p38β MAPK upregulates atrogin1/MAFbx by specific phosphorylation of C/EBPβ

Abstract

Background: The p38 mitogen-activated protein kinases (MAPK) family plays pivotal roles in skeletal muscle metabolism. Recent evidence revealed that p38α and p38β exert paradoxical effects on muscle protein homeostasis. However, it is unknown why p38β, but not p38α, is capable of mediating muscle catabolism via selective activation of the C/EBPβ that upregulates atrogin1/MAFbx.

Methods: Tryptic phosphopeptide mapping was carried out to identify p38α- and p38β-mediated phosphorylation sites in C/EBPβ. Chromosome immunoprecipitation (ChIP) assay was used to evaluate p38α and p38β effect on C/EBPβ binding to the atrogin1/MAFbx promoter. Overexpression or siRNA-mediated gene knockdown of p38α and p38β, and site-directed mutagenesis or knockout of C/EBPβ, were used to analyze the roles of these kinases in muscle catabolism in C2C12 myotubes and mice.

Results: Cellular expression of constitutively active p38α or p38β resulted in phosphorylation of C/EBPβ at multiple serine and threonine residues; however, only p38β phosphorylated Thr-188, which had been known to be critical to the DNA-binding activity of C/EBPβ. Only p38β, but not p38α, activated C/EBPβ-binding to the atrogin1/MAFbx promoter. A C/EBPβ mutant in which Thr-188 was replaced by alanine acted as a dominant-negative inhibitor of atrogin1/MAFbx upregulation induced by either p38β or Lewis lung carcinoma (LLC) cell-conditioned medium (LCM). In addition, knockdown of p38β specifically inhibited C/EBPβ activation and atrogin1/MAFbx upregulation induced by LCM. Finally, expression of active p38β in mouse tibialis anterior specifically induced C/EBPβ phosphorylation at Thr-188, atrogin1/MAFbx upregulation and muscle mass loss, which were blocked in C/EBPβ-null mice.

Conclusions: The α and β isoforms of p38 MAPK are capable of recognizing distinct phosphorylation sites in a substrate. The unique capacity of p38β in mediating muscle catabolism is due to its capability in phosphorylating Thr-188 of C/EBPβ.

Figure 1

Isolation of C/EBPβ phosphorylated by p38α or p38β MAPK for tryptic phosphopeptide mapping. HEK293T cells were co-transfected with plasmids encoding C/EBPβ (LAP fused with the FLAG tag) and constitutively active p38α or p38β (fused with the HA tag). After 48 h incubation the cells were lysed. Expression of the transfected plasmids was verified by western blot analysis of HA and FLAG. Activity of over-expressed p38α and p38β was evaluated by western blot analysis of ATF2 activation (A). Overexpressed C/EBPβ was pulled down with FLAG-M2 magnetic beads and separated by SDS-PAGE. The gel was stained with Coommassie Blue R-250 (B). The C/EBPβ band was then cut out and analyzed by tryptic phosphopeptide mapping utilizing mass spectrometry for phosphorylated amino acid residues. MAPK, mitogen-activated protein kinase.

Figure 2

Expression of constitutively active p38β in C2C12 myotubes resulted in specific phosphorylation of C/EBPβ at Thr-188 and upregulation of atrogin1/MAFbx. C2C12 myoblasts were transfected with a plasmid encoding constitutively active p38α, p38β or the empty vector (control). The myoblasts were allowed to differentiate for 96 h to form myotubes that were then harvested and analyzed for ATF2 activation, Thr-188 phosphorylation in C/EBPβ and level of atrogin1/MAFbx using western blotting.

