Southern Research Institute, Birmingham, AL, USA.
Int J Oncol. 2015 Jan;46(1):395-406. doi: 10.3892/ijo.2014.2728. Epub 2014 Oct 23.
Constitutive levels of Cks1 protein are very high in mammary carcinoma tissue and in breast tumor cell lines. However, despite being transcribed at relatively high levels, Cks1 protein is very low in normal mammary tissue. Also, basal Cks1 is barely detectable in primary human mammary epithelial cells (HMECs). Epoximicin, a proteasome inhibitor, induced detectable endogenous Cks1 in HMECs, and upregulated it above the basal level in MCF-7 breast cancer cells. Interestingly, transiently transfected Cks1 is remarkably unstable and accumulates only upon proteasomal blockade in multiple cell lines even when driven by the strong CMV promoter-enhancer. We examined the stability of site-directed Cks1 mutants in order to identify the structural determinants of its turnover in cancer cells. Since protein turnover is regulated by phosphorylation, and phosphoproteomic studies reveal phosphorylated tyrosines in Cks1, we replaced its five conserved tyrosines (Y) with phenylalanine (F), both individually and in combinations. We find that like wild-type, all transiently transfected mutant Cks1 vectors, even when driven by the CMV promoter-enhancer, expressed detectable protein only in cells treated with epoximicin. However, turnover of the Y8F, Y12F and Y19F Cks1 mutants was more rapid than that of wild-type, Y7F and Y57F. Since lysines are modified by ubiquitination or acetylation we also examined the consequences of lysine to arginine (K-R) substitutions on Cks1 proteasomal turnover. We found that the individual mutations K4R, K26R, K30R, and K34R slowed Cks1 turnover, while the K79R mutation or the combined mutation K75-76-78-79R increased turnover. Taken together, regulation of Cks1 protein stability is crucially dependent on specific tyrosine and lysine residues which are potential sites for post-translational modifications.
在乳腺癌组织和乳腺癌细胞系中，Cks1 蛋白的组成型水平非常高。然而，尽管 Cks1 蛋白转录水平相对较高，但在正常乳腺组织中其含量非常低。此外，在原代人乳腺上皮细胞（HMEC）中，基底 Cks1 几乎检测不到。环氧米星，一种蛋白酶体抑制剂，诱导 HMEC 中可检测的内源性 Cks1，并在上皮性乳腺癌细胞 MCF-7 中使其水平高于基础水平。有趣的是，瞬时转染的 Cks1 非常不稳定，仅在多种细胞系中通过蛋白酶体阻断时才会积累，即使在强 CMV 启动子增强子的驱动下也是如此。我们检查了定点突变 Cks1 的稳定性，以确定其在癌细胞中周转率的结构决定因素。由于蛋白质周转率受磷酸化调节，磷酸蛋白质组学研究揭示 Cks1 中有磷酸化的酪氨酸，因此我们用苯丙氨酸（F）替换了其五个保守的酪氨酸（Y），分别进行了替换，以及组合替换。我们发现，与野生型一样，所有瞬时转染的突变 Cks1 载体，即使在 CMV 启动子增强子的驱动下，仅在用环氧米星处理的细胞中才能表达可检测的蛋白。然而，与野生型相比，Y8F、Y12F 和 Y19F Cks1 突变体的周转率更快，Y7F 和 Y57F 则较慢。由于赖氨酸可被泛素化或乙酰化修饰，我们还检查了赖氨酸到精氨酸（K-R）取代对 Cks1 蛋白酶体周转率的影响。我们发现，单个突变 K4R、K26R、K30R 和 K34R 会减缓 Cks1 的周转率，而 K79R 突变或 K75-76-78-79R 的组合突变会增加周转率。总之，Cks1 蛋白稳定性的调节取决于特定的酪氨酸和赖氨酸残基，这些残基是翻译后修饰的潜在位点。
