Phosphorylation at Ser727 in signal transducer and activator of transcription 1 (STAT1) is essential for its activation and signal transduction. However, the upstream kinases responsible for phosphorylating Ser727 are still elusive. Here, we provide evidence showing that UVA-induced mitogen-activated protein kinase (MAPK) signaling pathways lead to STAT1 Ser727 phosphorylation. Our experimental results show that UVA-induced Ser727 phosphorylation of STAT1 was, to different degrees, diminished by PD98059 and U0126, two specific inhibitors of MEKs, and SB202190 and PD169316, inhibitors of p38 kinase and c-Jun N-terminal kinases (JNKs), respectively. STAT1 phosphorylation was also blocked by a dominant negative mutant of p38β kinase or JNK1, JNK1- or JNK2-deficiency, or an N-terminal or C-terminal kinase-dead mutant of mitogen- and stress-activated protein kinase 1 (MSK1), a downstream kinase closer to p38 kinase and extracellular signal-regulated kinases (ERKs). In vitro kinase assays using the combined STAT1 proteins as substrates from immunoprecipitation and glutathione S-transferase pull down show that active ERK1, JNK1, p38 kinase, MEK1 and MSK1 stimulated phosphorylation of STAT1 (Ser727) indirectly through an unidentified factor or a downstream kinase. Overall, our data indicate that phosphorylation of STAT1 at Ser727 occurs through diverse MAPK cascades including MEK1, ERKs, p38 kinase, JNKs and MSK1 in the cellular response to UVA.
Bibliographical noteFunding Information:
This work was supported in part by the Hormel Foundation and National Institutes of Health grants CA77646 and CA81064. We are very grateful to Dr Dario R.Alessi for providing the pCMV5-FLAG vector (CMVS), pCMV5-FLAG-wild-type MSK1 (MSK1wt), pCMV5-FLAG-MSK1-A195/N-terminal kinase-dead (MSK1-Nd), or pCMV5-FLAG-MSK1-A565/C-terminal kinase-dead (MSK1-Cd) (37). We thank Dr Nanyue Chen for preparation and identification of Jnk‡=‡, Jnk1ÿ=ÿ or Jnk2ÿ=ÿ, as well as Egfr‡=‡ and Egfrÿ=ÿ fibroblasts (30,32). We thank Dr Ann M.Bode for editorial assistance and Ms Andria Hansen for her secretarial assistance.