Skp2-mediated p27(Kip1) degradation during S/G2 phase progession of a adipocyte hyperplasia
Skp2-mediated p27(Kip1) degradation during S/G2 phase progession of a adipocyte hyperplasia
复制标题
DOI：
10.1002/jcp.20915
复制
发表时间：
2007-04-01
影响因子：
5.6
通讯作者：
Morrison, Ron F.
中科院分区：
文献类型：
--
作者：
Auld, Corinth A.;Fernandes, Karishma M.;Morrison, Ron F.
p27（Kip I）是Cdk 2活性和G（1）/S转换的重要调节因子，以细胞类型和条件特异性的方式受到严格调节，以整合控制细胞周期进程的促有丝分裂和分化信号。我们发现在脂肪细胞分化的早期阶段，密度阻滞的3 T3-LI前脂肪细胞同步重新进入细胞周期，p27蛋白水平从G中期到G（2）晚期逐渐下降。p27蛋白积累的这种急剧下降至少部分是由于蛋白稳定性的降低。26 S蛋白酶体的特异性抑制剂被证明可以完全阻断整个G（1）中p27蛋白水平的降低，增加泛素化p27蛋白的丰度，并抑制G（1）/S转换，导致G（1）停滞。进一步证明，在S期进展过程中，Cdk 2使p27在苏氨酸187上磷酸化，并且磷酸化的p27被多聚泛素化和降解。此外，我们证明Skp 2和CksI在S/G（2）期进展过程中显著增加，伴随着p27蛋白的最大下降。用RNA干扰完全敲除Skp 2可部分阻止p27降解，相当于用Cdk 2阻断观察到的降解，这表明SCFSkp 2 E3连接酶和其他蛋白酶体依赖性机制有助于前脂肪细胞复制期间p27降解。有趣的是，Skp 2介导的p27降解对于G（1）/S或S/G（2）转变不是必需的，因为前脂肪细胞在脂肪细胞增生期间从静止转变为增殖。最后，证据表明，升高的p27蛋白在Skp 2的情况下被中和的螯合p27蛋白到细胞周期蛋白DI/Cdk 4复合物。
p27(Kip I), an important regulator of Cdk2 activity and G(1)/S transition, is tightly regulated in a cell-type and condition-specific manner to integrate mitogenic and differentiation signals governing cell cycle progression. We show that p27 protein levels progressively declined from mid-G, through late-G(2) phase as density-arrested 3T3-LI preadipocytes synchronously reentered the cell cycle during early stages of adipocyte differentiation. This dramatic fall in p27 protein accumulation was due, at least in part, to a decrease in protein stability. Specific inhibitors of the 26S proteasome were shown to completely block the decrease in p27 protein levels throughout G(1), increase the abundance of ubiquitylated p27 protein, and inhibit G(1)/S transition resulting in G(1) arrest. It is further demonstrated that p27 was phosphorylated on threonine 187 during S phase progression by Cdk2 and that phosphorylated p27 was polyubiquitylated and degraded. Furthermore, we demonstrate that Skp2 and CksI dramatically increased during S/G(2) phase progression concomitantly with the maximal fall in p27 protein. Complete knockdown of Skp2 with RNA interference partially prevented p27 degradation equivalent to that observed with Cdk2 blockade suggesting that the SCFSkp2 E3 ligase and other proteasome-dependent mechanisms contribute to p27 degradation during preadipocyte replication. Interestingly, Skp2-mediated p27 degradation was not essential for G(1)/S or S/G(2) transition as preadipocytes shifted from quiescence to proliferation during adipocyte hyperplasia. Finally, evidence is presented suggesting that elevated p27 protein in the absence of Skp2 was neutralized by sequestration of p27 protein into Cyclin DI/Cdk4 complexes.