Tyrosine phosphorylation modulates binding preference to cyclin-dependent kinases and subcellular localization of P27Kip1 in the acute promyelocytic leukemia cell line NB4

We have investigated the role of tyrosine phosphorylation of the cyclin-dependent kinase (cdk) inhibitor p27(Kip1) using the acute promyelocytic leukemia cell line NB4 together with granulocyte colony-stimulating factor (G-CSF). Short-term G-CSF stimulation resulted in a rapid tyrosine dephosphorylation of p27(Kip1) accompanied by a change in its binding preferences to cdks. On G-CSF stimulation, p27(Kip1) dissociated from cdk4 and associated with cdk2. Binding assays with recombinant p27(Kip1) confirmed that tyrosine-phosphorylated p27(Kip1) preferentially bound to cdk4, whereas unphosphorylated protein preferentially associated with cdk2. In addition, studies with p27(Kip1) point mutations revealed a decisive role of Tyr88 and Tyr89 in binding to cdk4. Furthermore, phosphorylation of Tyr88 and Tyr89 was accompanied by strong nuclear translocation of p27(Kip1). Taken together, this report provides the first evidence that tyrosine phosphorylation of p27(Kip1) plays a crucial role in binding to cdks and its subcellular localization. Moreover, both effects are mediated by application of G-CSF.