title: Endoplasmic reticulum-associated ubiquitin-conjugating enzyme Ube2j1 is a novel substrate of MK2 (MAPKAP kinase-2) involved in MK2-mediated TNFα production.
abstract: The p38 MAPK (mitogen-activated protein kinase)/MK2 [MAPKAP (MAPK-activated protein) kinase-2] signalling pathway is a major regulator of stress- and cytokine-induced gene expression at the transcriptional and post-transcriptional level. Using phosphoproteomics we identified the ER (endoplasmic reticulum)-associated ubiquitin-conjugating enzyme Ube2j1 as a potential substrate of MK2. We demonstrate that Ube2j1 is phosphorylated in a cytokine-, cytosolic stress- and LPS (lipopolysaccharide)-induced manner. The cytosolic stress-induced phosphorylation of Ube2j1 proceeds at Ser(184), a site described previously to be phosphorylated in response to ER stress, which is located in a perfect MK2 consensus motif. The cytosolic stress-induced phosphorylation of Ube2j1, but not its ER-stress-induced phosphorylation is sensitive to p38/MK2 inhibitors and abrogated in MK2/MK3-deficient cells. In a pull-down assay we demonstrate the interaction of MK2 with Ube2j1 in HEK (human embryonic kidney)-293T cells. Furthermore, MK2 is able to phosphorylate recombinant Ube2j1, but not the S184A mutant in an in vitro kinase assay. These findings strongly suggest that MK2 directly phosphorylates Ube2j1 at Ser(184) upon p38-activating stress in vivo. However, ectopically expressed Ube2j1-S184A mutant displays ubiquitinating activity towards the model substrate ER-synthesized T-cell receptor-α similar to that of the wild-type protein. Interestingly, Ube2j1 is phosphorylated in response to LPS also in macrophages and contributes to MK2-dependent TNFα biosynthesis by a so far unknown mechanism.
authors.affiliation: *Institute of Physiological Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.
authors.forename: Manoj B
authors.initials: MB
authors.lastname: Menon
authors.forename: Christopher
authors.initials: C
authors.lastname: Tiedje
authors.forename: Juri
authors.initials: J
authors.lastname: Lafera
authors.forename: Natalia
authors.initials: N
authors.lastname: Ronkina
authors.forename: Timo
authors.initials: T
authors.lastname: Konen
authors.forename: Alexey
authors.initials: A
authors.lastname: Kotlyarov
authors.forename: Matthias
authors.initials: M
authors.lastname: Gaestel
mesh_terms: D000818:Animals; D002460:Cell Line; D059865:Endoplasmic Reticulum Stress; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000276:immunology / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D059748:Proteolysis; D013312:Stress, Physiological; D051193:Toll-Like Receptors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D054875:Ubiquitination; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000099190:MAP-Kinase-Activated Kinase 2
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D044763:Ubiquitin-Conjugating Enzymes; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2; C585487:Ube2j1 protein, mouse; C098416:MAP-kinase-activated kinase 3