Rao Prema S, Satelli Arun, Zhang Sheng, Srivastava Sanjay K, Srivenugopal Kalkunte S, Rao U Subrahmanyeswara
Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 Coulter Rd., Amarillo, TX 79106, USA.
Proteomics. 2009 May;9(10):2776-87. doi: 10.1002/pmic.200800847.
RNF2, a member of polycomb group (PcG) proteins, is involved in chromatin remodeling. However, mechanisms that regulate RNF2 function are unknown. To identify such mechanisms, RNF2 was expressed in HEK-293 cells and analyzed by 2-D electrophoresis. RNF2 was resolved into at least seven protein spots, migrating toward the lower pI from its expected pI of 6.38, suggesting that RNF2 undergoes post-translational modifications. Western blotting indicated that majority of these RNF2 spots contained phosphoserine(s), which were completely dephosphorylated upon treatment with a phosphatase. SB203580, a specific inhibitor of p38 MAPK, inhibited RNF2 phosphorylation at one site. On the other hand, PD98059, an inhibitor of MEK1/2, inhibited majority of the phosphorylation events in RNF2. Mass spectrometry analysis identified that RNF2 expressed in Sf9 insect cells undergoes co-translational excision of (1)Met coupled to N-acetylation of (2)Ser, and phosphorylation of (41)Ser. Interestingly, (41)Ser is a predicted p38/MAPK phosphorylation site, consistent with the loss of phosphorylation induced by SB203580. Further analysis indicated that RNF2 phosphorylation differentially modulates the expression of transcription factors and histone 2B acetylation. These results provide first evidence for phosphorylation of RNF2, and suggest that the mitogen activated protein kinases including p38 MAPK and ERK1/2 regulate growth, stress response, differentiation and other cellular processes, through phosphorylation of RNF2.
RNF2是多梳蛋白家族（PcG）的成员之一，参与染色质重塑。然而，调节RNF2功能的机制尚不清楚。为了确定这些机制，RNF2在HEK-293细胞中表达并通过二维电泳进行分析。RNF2被分离为至少七个蛋白点，从预期的6.38的等电点向较低的等电点迁移，这表明RNF2经历了翻译后修饰。蛋白质印迹表明，这些RNF2斑点中的大多数含有磷酸丝氨酸，在用磷酸酶处理后这些磷酸丝氨酸被完全去磷酸化。p38丝裂原活化蛋白激酶（MAPK）的特异性抑制剂SB203580抑制了RNF2在一个位点的磷酸化。另一方面，MEK1/2的抑制剂PD98059抑制了RNF2中的大多数磷酸化事件。质谱分析确定，在Sf9昆虫细胞中表达的RNF2经历了（1）甲硫氨酸的共翻译切除并与（2）丝氨酸的N-乙酰化以及（41）丝氨酸的磷酸化偶联。有趣的是，（41）丝氨酸是一个预测的p38/MAPK磷酸化位点，这与SB203580诱导的磷酸化缺失一致。进一步的分析表明，RNF2的磷酸化差异调节转录因子的表达和组蛋白2B的乙酰化。这些结果为RNF2的磷酸化提供了首个证据，并表明包括p38 MAPK和ERK1/2在内的丝裂原活化蛋白激酶通过RNF2的磷酸化来调节生长、应激反应、分化和其他细胞过程。
