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MAPK substrate phosphoproteomics screen identifies GRAB as a novel MKP‐5‐regulated MAPK substrate

The mitogen‐activated protein kinases (MAPKs) have been shown to regulate skeletal muscle function. Previously, we showed that MAPK phosphatase‐5 (MKP5) negatively regulates regeneration of skeletal muscle through inhibition of p38 MAPK and c‐Jun N‐terminal kinase (JNK) activities. However, the identity and contribution of MKP5‐dependent MAPK targets in the regulation of skeletal muscle function and regenerative myogenesis has not been established. In order to identify MKP5‐dependent MAPK substrates in skeletal muscle, we performed a global differential phospho‐MAPK substrate screen in regenerating skeletal muscles of wild type and Mkp5−/− mice. The results of this screen identified several MKP5‐dependent MAPK substrates that were differentially phosphorylated during muscle regeneration. We discovered a novel MKP5‐regulated MAPK substrate, called guanine nucleotide exchange factor for Rab3A (GRAB) that was hyper phosphorylated by 8‐fold in skeletal muscles of Mkp5−/− mice as compared with wild type mice. We identified that GRAB serine 169 is a novel site of phosphorylation mediated by JNK. Myoblasts overexpressing a phosphorylation‐defective mutant of GRAB (GRAB‐S169A) did not impair myoblast proliferation but inhibited the ability of C2C12 myoblasts to differentiate. We found that GRAB phosphorylation at Ser169 was required for the secretion of the pro‐myogenic cytokine interleukin 6 (IL‐6). Consistent with this observation, MKP5‐deficient mice exhibited increased circulating expression of IL‐6 as compared with wild type mice. Collectively, these data demonstrate a novel mechanism whereby MKP5‐mediated regulation of MAPK activity negatively regulates phosphorylation of GRAB which in turn is responsible for the secretion of the pro‐myogenic cytokine, IL‐6. These data are consistent with the notion that MKP5 serves as an essential regulator of MAPK‐dependent signaling in skeletal muscle.