Ataxia telangiectasia mutated proteins, MAPKs, and RSK2 are involved in the phosphorylation of STAT3

Phosphorylation at Ser⁷²⁷ is known to be required for complete activation of STAT3 by diverse stimuli including UV irradiation, but the kinase(s) responsible for phosphorylating STAT3 (Ser⁷²⁷) is still not well discerned. In the present study, we observed that activation of ATM is required for a UVA-stimulated increase in Ser⁷²⁷ 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 Ser⁷²⁷ 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 protein, a dominant negative form of STAT3. Taken together, our results demonstrate that the STAT3 phosphorylation at Ser⁷²⁷ 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 (Ser⁷²⁷) was further determined to be required for basal and UVA-stimulated STAT3 transcriptional activities.