Tyrosine phosphorylation of p27Kip1 affects its ability to act as an inhibitor of cyclin D-cdk4.

Abstract

The tumor suppressor p27 controls cell cycle progression by regulating the activity of cyclin-cdk complexes. These serine/threonine kinases phosphorylate various substrates leading to the transition from one cell cycle phase to another. While p27 is always an inhibitor of cdk2, its ability to inhibit cdk4 depends on the cell type and whether the cell is proliferating or growth arrested. We have demonstrated that while p27 associates with cyclin D-cdk4, its ability to inhibit, or alternatively, not to inhibit this complex is dependent on the absence or presence of specific tyrosine phosphorylation that interconverts p27 from a bound, inhibitor to a bound, non-inhibitor under different growth conditions. In proliferating (A) cells, p27 is tyrosine phosphorylated and binds to cyclin D-cdk4 without inhibiting its kinase activity. In contact arrested (G0) cells, however, p27 is not tyrosine phosphorylated and binds and inhibits cyclin D-cdk4. Tyrosine phosphorylation within the 3-10 helix of p27 may force the C-terminal tail of p27 from the ATP binding site of cdk4, allowing p27 to associate in a bound, non-inhibitory mode. Lack of tyrosine phosphorylation would reestablish p27’s contact with cdk4, physically blocking the ATP binding site. Residues Y88 and Y89 of p27 are found within two consensus SH2 motifs. We have not identified the growth state dependent tyrosine kinase, but several tyrosine kinases, such as Abl, Lyn, Src, and Yes, can phosphorylate p27. We explored the role of p27 tyrosine phosphorylation in three physiological conditions: upon release from quiescence, during cancer progression, and during differentiation. Our data demonstrated that as cells exit G0 and enter early G1, tyrosine phosphorylation of p27 correlated with cdk4 kinase activity. Cdk2 kinase activity was only detected hours later when p27 levels decreased. This suggested that tyrosine phosphorylation of p27 might help to ensure the temporal order of cdk activation. In pancreatic cancer cells, we demonstrated that Src tyrosine kinase inhibitors decreased p27 tyrosine phosphorylation and both cyclin D-cdk4/6 and cyclin A/E-cdk2 kinase activity. Cyclin A/E-cdk2 kinase activity was inhibited by p27’s increased association with the complex, while cyclin D-cdk4/6 complexes were inhibited by a decrease in tyrosine phosphorylation of p27. This suggested that in some cancer cell lines, decreasing p27 tyrosine phosphorylation correlated with a decrease in cyclin D-cdk4/6 kinase activity. In differentiation, the role of p27 tyrosine phosphorylation was less conclusive.