Altered p27Kip1 phosphorylation, localization, and function in human epithelial cells resistant to transforming growth factor β-mediated G1 arrest

p27(Kip1) is an important effector of G(1) arrest by transforming growth factor beta (TGF-beta). Investigations in a human mammary epithelial cell (HMEC) model, including cells that are sensitive (184(S)) and resistant (184A1L5(R)) to G(1) arrest by TGF-beta revealed aberrant p27 regulation in the resistant cells. Cyclin El-cyclindependent kinase 2 (cdk2) and cyclin A-cdk2 activities were increased, and p27-associated kinase activity was detected in 184A1L5(R) cells. p27 from 184A1L5(R) cells was localized to both nucleus and cytoplasm, showed an altered profile of phosphoisoforms, and had a reduced ability to bind and inhibit cyclin El-cdk2 in vitro when compared to p27 from the sensitive 184s cells. In proliferating 184A1L5R cells, more p27 was associated with cyclin Dl-cdk4 complexes than in 184s. While TGF-beta[3 inhibited the formation of cyclin D1-cdk4-p27 complexes in 184s cells, it did not inhibit the assembly of cyclin Dl-udk4-p27 complexes in the resistant 184A1L5(R) cells. p27 phosphorylation changed during cell cycle progression, with cyclin El-bound p27 in Go showing a different phosphorylation pattern from that of cyclin D1-bound p27 in mid-G(1). These data suggest a model in which TGF-beta modulates p27 phosphorylation from its cyclin D1-bound assembly phosphoform to an alternate form that binds tightly to inhibit cyclin El-cdk2. Altered phosphorylation of p27 in the resistant 184A1L5(R) cells may favor the binding of p27 to cyclin Dl-cdk4 and prevent its accumulation in cyclin El-cdk2 in response to TGF-beta.