Venture Business Laboratory, Center for Innovation, Kanazawa University, Kakuma, Kanazawa 920-1192, Ishikawa, Japan.
J Cell Sci. 2011 Aug 15;124(Pt 16):2816-25. doi: 10.1242/jcs.083931.
Cdc25A, which is one of the three mammalian CDK-activating Cdc25 protein phosphatases (Cdc25A, B and C), is degraded through SCF(βTrCP)-mediated ubiquitylation following genomic insult; however, the regulation of the stability of the other two Cdc25 proteins is not well understood. Previously, we showed that Cdc25B is primarily degraded by cellular stresses that activate stress-activated MAPKs, such as Jun NH(2)-terminal kinase (JNK) and p38. Here, we report that Cdc25B was ubiquitylated by SCF(βTrCP) E3 ligase upon phosphorylation at two Ser residues in the βTrCP-binding-motif-like sequence D(94)AGLCMDSPSP(104). Point mutation of these Ser residues to alanine (Ala) abolished the JNK-induced ubiquitylation by SCF(βTrCP), and point mutation of DAG to AAG or DAA eradicated both βTrCP binding and ubiquitylation. Further analysis of the mode of βTrCP binding to this region revealed that the PEST-like sequence from E(82)SS to D(94)AG is crucially involved in both the βTrCP binding and ubiquitylation of Cdc25B. Furthermore, the phospho-mimetic replacement of all 10 Ser residues in the E(82)SS to SPSP(104) region with Asp resulted in βTrCP binding. Collectively, these results indicate that stress-induced Cdc25B ubiquitylation by SCF(βTrCP) requires the phosphorylation of S(101)PS(103)P in the βTrCP-binding-motif-like and adjacent PEST-like sequences.
Cdc25A 是三种哺乳动物 CDK 激活 Cdc25 蛋白磷酸酶之一（Cdc25A、B 和 C），在基因组损伤后通过 SCF（βTrCP）介导的泛素化降解；然而，其他两种 Cdc25 蛋白的稳定性调节尚不清楚。先前，我们表明 Cdc25B 主要通过激活应激激活 MAPK（如 Jun NH2-末端激酶（JNK）和 p38）的细胞应激降解。在这里，我们报告 Cdc25B 在两个 Ser 残基在 βTrCP 结合基序样序列 D（94）AGLCMDSPSP（104）处被磷酸化后被 SCF（βTrCP）E3 连接酶泛素化。这些 Ser 残基突变为丙氨酸（Ala）可消除 JNK 诱导的 SCF（βTrCP）泛素化，而 DAG 突变为 AAG 或 DAA 则消除了 βTrCP 结合和泛素化。对该区域的 βTrCP 结合模式的进一步分析表明，从 E（82）SS 到 D（94）AG 的 PEST 样序列对于 Cdc25B 的 βTrCP 结合和泛素化至关重要。此外，用 Asp 替换 E（82）SS 到 SPSP（104）区域中的 10 个 Ser 残基的磷酸模拟替换导致 βTrCP 结合。总之，这些结果表明，应激诱导的 Cdc25B 通过 SCF（βTrCP）的泛素化需要βTrCP 结合基序样和相邻 PEST 样序列中 S（101）PS（103）P 的磷酸化。
