Lee Jeong Goo, Kay EunDuck P
Doheny Eye Institute, Keck School of Medicine, University of Southern California, 1450 San Pablo Street, Los Angeles, CA 90033, USA.
Invest Ophthalmol Vis Sci. 2008 Jan;49(1):189-96. doi: 10.1167/iovs.07-0855.
p27(Kip1) (p27) is an important regulator of G(1) progression. For cells to proliferate, p27 must undergo proteolysis. FGF-2 enables phosphorylation of p27 at both the Thr-187 and Ser-10 sites, an event that is prerequisite for polyubiquitination. This study was undertaken to determine whether degradation of the two phosphorylated p27s is mediated by a distinct ubiquitin E3 ligase complex at different subcellular locations.
Expression of p27, KPC1, KPC2, Skp1, Skp2, and Cul1 was analyzed by immunoblot analysis. Association of p27 with ubiquitin E3 ligase was determined with coimmunoprecipitation followed by immunoblot analysis. Inhibitors were used to inhibit proteasomal degradation and nuclear export of the phosphorylated p27. DNA synthesis was measured by BrdU incorporation into DNA.
Among ubiquitin ligase complex proteins, Cul1, KPC1, and KPC2 were constitutively expressed, whereas expression of Skp1 and Skp2 was temporally induced by FGF-2. Skp1, Skp2, and Cul1 were involved in polyubiquitination of phosphorylated p27 at Thr-187 (pp27Thr187) in nuclei. Maximum association of pp27Thr187 with the ubiquitin E3 ligase occurred 24 hours after FGF-2 stimulation. pp27Ser10 used the cytoplasmic ubiquitin E3 ligases KPC1 and KPC2, with maximum protein interaction observed at 8 hours. MG132 effectively blocked degradation of both pp27Thr187 and pp27Ser10, whereas leptomycin B blocked the nuclear export of pp27Ser10. Both inhibitors blocked BrdU incorporation into DNA.
The findings demonstrate distinct polyubiquitination pathways for pp27Thr187 and pp27Ser10; the former is ubiquitinated through the nuclear ubiquitin E3 ligase system during late G(1) phase; the latter by cytosolic ubiquitin E3 ligase during early G(1) phase.
p27（Kip1）（p27）是G1期进程的重要调节因子。细胞增殖时，p27必须经历蛋白水解过程。成纤维细胞生长因子-2（FGF-2）可使p27的苏氨酸-187（Thr-187）和丝氨酸-10（Ser-10）位点发生磷酸化，这是多聚泛素化的前提条件。本研究旨在确定两种磷酸化p27的降解是否由不同亚细胞位置的独特泛素E3连接酶复合物介导。
通过免疫印迹分析检测p27、KPC1、KPC2、Skp1、Skp2和Cul1的表达。通过免疫共沉淀结合免疫印迹分析确定p27与泛素E3连接酶的结合情况。使用抑制剂抑制磷酸化p27的蛋白酶体降解和核输出。通过将5-溴脱氧尿嘧啶核苷（BrdU）掺入DNA来测量DNA合成。
在泛素连接酶复合物蛋白中，Cul1、KPC1和KPC2组成性表达，而Skp1和Skp2的表达受FGF-2的时间诱导。Skp1、Skp2和Cul1参与细胞核中磷酸化p27苏氨酸-187（pp27Thr187）的多聚泛素化。FGF-2刺激后24小时，pp27Thr187与泛素E3连接酶的结合达到最大值。磷酸化p27丝氨酸-10（pp27Ser10）利用细胞质泛素E3连接酶KPC1和KPC2，在8小时时观察到最大蛋白相互作用。MG132有效阻断pp27Thr187和pp27Ser10的降解，而雷帕霉素B阻断pp27Ser10的核输出。两种抑制剂均阻断BrdU掺入DNA。
研究结果表明pp27Thr187和pp27Ser10存在不同的多聚泛素化途径；前者在G1期晚期通过细胞核泛素E3连接酶系统进行泛素化；后者在G1期早期由细胞质泛素E3连接酶进行泛素化。
