A Phosphoproteomic Screen Identifies a Guanine Nucleotide Exchange Factor for Rab3A Protein as a Mitogen-activated Protein (MAP) Kinase Phosphatase-5-regulated MAP Kinase Target in Interleukin 6 (IL-6) Secretion and Myogenesis

Abstract

Edited by Henrik G. DohlmanThe mitogen-activated protein kinases (MAPKs) have been shown to regulate skeletal muscle function. Previously, we showed that MAPK phosphatase-5 (MKP-5) negatively regulates myogenesis and regeneration of skeletal muscle through inhibition of p38 MAPK and c-Jun N-terminal kinase (JNK). However, the identity and contribution of MKP-5-regulated MAPK targets in the control of skeletal muscle function and regenerative myogenesis have not been established. To identify MKP-5-regulated MAPK substrates in skeletal muscle, we performed a global differential phospho-MAPK substrate screen in regenerating skeletal muscles of wild type and MKP-5-deficient mice. We discovered a novel MKP-5-regulated MAPK substrate called guanine nucleotide exchange factor for Rab3A (GRAB) that was hyperphosphorylated on a phospho-MAPK motif in skeletal muscle of MKP-5-deficient mice. GRAB was found to be phosphorylated by JNK on serine 169. Myoblasts overexpressing a phosphorylation-defective mutant of GRAB containing a mutation at Ser-169 to Ala-169 (GRAB-S169A) inhibited the ability of C2C12 myoblasts to differentiate. We found that GRAB phosphorylation at Ser-169 was required for the secretion of the promyogenic cytokine interleukin 6 (IL-6). Consistent with this observation, MKP-5-deficient mice exhibited increased circulating IL-6 expression as compared with wild type mice. Collectively, these data demonstrate a novel mechanism whereby MKP-5-mediated regulation of JNK negatively regulates phosphorylation of GRAB, which subsequently controls secretion of IL-6. These data support the notion that MKP-5 serves as a negative regulator of MAPKdependent signaling of critical skeletal muscle signaling pathways. MAPK phosphatases (MKPs)2 are members of the dual specificity protein-tyrosine phosphatase family that specifically dephosphorylate the MAPKs (1). We have shown that the MKPs can both positively and negatively regulate myogenesis through coordinate MAPK dephosphorylation (2-5). In particular, we have shown that loss of MKP-5 expression in mice results in enhanced satellite cell proliferation and differentiation, which contribute to the increased capacity of skeletal muscle to regenerate (3). Furthermore, genetic ablation of MKP-5 in a model of dystrophic muscle disease abrogates the dystrophic phenotype, suggesting an important role for MKP-5 in skeletal muscle disease progression (3). Thus, understanding the signaling pathway(s) regulated by MKP-5 in skeletal muscle homeostasis might provide new insight into the mechanisms of both skeletal muscle growth and the progression of dystrophic muscle disease.The maintenance of skeletal muscle function is a dynamic process that requires the interplay between skeletal muscle and trophic factors. An imbalance in the equilibrium between the factors that positively and negatively regulate skeletal muscle function is associated with diseases such as atrophy, cancer cachexia, muscular dystrophies, and neuromuscular disorders. Skeletal muscle produces and secretes a variety ...

Dual-specificity MAP kinase phosphatases in health and disease

“… [ 187 ] Protection from DSS-induced intestinal inflammation. [ 191 ] DUSP16 /MKP-7 Impaired GM-CSF-driven proliferation of bone marrow progenitors. [ 195 ] Increased CD4+ T-cell proliferation & a reduced Th17 cell population.…”

“…Interestingly, the effects of DUSP10 /MKP-5 deletion on myogenesis appear to be due to two main effects. Firstly, deletion of DUSP10 /MKP-5 increases MAPK-dependent phosphorylation of guanine nucleotide exchange factor for the Ras-related protein Rab-3A Rab3A (GRAB) at Serine 169, a site required for secretion of the promyogenic cytokine IL-6 [ 191 ]. Secondly, in the absence of DUSP10 /MKP-5 increased JNK and p38 mediated phosphorylation and activation of STAT3, increases the expression of the anti-apoptotic protein B-cell lymphoma 2 (Bcl2) thus preventing apoptosis during regenerative myogenesis and also leads to improved antioxidant defence capacity due to a sustained increase in catalase expression that protects mitochondrial function [ 192 ].…”

Loss of MKP-5 promotes myofiber survival by activating STAT3/Bcl-2 signaling during regenerative myogenesis

“…In this regard, our group recently demonstrated that MKP-5 regulates myogenesis by mediating MAPK-dependent phosphorylation of the guanine-nucleotide exchange factor for Rab3A (GRAB), which is responsible for the secretion of IL-6. MKP-5-deficient mice, as a result, express increased circulating levels of IL-6 and increased STAT3 activation [ 33 ]. Activated STAT3 couples to the apoptotic pathway by upregulating the anti-apoptotic transcription factor, Bcl-2 [ 34 , 35 ].…”

Regulation of Rab8 in Toll-Like Receptor Signalling Pathways

“…Multiple studies have implicated different kinases that regulate GRAB GEF function in various systems (Figure 1.8). For instance, the serine kinase c-Jun N-terminal kinase (JNK) has been identified to phosphorylate Ser 169 of GRAB in myocytes, promoting the secretion of the important muscle repair cytokine IL-6, however, in this study GRAB GEF function has not yet been tested (Lee and Min et al 2017). Interestingly in another publication, GRAB phosphorylation at Ser 169/180 by the neuron specific proline-directed serine/threonine membrane bound kinase Cdk5 is shown to actually decrease its GEF binding affinity to GDP-bound Rab8, modulating axon outgrowth (Furusawa and Asada et al 2016).…”

“…While it is unknown if GRAB also exists in an autoinhibitory state like Rabin8, it is clear that kinases are important regulators for both of these Rab8 GEFs. (Chiba and Amagai et al 2013, Wang and Ren et al 2015, Furusawa and Asada et al 2016, Lee and Min et al 2017.…”