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

Sandra Ciarallo, Venkateswaran Subramaniam, Wesley Hung, Jin Hwa Lee, Rouslan Kotchetkov, Charanjit Sandhu, Andrea Milic, Joyce M. Slingerland

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Abstract

p27Kip1 is an important effector of G1 arrest by transforming growth factor β(TGF-β). Investigations in a human mammary epithelial cell (HMEC) model, including cells that are sensitive (184s) and resistant (184A1L5R) to G1 arrest by TGF-β, revealed aberrant p27 regulation in the resistant cells. Cyclin El-cyclin-dependent kinase 2 (cdk2) and cyclin A-cdk2 activities were increased, and p27-associated kinase activity was detected in 184A1L5R cells. p27 from 184A1L5R 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 184s cells. In proliferating 184A1L5R cells, more p27 was associated with cyclin D1-cdk4 complexes than in 184s. While TGF-β inhibited the formation of cyclin D1-cdk4-p27 complexes in 184s cells, it did not inhibit the assembly of cyclin Dl-cdk4-p27 complexes in the resistant 184ALL5R cells. p27 phosphorylation changed during cell cycle progression, with cyclin E1-bound p27 in G0 showing a different phosphorylation pattern from that of cyclin D1-bound p27 in mid-G1. 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 184ALL5R cells may favor the binding of p27 to cyclin D1-cdk4 and prevent its accumulation in cyclin E1-cdk2 in response to TGF-β.

Original languageEnglish (US)
Pages (from-to)2993-3002
Number of pages10
JournalMolecular and cellular biology
Volume22
Issue number9
DOIs
StatePublished - May 6 2002

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ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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