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 ERK3 and ERK4 have been identified as activators of MK5, the kinases that activate MK5 remain controversial. 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: treating 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 myofibroblasts, phosphothreonine-182 MK5 (pT182-MK5) immunoreactivity, representing phosphorylation of MK5 within its activation loop, was predominantly nuclear but increased in intensity and relocated to the cytoplasm in response to serum, sorbitol, angiotensin II, TGFβ, or H2O2: this was prevented upon inhibition of p38α/β. 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. These observations suggest that p38α and/or p38β are mediators of T182-MK5 phosphorylation in cardiac myofibroblasts.