Zhang Yiguo, Cho Yong-Yeon, Petersen Brandon L, Bode Ann M, Zhu Feng, Dong Zigang
Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA.
J Biol Chem. 2003 Apr 11;278(15):12650-9. doi: 10.1074/jbc.M210368200. Epub 2003 Jan 31.
Phosphorylation at Ser(727) is known to be required for complete activation of STAT3 by diverse stimuli including UV irradiation, but the kinase(s) responsible for phosphorylating STAT3 (Ser(727)) is still not well discerned. In the present study, we observed that activation of ATM is required for a UVA-stimulated increase in Ser(727) phosphorylation of STAT3 as well as in activation and phosphorylation of p90 ribosomal protein S6 kinases (RSKs). Moreover, UVA-stimulated activation of upstream kinases, such as c-Jun N-terminal kinases (JNKs) and ERKs, involved in mediating phosphorylation of RSKs and STAT3 was defective or delayed in ATM-deficient cells. Furthermore, we provide evidence that RSK2-deficient cells were defective for UV-induced Ser(727) phosphorylation of STAT3, and the defect was restored after ectopic expression of transfected full-length RSK2. In vitro experiments showed that active RSK2 and JNK1 induce the phosphorylation of STAT3 precipitates from immunoprecipitation but not from glutathione S-transferase (GST) pull-down. Interestingly, the GST fusion STAT3 proteins mixed together with STAT3 immunoprecipitates can be phosphorylated by JNK. However, the in vitro phosphorylation of STAT3 was reduced by the GST-STAT3 beta protein, a dominant negative form of STAT3. Taken together, our results demonstrate that the STAT3 phosphorylation at Ser(727) is triggered by active RSK2 or JNK1 in the presence of a downstream kinase or a cofactor, and thereby the intracellular phosphorylation process is stimulated through a signaling pathway involving ATM, MAPKs, RSK2, and an as yet unidentified kinase or cofactor. Additionally, RSK2-mediated phosphorylation of STAT3 (Ser(727)) was further determined to be required for basal and UVA-stimulated STAT3 transcriptional activities.
已知包括紫外线照射在内的多种刺激可使STAT3完全激活，而这需要Ser(727)位点发生磷酸化，但负责使STAT3（Ser(727)）磷酸化的激酶仍未完全明确。在本研究中，我们观察到，UVA刺激引起的STAT3的Ser(727)磷酸化增加以及p90核糖体蛋白S6激酶（RSKs）的激活和磷酸化需要ATM激活。此外，参与介导RSKs和STAT3磷酸化的上游激酶，如c-Jun氨基末端激酶（JNKs）和细胞外信号调节激酶（ERKs），在UVA刺激下的激活在ATM缺陷细胞中存在缺陷或延迟。此外，我们提供的证据表明，RSK2缺陷细胞在紫外线诱导的STAT3的Ser(727)磷酸化方面存在缺陷，转染全长RSK2的异位表达后该缺陷得以恢复。体外实验表明，活性RSK2和JNK1可诱导免疫沉淀法获得的STAT3沉淀发生磷酸化，但不能诱导谷胱甘肽S-转移酶（GST）下拉法获得的STAT3沉淀发生磷酸化。有趣的是，与STAT3免疫沉淀产物混合在一起的GST融合STAT3蛋白可被JNK磷酸化。然而，GST-STAT3β蛋白（一种STAT3的显性负性形式）可降低STAT3的体外磷酸化。综上所述，我们的结果表明，在存在下游激酶或辅因子的情况下，活性RSK2或JNK1可触发STAT3在Ser(727)位点的磷酸化，从而通过涉及ATM、丝裂原活化蛋白激酶（MAPKs）、RSK2以及一种尚未确定的激酶或辅因子的信号通路刺激细胞内磷酸化过程。此外，还进一步确定RSK2介导的STAT3（Ser(727)）磷酸化是基础和UVA刺激的STAT3转录活性所必需的。
