Zhang Y, Dong Z, Nomura M, Zhong S, Chen N, Bode A M, Dong Z
Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA.
J Biol Chem. 2001 Jun 15;276(24):20913-23. doi: 10.1074/jbc.M009047200. Epub 2001 Mar 28.
Ultraviolet light A (UVA) plays an important role in the etiology of human skin cancer, and UVA-induced signal transduction has a critical role in UVA-induced skin carcinogenesis. The upstream signaling pathways leading to p70(S6K) phosphorylation and activation are not well understood. Here, we observed that UVA induces phosphorylation and activation of p70(S6K). Further, UVA-stimulated p70(S6K) activity and phosphorylation at Thr(389) were blocked by wortmannin, rapamycin, PD98059, SB202190, and dominant negative mutants of phosphatidylinositol (PI) 3-kinase p85 subunit (DNM-Deltap85), ERK2 (DNM-ERK2), p38 kinase (DNM-p38), and JNK1 (DNM-JNK1) and were absent in Jnk1-/- or Jnk2-/- knockout cells. The p70(S6K) phosphorylation at Ser(411) and Thr(421)/Ser(424) was inhibited by rapamycin, PD98059, or DNM-ERK2 but not by wortmannin, SB202190, DNM-Deltap85, or DNM-p38. However, Ser(411), but not Thr(421)/Ser(424) phosphorylation, was suppressed in DNM-JNK1 and abrogated in Jnk1-/- or Jnk2-/- cells. In vitro assays indicated that Ser(411) on immunoprecipitated p70(S6K) proteins is phosphorylated by active JNKs and ERKs, but not p38 kinase, and Thr(421)/Ser(424) is phosphorylated by ERK1, but not ERK2, JNKs, or p38 kinase. Moreover, p70(S6K) co-immunoprecipitated with PI 3-kinase and possibly PDK1. The complex possibly possessed a partial basal level of phosphorylation, but not at MAPK sites, which was available for its activation by MAPKs in vitro. Thus, these results suggest that activation of MAPKs, like PI 3-kinase/mTOR, may be involved in UVA-induced phosphorylation and activation of p70(S6K).
紫外线A（UVA）在人类皮肤癌的病因学中起重要作用，且UVA诱导的信号转导在UVA诱导的皮肤癌发生中起关键作用。导致p70（S6K）磷酸化和激活的上游信号通路尚未完全明确。在此，我们观察到UVA诱导p70（S6K）磷酸化和激活。此外，渥曼青霉素、雷帕霉素、PD98059、SB202190以及磷脂酰肌醇（PI）3激酶p85亚基的显性负性突变体（DNM-Deltap85）、ERK2（DNM-ERK2）、p38激酶（DNM-p38）和JNK1（DNM-JNK1）可阻断UVA刺激的p70（S6K）活性及苏氨酸（Thr）389位点的磷酸化，且在Jnk1-/-或Jnk2-/-基因敲除细胞中不存在这种情况。雷帕霉素、PD98059或DNM-ERK2可抑制丝氨酸（Ser）411、苏氨酸（Thr）421/丝氨酸（Ser）424位点的p70（S6K）磷酸化，但渥曼青霉素、SB202190、DNM-Deltap85或DNM-p38则不能。然而，DNM-JNK1可抑制Ser411位点的磷酸化，但不能抑制Thr421/Ser424位点的磷酸化，且在Jnk1-/-或Jnk2-/-细胞中该位点的磷酸化被消除。体外实验表明，免疫沉淀的p70（S6K）蛋白上的Ser411位点可被活性JNKs和ERKs磷酸化，但不能被p38激酶磷酸化，而Thr421/Ser424位点可被ERK1磷酸化，但不能被ERK2、JNKs或p38激酶磷酸化。此外，p70（S6K）与PI 3激酶共免疫沉淀，可能还与PDK1共沉淀。该复合物可能具有部分基础水平的磷酸化，但在丝裂原活化蛋白激酶（MAPK）位点没有，这使其在体外可被MAPKs激活。因此，这些结果表明，与PI 3激酶/mTOR一样，MAPKs的激活可能参与UVA诱导的p70（S6K）磷酸化和激活。
