Department of Biochemistry and Molecular Biology and the Stanley S Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
J Biol Chem. 2010 Dec 31;285(53):41300-9. doi: 10.1074/jbc.M110.169136. Epub 2010 Nov 1.
In CHO cells, CDK1/2-dependent phosphorylation of Ubc2/Rad6 at Ser(120) stimulates its ubiquitin conjugating activity and can be replicated by a S120D point mutant (Sarcevic, B., Mawson, A., Baker, R. T., and Sutherland, R. L. (2002) EMBO J. 21, 2009-2018). In contrast, we find that ectopic expression of wild type Ubc2b but not Ubc2bS120D or Ubc2bS120A in T47D human breast cancer cells specifically stimulates N-end rule-dependent degradation but not the Ubc2-independent unfolded protein response pathway, indicating that the former is E2 limiting in vivo and likely down-regulated by Ser(120) phosphorylation, as modeled by the S120D point mutation. In vitro kinetic analysis shows the in vivo phenotype of Ubc2bS120D and Ubc2bS120A is not due to differences in activating enzyme-catalyzed E2 transthiolation. However, the Ser(120) mutants possess marked differences in their abilities to support in vitro conjugation by the N-end rule-specific E3α/Ubr1 ligase that presumably accounts for their in vivo effects. Initial rate kinetics of human E3α-catalyzed conjugation of the human α-lactalbumin N-end rule substrate shows Ubc2bS120D is 20-fold less active than wild type E2, resulting from an 8-fold increase in K(m) and a 2.5-fold decrease in V(max), the latter reflecting a decreased ability to support the initial step in target protein conjugation; Ubc2bS120A is 8-fold less active than wild type E2 due almost exclusively to a decrease in V(max), reflecting a defect in polyubiquitin chain elongation. These studies suggest a mechanism for the integrated regulation of diverse ubiquitin-dependent signaling pathways through E2 phosphorylation that yields differential effects on its cognate ligases.
在 CHO 细胞中，CDK1/2 依赖性磷酸化 Ubc2/Rad6 丝氨酸 120（Ser120）可刺激其泛素缀合活性，并可由 S120D 点突变体复制（Sarcevic，B.，Mawson，A.，Baker，R.T.，和 Sutherland，R.L.（2002）EMBO J. 21，2009-2018）。相比之下，我们发现，在 T47D 人乳腺癌细胞中外源性表达野生型 Ubc2b 但不是 Ubc2bS120D 或 Ubc2bS120A 特异性地刺激 N 末端规则依赖性降解但不是 Ubc2 非依赖性未折叠蛋白反应途径，表明前者在体内是 E2 限制的，可能通过 Ser120 磷酸化下调，如 S120D 点突变所模拟。体内动力学分析显示，Ubc2bS120D 和 Ubc2bS120A 的体内表型不是由于激活酶催化的 E2 转硫反应的差异。然而，Ser120 突变体在支持 N 末端规则特异性 E3α/Ubr1 连接酶体外缀合的能力上存在明显差异，这可能解释了它们的体内效应。人 E3α 催化的人α-乳白蛋白 N 末端规则底物的人 Ubc2b 的初始速率动力学显示，Ubc2bS120D 的活性比野生型 E2 低 20 倍，这是由于 K(m)增加 8 倍和 V(max)降低 2.5 倍的结果，后者反映了在靶蛋白缀合的初始步骤中支持能力降低；Ubc2bS120A 的活性比野生型 E2 低 8 倍，几乎完全是由于 V(max)降低，反映了多泛素链延伸缺陷。这些研究表明，E2 磷酸化通过对其同源连接酶产生不同的影响，从而对不同的泛素依赖性信号通路进行整合调节的机制。
