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Altered p27(Kip1) phosphorylation, localization, and function in human epithelial cells resistant to transforming
Sandra Ciarallo1, Venkateswaran Subramaniam, Wesley Hung
1Molecular and Cell Biology, Sunnybrook & Women's College Health Sciences Centre, University of Toronto, Toronto, Canada.
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.
Insights
Transforming growth factor beta (TGF-beta) normally arrests cell growth by regulating p27(Kip1). Resistant cells show altered p27 phosphorylation, preventing TGF-beta
Area of Science:
- Cell Cycle Regulation
- Molecular Biology
- Cancer Research
Background:
- p27(Kip1) is a key regulator of G1 cell cycle arrest, particularly in response to transforming growth factor beta (TGF-beta).
- Aberrant regulation of p27(Kip1) is implicated in cell proliferation and cancer development.
- Understanding the molecular mechanisms of TGF-beta-induced cell cycle arrest is crucial for therapeutic strategies.
Purpose of the Study:
- To investigate the aberrant regulation of p27(Kip1) in human mammary epithelial cells (HMEC) resistant to TGF-beta-induced G1 arrest.
- To elucidate the role of p27(Kip1) phosphorylation and its association with cyclin-dependent kinases (CDKs) in TGF-beta resistance.
- To propose a model for how TGF-beta modulates p27(Kip1) activity through phosphorylation.
Main Methods:
- Comparative analysis of p27(Kip1) regulation in TGF-beta-sensitive (184(S)) and resistant (184A1L5(R)) HMEC lines.
- Assays for cyclin E1-cdk2 and cyclin A-cdk2 activities, and p27-associated kinase activity.
- Immunolocalization, phosphoisoform analysis, in vitro binding and inhibition assays, and analysis of p27 complex formation with cyclin D1-cdk4.
Main Results:
- Resistant 184A1L5(R) cells exhibited increased cyclin E1-cdk2 and cyclin A-cdk2 activities and detected p27-associated kinase activity.
- p27 from resistant cells showed altered localization, phosphoisoform profile, and reduced ability to inhibit cyclin E1-cdk2 in vitro.
- In resistant cells, p27 preferentially associated with cyclin D1-cdk4, and TGF-beta failed to inhibit cyclin D1-cdk4-p27 complex formation.
Conclusions:
- Altered p27(Kip1) phosphorylation in TGF-beta-resistant cells favors its association with cyclin D1-cdk4, hindering its inhibition of cyclin E1-cdk2.
- TGF-beta's mechanism involves modulating p27(Kip1) phosphorylation to promote its inhibitory interaction with cyclin E1-cdk2.
- Dysregulation of p27(Kip1) phosphorylation is a key factor in resistance to TGF-beta-mediated cell cycle arrest.