Posttranslational modifications of p27kip1 determine its binding specificity to different cyclins and cyclin-dependent kinases in vivo.

Using 2-dimensional gel electrophoresis (2D-gel) analysis, we show here that cell-cycle entry is associated with a significant increase in p27(kip1) phosphorylation in human primary B cells. A similar pattern of increase in p27(kip1) phosphorylation was also seen in 2 fast-growing tumor cell lines, Burkitt lymphoma cell line BL40 and breast carcinoma cell line Cal51, where inactive p27(kip1) is expressed at high levels. Detailed analysis revealed for the first time that different cyclins and cyclin-dependent kinases (cdk's) interact with distinct posttranslationally modified isoforms of p27(kip1) in vivo. Cyclin E but not cyclin A selectively interacts with phosphorylated p27(kip1) isoforms, while cyclin D1 and D2 favor unphosphorylated p27(kip1) isoforms in vivo. Interestingly, cyclin D3 and cdk4 selectively interact with phosphorylated p27(kip1) in BL40 cells. Among all D-type cyclin/cdk4 and cdk6 complexes, cyclin D3/cdk4 is most active in sequestering the inhibitory activity of p27(kip1) in vitro in a cyclinE/cdk2 kinase assay. This novel feature of the binding specificity of p27(kip1) to cyclins and cdk's in vivo is interpreted in the context of overexpression of cyclin D3 in the presence of high levels of p27(kip1) in human B-cell lymphomas with adverse clinical outcome.