Centre de Recherche en Cancérologie de l'Université Laval, Department of Molecular Biology, Medical Biochemistry and Pathology, Hôpital Hôtel-Dieu de Québec, Québec City, Québec, Canada G1R 2J6.
Oncogene. 2010 Mar 11;29(10):1486-97. doi: 10.1038/onc.2009.443. Epub 2009 Dec 7.
Werner's syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCdelta and PKCbetaII in the membrane fraction of cells. In contrast, different DNA-damaging treatments known to activate PKCs did not induce RACK1/PKCs association in cells. Overall, our results indicate that a depletion of the WRN protein in normal fibroblasts causes the activation of several PKCs through translocation and association of RACK1 with such kinases.
沃纳综合征（WS）是一种罕见的常染色体疾病，其特征是多种与年龄相关的病理表现提前发生。WS 患者缺陷的蛋白（WRN）是一种参与 DNA 修复、复制、转录和端粒维持的解旋酶/核酸外切酶。在这项研究中，我们表明，正常人类成纤维细胞中 WRN 蛋白的敲低会诱导几种蛋白激酶 C（PKC）酶的磷酸化和激活。使用串联亲和纯化策略，我们发现 WRN 与蛋白激酶 C 受体 1（RACK1）物理和功能相互作用，RACK1 是一种高度保守的锚定蛋白，参与多种生物学过程，如细胞生长和增殖。RACK1 与 WRN 的 RQC 结构域强烈结合，与酸性重复区弱结合。纯化的 RACK1 对 WRN 的解旋酶活性没有影响，但在体外选择性抑制 WRN 的核酸外切酶活性。有趣的是，敲低 RACK1 会增加细胞内 DNA 断裂的频率。WRN 蛋白的耗竭反过来导致核内 RACK1 的一部分从核内易位到细胞膜部分与 PKCdelta 和 PKCbetaII 结合并激活它们。相比之下，已知激活 PKCs 的不同 DNA 损伤处理并没有在细胞中诱导 RACK1/PKCs 结合。总的来说，我们的结果表明，正常成纤维细胞中 WRN 蛋白的耗竭通过 RACK1 与这些激酶的易位和结合导致几种 PKC 的激活。
