Tyrosine dephosphorylation of p27^Kip1 alters the binding to cdks. (A) NB4 cells were stimulated with G-CSF and harvested at different time points. Immunoprecipitations with cdk2 and cdk4 were performed and probed by Western blotting with antibodies as indicated. A total of 50 or 100 μg NB4 lysate from untreated cells was also loaded as a control. (B) p27^Kip1 immunoprecipitate from NB4 lysate was incubated with LAR-PTP. The Western blot was probed with antibodies to cdk4 and p27^Kip1. Binding of cdk4 to p27^Kip1 (treated with LAR-PTP or untreated) was detected. (C) GST-p27^Kip1 fusion proteins (p27^Kip1 wt and tyrosine to phenylalanine mutations Y74F, Y88F, Y89F, Y88/89F, Y74/88/89F) were expressed in E coli strains DH5α (pTyr -: not tyrosine-phosphorylated) and TKX1 (pTyr +: tyrosine-phosphorylated). After incubation of the fusion proteins in NB4 lysate together with GSH agarose for 15 minutes at 4°C, the protein complexes were separated by PAGE and blotted and binding of the p27^Kip1 wt or point mutations with cdk2 and cdk4 was evaluated by detection with the appropriate antibodies. (D) Plasmids containing p27^Kip1 wt sequence as well as point mutations (with or without Abl-PP sequence) were transiently expressed in NIH/3T3 cells. p27^Kip1 immunoprecipitations were performed and probed with antibodies as indicated. As control for Abl-PP expression, a total of 100 μg lysate was also detected. (E) Lysate from transfected NIH/3T3 cells was immunoprecipitated and probed as indicated.