Endoplasmic reticulum-associated ubiquitin conjugating enzyme- Ube2j1 is a novel substrate of MAPKAP kinase-2 involved in MK2-mediated TNF{alpha} production

The p38 MAPK/MK2 signalling pathway is a major regulator of stress- and cytokine-induced gene expression at the transcriptional and post-transcriptional level. By phospho-proteomics we identified the ER-associated ubiquitin-conjugating enzyme Ube2j1 as a potential substrate of MK2. We demonstrate that Ube2j1 is phosphorylated in a cytokine-, cytosolic stress- and LPS-induced manner. The cytosolic stress-induced phosphorylation of Ube2j1 proceeds at serine 184, a site previously described 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 MK2/3-deficient cells. In a pull down assay we demonstrate interaction of MK2 with Ube2j1 in HEK 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 serine 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.

Source: BJ Cell - September 11, 2013 Category: Biochemistry Authors: M B Menon, C Tiedje, J Lafera, N Ronkina, T Konen, A Kotlyarov, M Gaestel Tags: BJ Signal Source Type: research