Altered p27(Kip1) phosphorylation, localization, and function in human epithelial cells resistant to transforming growth factor beta-mediated G(1) 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 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.
Ciarallo
Sandra
S
Molecular and Cell Biology, Sunnybrook & Women's College Health Sciences Centre, University of Toronto, Toronto, Canada.
Subramaniam
Venkateswaran
V
Hung
Wesley
W
Lee
Jin-Hwa
JH
Kotchetkov
Rouslan
R
Sandhu
Charanjit
C
Milic
Andrea
A
Slingerland
Joyce M
JM
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*
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