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>Altered p27~(Kip1) Phosphorylation, Localization, and Function in Human Epithelial Cells Resistant to Transforming Growth Factor β-Mediated G_1 Arrest
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Altered p27~(Kip1) Phosphorylation, Localization, and Function in Human Epithelial Cells Resistant to Transforming Growth Factor β-Mediated G_1 Arrest
p27~(Kip1) is an important effector of G_1 arrest by transforming growth factor β (TGF-β). 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-β, revealed aberrant p27 regulation in the resistant cells. Cyclin E1-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 E1-cdk2 in vitro when compared to p27 from the sensitive 184~S cells. In proliferating 184A1L5~R cells, more p27 was associated with cyclin D1-cdk4 complexes than in 184~S. While TGF-β inhibited the formation of cyclin D1-cdk4-p27 complexes in 184~S cells, it did not inhibit the assembly of cyclin D1-cdk4-p27 complexes in the resistant 184A1L5~R cells. p27 phosphorylation changed during cell cycle progression, with cyclin E1-bound p27 in G_0 showing a different phosphorylation pattern from that of cyclin D1-bound p27 in mid-G_1. These data suggest a model in which TGF-β modulates p27 phosphorylation from its cyclin D1-bound assembly phosphoform to an alternate form that binds tightly to inhibit cyclin E1-cdk2. Altered phosphorylation of p27 in the resistant 184A1L5~R cells may favor the binding of p27 to cyclin D1-cdk4 and prevent its accumulation in cyclin E1-cdk2 in response to TGF-β.
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