Figure 3

p38β is critical to LCM-induced phosphorylation of C/EBPβ at Thr-188 and upregulation of atrogin1/MAFbx. C2C12 myoblasts were transfected with siRNA as indicated. After differentiation, myotubes were treated with LCM or control medium. In 1 h, levels of p38α and p38β, and phosphorylation of C/EBPβ at Thr-188 were evaluated by western blotting (A). In 8 h, levels of atrogin1/MAFbx were evaluated by western blotting (B). Optical density of the bands that represent C/EBP phosphorylated at Thr-188 or atrogin1/MAFbx was analyzed by ANOVA. *denotes a difference from control without LCM treatment and †denotes a difference from control with LCM treatment (P <0.05). LCM, Lewis lung carcinoma cell-conditioned medium.

Figure 4

p38β specifically activates C/EBPβ-binding to the atrogin1/MAFbx promoter. (A) C2C12 myoblasts were transfected with a plasmid encoding constitutively active p38α, p38β or the empty vector (control). After differentiation, ChIP assay was carried out to evaluate C/EBPβ binding in myotubes to a 190-base pair fragment of the atrogin1/MAFbx promoter that contains the previously identified C/EBPβ-responsive cis-enhancer element [17] using control and the target PCR primers. Pre-immune IgG used as the control to the antibody against C/EBPβ did not pull down the target fragment (data not shown). (B) C2C12 myoblasts were transfected with siRNA as indicated. After differentiation, myotubes were treated with LCM or control medium for 1 h and ChIP assay was carried out to evaluate C/EBPβ binding to the atrogin1/MAFbx promoter. ChIP, chromosome immunoprecipitation; LCM, Lewis lung carcinoma cell-conditioned medium.

Figure 5

C/EBPβ phosphorylation at Thr-188 is crucial for atrogin1/MAFbx upregulation by p38β or LCM. (A) C2C12 myoblasts were transfected with a plasmid encoding constitutively active p38α, p38β or empty vector. A plasmid encoding FLAG-tagged C/EBPβ mutant in which Thr-188 was replaced with alanine (LAP-T188A) or the control vector was co-transfected as indicated. After differentiation, myotubes were lysed and analyzed by western blotting for the expression of LAP-T188A (with antibodies against FLAG and C/EBPβ) and atrogin1/MAFbx. (B) C2C12 myoblasts were transfected with LAP-T188A or the empty vector as control. After differentiation, myotubes were treated with LCM or control medium for 8 h. Lysate of myotubes was analyzed by western blotting for the expression of LAP-T188A (with antibodies against FLAG and C/EBPβ) and atrogin1/MAFbx. Optical density of the bands that represent atrogin1/MAFbx was analyzed by ANOVA. *denotes a difference from control without LCM treatment and †denotes a difference from control with LCM treatment (P <0.05). LCM, Lewis lung carcinoma cell-conditioned medium.

Figure 6

Expression of constitutively active p38β in mouse muscle induces C/EBPβ phosphorylation at Thr-188. Plasmids encoding constitutively active p38α or p38β were transfected into TA of the right leg of wild type or C/EBPβ^−/− mice, and the empty vector into the left leg. In 14 days TA samples were collected, weighed and lysed. Expression of the p38 isoforms was verified by western blot analysis of HA tag expression and ATF2 activation. A representative blot is shown (A). The TA lysate was subjected to western blot analysis of C/EBPβ phosphorylation at Thr-188 and FoxO1/3 activation. Representative blots and densitometry data are shown. Optical density of the bands that represent various proteins was measured. Levels of protein phosphorylation were normalized to that of total proteins. *denotes a difference (P <0.05) based on Student t test (B). TA, tibialis anterior.

Figure 7

Expression of constitutively active p38β in mouse muscle induces atrogin1/MAFbx upregulation and muscle mass loss in a C/EBPβ-dependent manner. The TA lysate derived from Figure 6 was further analyzed for level of atrogin1/MAFbx by western blotting. Representative blots and densitometry data are shown (A). The weight of TA transfected with a plasmid encoding an active p38 isoform was compared to that of TA transfected with empty vector (control) from the same mouse (B). *denotes a difference (P <0.05) based on ANOVA. TA, tibialis anterior.