Journal: Molecular and Cellular Biology
Article Title: c-Jun N-Terminal Kinase Contributes to Aberrant Retinoid Signaling in Lung Cancer Cells by Phosphorylating and Inducing Proteasomal Degradation of Retinoic Acid Receptor ?
Figure Lengend Snippet: JNK phosphorylates RARα and promotes ubiquitination. (A) JNK phosphorylates RARα in intact cells. HeLa cells were transiently cotransfected with expression vectors containing Flag-tagged RARα and JNK1, labeled with [ 32 P]orthophosphate, treated (+) or not treated (−) with UV radiation, and lysed. RARα was immunoprecipitated (IP) from cell lysates with anti-Flag antibodies, and the immunoprecipitate was visualized by autoradiography (top) or subjected to Western blotting with anti-RARα antibodies (bottom). (B) JNK phosphorylates RARα in vitro. JNK and p38 kinases were immunoprecipitated from HeLa cells after UV irradiation to activate stress pathways and subjected to in vitro kinase assays with GST-RARα, GST-c-Jun, and GST-ATF2 as substrates. GST-c-Jun and GST-ATF2 were positive controls for JNK and p38, respectively. (C) RARα and JNK1 physically interact in vivo. COS-1 cells were transiently cotransfected with expression vectors containing RARα and HA-tagged JNK1 or empty vector (−) and lysed, and the lysates were subjected to immunoprecipitation with RARα antibodies, followed by Western blotting with anti-HA and RAR antibodies. (D) RARα degradation is JNK dependent. GST or GST-RARα was incubated with (lanes 1, 2, 4, and 5) or without (lane 3) active JNK1. Afterwards, the mixture was subjected to an ubiquitination reaction in the presence of an S-100 fraction of HeLa extract, His-ubiquitin (lanes 1, 2, 3, and 5), and ATP (lanes 1 to 4). In lane 5, ATP was replaced by AMP-PNP, which supports ubiquitination but not kinase reactions.
Article Snippet: We purchased and SB203580 (Calbiochem, San Diego, Calif.); His-ubiquitin, MG132, all- trans RA, 9- cis RA, E- 4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl]benzoic acid (TTNPB), ATP, triphosphate 5′-adenylic acid (AMP-PNP), phorbol myristate acetate (PMA), insulin-like growth factor 1, creatine kinase, and phosphocreatine (Sigma-Aldrich, St. Louis, Mo.); lactacystin and Z-VAD-FMK (Biomol, Plymouth Meeting, Pa.); [γ-32 P]ATP, [32 P]orthophosphate, and [35 S]methionine (ICN Biomedicals, Inc., Costa Mesa, Calif.); polyclonal antibodies against human RARα, RARβ, RARγ, RXRα, MKK4, JNK, and poly-ADP ribose polymerase (Santa Cruz Biotechnology, Santa Cruz, Calif.); U0126 and polyclonal antibodies against human phospho-JNK (Thr183/Tyr185), phospho-c-Jun (Ser63), c-Jun, phospho-Erk (Thr202/Tyr204), Erk, phospho-Akt (Ser473), and Akt (Cell Signaling Technology, Beverly, Mass.); monoclonal antibodies against Flag epitope and actin (Sigma-Aldrich); anti-HA antibody (Roche Molecular Biochemicals, Indianapolis, Ind.); and purified recombinant glutathione S -transferase (GST)-ATF2, GST-c-Jun, and HSP27 (Upstate Biotechnology, Lake Placid, N.Y.).
Techniques: Expressing, Labeling, Immunoprecipitation, Autoradiography, Western Blot, In Vitro, Irradiation, In Vivo, Plasmid Preparation, Incubation