Ciarallo Sandra, Subramaniam Venkateswaran, Hung Wesley, Lee Jin-Hwa, Kotchetkov Rouslan, Sandhu Charanjit, Milic Andrea, Slingerland Joyce M
Molecular and Cell Biology, Sunnybrook & Women's College Health Sciences Centre, University of Toronto, Toronto, Canada.
Mol Cell Biol. 2002 May;22(9):2993-3002. doi: 10.1128/MCB.22.9.2993-3002.2002.
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.
p27（Kip1）是转化生长因子β（TGF-β）介导G1期阻滞的重要效应分子。在人乳腺上皮细胞（HMEC）模型中进行的研究，包括对TGF-β介导的G1期阻滞敏感（184(S)）和耐药（184A1L5(R)）的细胞，揭示了耐药细胞中p27调控异常。细胞周期蛋白E1 - 细胞周期蛋白依赖性激酶2（cdk2）和细胞周期蛋白A - cdk2活性增加，并且在184A1L5(R)细胞中检测到与p27相关的激酶活性。与敏感的184(S)细胞中的p27相比，184A1L5(R)细胞中的p27定位于细胞核和细胞质，显示出磷酸异构体谱改变，并且在体外结合和抑制细胞周期蛋白E1 - cdk2的能力降低。在增殖的184A1L5(R)细胞中，与184(S)细胞相比，更多的p27与细胞周期蛋白D1 - cdk4复合物相关。虽然TGF-β抑制184(S)细胞中细胞周期蛋白D1 - cdk4 - p27复合物的形成，但它不抑制耐药的184A1L5(R)细胞中细胞周期蛋白D1 - cdk4 - p27复合物的组装。p27磷酸化在细胞周期进程中发生变化，G0期与细胞周期蛋白E1结合的p27显示出与G1中期与细胞周期蛋白D1结合的p27不同的磷酸化模式。这些数据提示了一个模型，其中TGF-β将p27磷酸化从其与细胞周期蛋白D1结合的组装磷酸形式调节为紧密结合以抑制细胞周期蛋白E1 - cdk2的另一种形式。耐药的184A1L5(R)细胞中p27磷酸化的改变可能有利于p27与细胞周期蛋白D1 - cdk4的结合，并阻止其响应TGF-β在细胞周期蛋白E1 - cdk2中积累。
