Motti Maria Letizia, De Marco Carmela, Califano Daniela, Fusco Alfredo, Viglietto Giuseppe
Dipartimento di Biologia e Patologia Cellulare e Molecolare L Califano Facoltà di Medicina e Chirurgia, Università Federico II, via S. Pansini 5, Napoli, Italy.
Cell Cycle. 2004 Aug;3(8):1074-80. Epub 2004 Aug 7.
The localization of the cyclin-dependent kinase inhibitor p27(kip1) is dependent on the phosphorylation of one of three key amino acid residues: S10, T157 and T198. However, it was unclear whether endogenous p27(kip1) is phosphorylated at T198 in the living cell. In the present work we describe the generation and characterization of a polyclonal antibody able to recognize recombinant, transfected as well as endogenous T198-phosphorylated p27(kip1). Using this antibody, we demonstrate that: (1) endogenous p27(kip1) is phosphorylated at T198 in 4 breast cancer cells lines (MCF7, MDA-MB231, MDA- MB436 and MDA-MB468); (2) T198 phosphorylation is increased in breast cancer cells compared with normal mammary epithelial cells (HMEC); (3) T198-phosphorylated p27(kip1) is exclusively cytoplasmic; (4) T198 phosphorylation is dependent on the activity of the PI3K-PKB/Akt pathway, being it drastically reduced by the pharmacological PI3K inhibitor LY294002 or stimulated by the constitutive activation of PKB/Akt. Finally, in primary human breast carcinomas, cytoplasmic accumulation of T198-phosphorylated p27(kip1) parallels Akt activation. We conclude that in breast cancer cells p27(kip1) is phosphorylated at T198 in a PI3K/Akt dependent manner and that this phosphorylation may contribute to p27(kip1) cytoplasmic mislocalization observed in breast cancer.
细胞周期蛋白依赖性激酶抑制剂p27（kip1）的定位取决于三个关键氨基酸残基之一的磷酸化：S10、T157和T198。然而，尚不清楚内源性p27（kip1）在活细胞中是否在T198处发生磷酸化。在本研究中，我们描述了一种能够识别重组、转染以及内源性T198磷酸化p27（kip1）的多克隆抗体的产生和特性。使用该抗体，我们证明：（1）在4种乳腺癌细胞系（MCF7、MDA-MB231、MDA-MB436和MDA-MB468）中，内源性p27（kip1）在T198处发生磷酸化；（2）与正常乳腺上皮细胞（HMEC）相比，乳腺癌细胞中T198磷酸化增加；（3）T198磷酸化的p27（kip1）仅存在于细胞质中；（4）T198磷酸化依赖于PI3K-PKB/Akt途径的活性，药理学PI3K抑制剂LY294002可使其显著降低，而PKB/Akt的组成性激活则可刺激其增加。最后在原发性人乳腺癌中，T198磷酸化的p27（kip1）的细胞质积累与Akt激活平行。我们得出结论，在乳腺癌细胞中，p27（kip1）以PI3K/Akt依赖性方式在T198处发生磷酸化，这种磷酸化可能导致乳腺癌中观察到的p27（kip1）细胞质定位错误。
