title: Altered p27(Kip1) phosphorylation, localization, and function in human epithelial cells resistant to transforming growth factor beta-mediated G(1) arrest.
abstract: 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 E1-cyclin-dependent 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-beta 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-beta 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-beta.
authors.affiliation: Molecular and Cell Biology, Sunnybrook & Women's College Health Sciences Centre, University of Toronto, Toronto, Canada.
authors.forename: Sandra
authors.initials: S
authors.lastname: Ciarallo
authors.forename: Venkateswaran
authors.initials: V
authors.lastname: Subramaniam
authors.forename: Wesley
authors.initials: W
authors.lastname: Hung
authors.forename: Jin-Hwa
authors.initials: JH
authors.lastname: Lee
authors.forename: Rouslan
authors.initials: R
authors.lastname: Kotchetkov
authors.forename: Charanjit
authors.initials: C
authors.lastname: Sandhu
authors.forename: Andrea
authors.initials: A
authors.lastname: Milic
authors.forename: Joyce M
authors.initials: JM
authors.lastname: Slingerland
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D019938:Cyclin D1 / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D016193:G1 Phase / Q000187:drug effects*; D006801:Humans; D007150:Immunohistochemistry; D007525:Isoelectric Focusing; D046911:Macromolecular Substances; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D013696:Temperature; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D019927:Cyclin E; D046911:Macromolecular Substances; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases