Figure 1.

p50^Cdc37 in cells is tyrosine phosphorylated. A. Tyrosine phosphorylation of cellular p50^Cdc37 occurs only on amino acids Y4 and Y298. COS7 cells were transfected with indicated FLAG-tagged p50^Cdc37 constructs. Twenty-four hours after transfection, cells were treated with 100 µM bpv(phen) for 30 min (to inhibit tyrosine phosphatases), lysed with 1% SDS buffer, and boiled for 5 min. p50^Cdc37 proteins were precipitated with anti-FLAG antibody, and tyrosine phosphorylation was detected by blotting with antibody 4G10. Protein loading was monitored with anti-FLAG antibody. Note that p50^Cdc37-Y298E and p50^Cdc37-phosphoY298 have a similar mobility. B. p50^Cdc37 tyrosine phosphorylation status does not affect p50^Cdc37 serine phosphorylation. Indicated p50^Cdc37 proteins were expressed in COS7 cells and immunoprecipitated as described above andS13 phosphorylation was detected. C. p50^Cdc37 serine phosphorylation status affects p50^Cdc37 tyrosine phosphorylation. Indicated p50^Cdc37 proteins were expressed in COS7 cells and assessed as described above. Phosphorylation of Y4 and Y298 was assessed using site-specific anti-phospho-tyrosine antibodies (see Fig. S1 for antibody validation). D. p50^Cdc37 tyrosine phosphorylation is reduced by Src family kinase inhibition. 293H cells were transfected with indicated p50^Cdc37 plasmids. Twenty-four hours after transfection, cells were pretreated with indicated kinase inhibitors for 2 hours, and then with bpv(phen) for 20 min. Sample preparation and analysis were as described above. E. Yes kinase is responsible for p50^Cdc37 tyrosine phosphorylation. 293H cells were co-transfected with siRNA specific for the indicated Src family kinases and p50^Cdc37-Y4F or p50^Cdc37-Y298F. Two days after transfection, cells were lysed and samples were prepared and analyzed as above (see also Fig. S1).