Phosphorylation of interleukin (IL)-24 is required for mediating its anti-cancer activity

// Janani Panneerselvam 1, 4, * , Manish Shanker 6, 8, * , Jiankang Jin 6, 9, * , Cynthia D. Branch 6, 10 , Ranganayaki Muralidharan 1, 4 , Yan D. Zhao 3, 4 , Sunil Chada 7 , Anupama Munshi 2, 4 , Rajagopal Ramesh 1, 4, 5 1 Department of Pathology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA 2 Department of Radiation Oncology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA 3 Department of Biostatistics and Epidemiology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA 4 Stephenson Cancer Center, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA 5 Graduate Program in Biomedical Sciences, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA 6 Department of Thoracic & Cardiovascular Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA 7 DNASolve, Houston, Texas, USA 8 The University of Texas Dental School, Houston, Texas, USA 9 Department of Gastrointestinal Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 10 Department of Plastic Surgery, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA * These authors have contributed equally to this work Correspondence to: Rajagopal Ramesh, e-mail: rajagopal-ramesh@ouhsc.edu Keywords: IL-24, phosphorylation, lung cancer, cytokine Received: March 27, 2015      Accepted: May 06, 2015      Published: May 18, 2015 ABSTRACT Interleukin ( IL )- 24 is a tumor suppressor/cytokine gene that undergoes post-translational modifications (PTMs). Glycosylation and ubiquitination are important for IL-24 protein stabilization and degradation respectively. Little is known about IL-24 protein phosphorylation and its role in IL-24-mediated anti-tumor activities. In this study we conducted molecular studies to determine whether IL-24 phosphorylation is important for IL-24-mediated anti-cancer activity. Human H1299 lung tumor cell line that was stably transfected with a doxycycline (DOX)-inducible (Tet-on) plasmid vector carrying the cDNA of IL-24 -wild-type (IL-24 wt ) or IL-24 with all five phosphorylation sites replaced (IL-24 mt ) was used in the present study. Inhibition of tumor cell proliferation, cell migration and invasion, and induction of G2/M cell cycle arrest was observed in DOX-induced IL-24 wt -expressing cells but not in IL-24 mt -expressing cells. Secretion of IL-24 mt protein was greatly reduced compared to IL-24 wt protein. Further, IL-24 wt and IL-24 mt proteins markedly differed in their subcellular organelle localization. IL-24 wt but not IL-24 mt inhibited the AKT/mTOR signaling pathway. SiRNA-mediated AKT knockdown and overexpression of myristolyated AKT protein confirmed that IL-24 wt but not IL-24 mt mediated its anti-cancer activity by inhibiting the AKT signaling pathway. Our results demonstrate that IL-24 phosphorylation is required for inhibiting the AKT/mTOR signaling pathway and exerting its anti-cancer activities.