C, Myc-tagged Cool-1 Y442F, were serum-starved for 15 h and then stimulated with EGF for the indicated lengths of time. Immunofluorescence was performed on the samples using an anti-vinculin antibody to detect focal complexes. The arrows indicate cells with focal complexes. D, for each immunofluorescence slide, we counted 6 different fields for a total of 172 cells expressing wild-type Cool-1 and 147 cells expressing the Cool-1 Y442F mutant. Following EGF treatment, ∼80% of the cells expressing wild-type Cool-1 rounded-up and exhibited a loss of focal complexes (as visualized by anti-vinculin staining). For cells expressing the Cool-1 Y442F mutant, ∼20% of the cells were round and less attached whereas 80% of the cells still showed focal complexes even after EGF treatment. The error bars indicate ± S.D.