Inhibition of p70 S6 kinase (S6K1) activity by A77 1726, the active metabolite of leflunomide, induces autophagy through TAK1-mediated AMPK and JNK activation

// Xiulong Xu 1, 2, 3, 8 , Jing Sun 1, 2 , Ruilong Song 4 , Michelle E. Doscas 3 , Ashley J. Williamson 5 , Jingsong Zhou 6 , Jun Sun 7 , Xinan Jiao 8, 9 , Xiufan Liu 9, 10 , Yi Li 11 1 Institute of Comparative Medicine, Yangzhou University, Yangzhou 225009, Jiangsu Province, P. R. China 2 College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, Jiangsu Province, P. R. China 3 Department of Anatomy and Cell Biology Rush University Medical Center, Chicago, IL 60612, USA 4 Core Facility, Yangzhou University, Yangzhou 225009, Jiangsu Province, P. R. China 5 Rush Medical College, Chicago, IL 60612, USA 6 Department of Physiology, Kansas City University of Medicine and Biosciences, Kansas City, MO 64106, USA 7 Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA 8 Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou 225009, Jiangsu Province, China 9 Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou 225009, Jiangsu Province, China 10 Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China 11 Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX 77030, USA Correspondence to: Xiulong Xu, email: xxl@yzu.edu.cn , xxu@rush.edu Keywords: p70 S6 kinase ULK, autophagy, leflunomide, mTOR Received: August 10, 2016     Accepted: February 03, 2017     Published: March 31, 2017 ABSTRACT mTOR activation suppresses autophagy by phosphorylating ULK1 at S757 and suppressing its enzymatic activity. Here we report that feedback activation of mTOR in the PI-3 kinase pathway by two p70 S6 kinase (S6K1) inhibitors (PF-4708671 and A77 1726, the active metabolite of an immunosuppressive drug leflunomide) or by S6K1 knockdown did not suppress but rather induced autophagy. Suppression of S6K1 activity led to the phosphorylation and activation of AMPK, which then phosphorylated ULK1 at S555. While mTOR feedback activation led to increased phosphorylation of ULK1 at S757, this modification did not the disrupt ULK1-AMPK interaction nor dampen ULK1 S555 phosphorylation and the induction of autophagy. In addition, inhibition of S6K1 activity led to JNK activation, which also contributed to autophagy. 5Z-7-oxozeaenol, a specific inhibitor of TAK1, or TAK1 siRNA blocked A77 1726-induced activation of AMPK and JNK, and LC3 lipidation. Taken together, our study establishes S6K1 as a key player in the PI-3 kinase pathway to suppress autophagy through inhibiting AMPK and JNK in a TAK1-dependent manner.