p38 beta MAPK upregulates atrogin1/MAFbx by specific phosphorylation of C/EBP beta

Background: The p38 mitogen-activated protein kinases (MAPK) family plays pivotal roles in skeletal muscle metabolism. Recent evidence revealed that p38 alpha and p38 beta exert paradoxical effects on muscle protein homeostasis. However, it is unknown why p38 beta, but not p38 alpha, is capable of mediating muscle catabolism via selective activation of the C/EBP beta that upregulates atrogin1/MAFbx. Methods: Tryptic phosphopeptide mapping was carried out to identify p38 alpha- and p38 beta-mediated phosphorylation sites in C/EBP beta. Chromosome immunoprecipitation (ChIP) assay was used to evaluate p38 alpha and p38 beta effect on C/EBP beta binding to the atrogin1/MAFbx promoter. Overexpression or siRNA-mediated gene knockdown of p38 alpha and p38 beta, and site-directed mutagenesis or knockout of C/EBP beta, were used to analyze the roles of these kinases in muscle catabolism in C2C12 myotubes and mice. Results: Cellular expression of constitutively active p38 alpha or p38 beta resulted in phosphorylation of C/EBP beta at multiple serine and threonine residues; however, only p38 beta phosphorylated Thr-188, which had been known to be critical to the DNA-binding activity of C/EBP beta. Only p38 beta, but not p38 alpha, activated C/EBP beta-binding to the atrogin1/MAFbx promoter. A C/EBP beta mutant in which Thr-188 was replaced by alanine acted as a dominant-negative inhibitor of atrogin1/MAFbx upregulation induced by either p38 beta or Lewis lung carcinoma (LLC) cell-conditioned medium (LCM). In addition, knockdown of p38 beta specifically inhibited C/EBP beta activation and atrogin1/MAFbx upregulation induced by LCM. Finally, expression of active p38 beta in mouse tibialis anterior specifically induced C/EBP beta phosphorylation at Thr-188, atrogin1/MAFbx upregulation and muscle mass loss, which were blocked in C/EBP beta-null mice. Conclusions: The alpha and beta isoforms of p38 MAPK are capable of recognizing distinct phosphorylation sites in a substrate. The unique capacity of p38 beta in mediating muscle catabolism is due to its capability in phosphorylating Thr-188 of C/EBP beta.