Foster James S, Fernando Romaine I, Ishida Noriko, Nakayama Keiichi I, Wimalasena Jay
Department of Obstetrics and Gynecology, Graduate School of Medicine, Program in Comparative and Experimental Medicine, University of Tennessee Medical Center, Knoxville, Tennessee 37920, USA.
J Biol Chem. 2003 Oct 17;278(42):41355-66. doi: 10.1074/jbc.M302830200. Epub 2003 Aug 6.
The cyclin-dependent kinase (CDK) inhibitor p27Kip1 plays a key role in growth and development of the mammary epithelium and in breast cancer. p27Kip1 levels are regulated through ubiquitin/proteasome-mediated proteolysis, promoted by CDK2 and the F box protein Skp2 at the G1/S transition, and independent of Skp2 in mid-G1. We investigated the respective roles of Skp2 and subcellular localization of p27Kip1 in down-regulation of p27Kip1 induced in MCF-7 cells by estrogens. 17beta-Estradiol treatment increased Skp2 expression in MCF-7 cells; however, this increase was prevented by G1 blockade mediated by p16Ink4a or the CDK inhibitor roscovitine, whereas down-regulation of p27Kip1 was maintained. Exogenous Skp2 prevented growth arrest of MCF-7 cells by antiestrogen, coinciding with decreased p27Kip1 expression. Under conditions of G1 blockade, p27Kip1 was stabilized by inhibition of CRM1-dependent nuclear export with leptomycin B or by mutation of p27Kip1 (Ser10 --> Ala; S10A) interfering with CRM1/p27Kip1 interaction. Antisense Skp2 oligonucleotides and a dominant-interfering Cul-1(1-452) mutant prevented down-regulation of p27Kip1S10A, whereas Skp2 overexpression elicited its destruction in mitogen-deprived cells. Active mediators of the extracellular signal-regulated kinase (ERK) pathway including Raf-1caax induced cytoplasmic localization of p27Kip1 in antiestrogen-treated cells and prevented accumulation of p27Kip1 in these cells independent of Skp2 expression and coinciding with ERK activation. Genetic or chemical blockade of the ERK pathway prevented down-regulation and cytoplasmic localization of p27Kip1 in response to estrogen. Our studies indicate that estrogens elicit down-regulation of p27Kip1 in MCF-7 cells through Skp2-dependent and -independent mechanisms that depend upon subcellular localization of p27Kip1 and require the participation of mediators of the Ras/Raf-1/ERK signaling pathway.
细胞周期蛋白依赖性激酶（CDK）抑制剂p27Kip1在乳腺上皮细胞的生长发育以及乳腺癌中发挥关键作用。p27Kip1水平通过泛素/蛋白酶体介导的蛋白水解作用进行调节，在G1/S期转换时由CDK2和F盒蛋白Skp2促进，而在G1中期则独立于Skp2。我们研究了Skp2和p27Kip1的亚细胞定位在雌激素诱导的MCF-7细胞中p27Kip1下调过程中的各自作用。17β-雌二醇处理可增加MCF-7细胞中Skp2的表达；然而，这种增加被p16Ink4a介导的G1期阻滞或CDK抑制剂roscovitine所抑制，而p27Kip1的下调则持续存在。外源性Skp2可防止抗雌激素药物导致的MCF-7细胞生长停滞，同时p27Kip1表达降低。在G1期阻滞条件下，通过雷帕霉素B抑制CRM1依赖性核输出或通过干扰CRM1/p27Kip1相互作用的p27Kip1突变（Ser10→Ala；S10A）可使p27Kip1稳定。反义Skp2寡核苷酸和显性干扰性Cul-1(1-452)突变体可防止p27Kip1S10A的下调，而Skp2过表达则在有丝分裂原缺乏的细胞中引发其破坏。细胞外信号调节激酶（ERK）途径的活性介质，包括Raf-1caax，可在抗雌激素处理的细胞中诱导p27Kip1的细胞质定位，并在这些细胞中阻止p27Kip1的积累，这与Skp2表达无关且与ERK激活同时发生。ERK途径的基因或化学阻滞可防止雌激素诱导的p27Kip1下调和细胞质定位。我们的研究表明，雌激素通过Skp2依赖性和非依赖性机制在MCF-7细胞中引发p27Kip1的下调，这些机制取决于p27Kip1的亚细胞定位，并需要Ras/Raf-1/ERK信号通路介质的参与。
