Expression, subcellular localization, and phosphorylation of MK5 in adult cardiac ventricular fibroblasts

MAP kinase-activated protein kinase-5 (MK5) plays an important role in cardiac fibroblast function. Although p38 MAPK and atypical MAPKs and ERK3 and ERK4 have been identified as activators of MK5, the kinases that activate MK5 remain controversial. Here we examined the expression, subcellular distribution, and regulation of MK5 in cardiac ventricular myofibroblasts and myocytes. The copy numbers for MK5 and ERK4 mRNA were comparable in myocytes and myofibroblasts, whereas that of ERK3 was much higher in myofibroblasts. Interestingly, MK5 and ERK3 immunoreactivity was detected in myofibroblasts but not myocytes whereas ERK4 immunoreactivity was detected in myocytes: treating in myocytes with a proteasome inhibitor or hypertrophic agonists failed to rescue MK5 immunoreactivity. In myofibroblasts, MK5 and ERK3 immunoreactivity was predominantly nuclear and cytosolic, respectively. In serum-starved cardiac myofibroblasts, phosphothreonine-182 MK5 (pT182-MK5) immunoreactivity was predominantly nuclear but increased in intensity and relocated to the cytoplasm in response to serum, sorbitol, angiotensin II, TGFβ, or HO and this was prevented by inhibition of p38/. Phos-tag SDS-PAGE revealed multiple slower migrating bands of MK5 immunoreactivity, indicating phosphorylation of MK5 at multiple sites. Phos-tag PAGE also revealed MK5 phosphorylation was increased with fibroblast activation and in hearts exposed to a chronic increase in afterload. MK5 and ERK3 co-immunoprecipitated and proximity ligation assays revealed ERK3 and MK5 in close proximity in myofibroblast cytoplasmic compartment. Furthermore, p38/ inhibition decreased the abundance of MK5 immunoreactivity in ERK3 immunoprecipitates. Finally, deleting MK5 did not reduce the abundance of ERK3 immunoreactivity. These observations suggest that p38 and/or p38 are the primary mediators of T182-MK5 phosphorylation and hence MK5 activation in cardiac myofibroblasts.