Phosphorylation Of Mk5 At Threonine-182 In The Activation Loop Is Mediated By P38 Alpha/Beta In Cardiac Fibroblasts

MAP kinase-activated protein kinase-5 (MK5) is a protein serine/threonine kinase involved in fibroblast function. MK5 is activated by phosphorylation at threonine-182 (Thr182): p38α/β, ERK3, and ERK4 have been implicated. We examined the phosphorylation of MK5 in adult cardiac ventricular fibroblasts. In serum-starved cardiac myofibroblasts (fibroblasts maintained on a plastic substrate), phospho-MK5 Thr182 (pThr182) immunoreactivity was predominantly nuclear. In response to serum, sorbitol, angiotensinII, TGFβ, or H 2 O 2 , pThr182 immunoreactivity both increased in intensity and relocated to the cytoplasm and the perinuclear region. In each case, the p38α/β inhibitor, SB203580, prevented both the increase in intensity and redistribution of pThr182 immunoreactivity. Incontrast, siRNA-mediated knockdown of ERK3 resulted in a diffuse cytosolic distribution of pThr182 immunoreactivity but failed to attenuate the increase in intensity. On Phos-tag PAGE, the electrophoretic mobility of phosphorylated proteins is reduced. Phos-tag PAGE resolved MK5 immunoreactivity from actively dividing myofibroblasts into several slower-migrating bands that were absent following 1) pretreatment with phosphoprotein phosphatase or 2) including EDTA in the Phos-tag gels. In serum-stimulated myofibroblasts, SB203580 reduced both the abundance of lower-mobility forms of MK5 on Phos-tag PAGE and the abundance of MK5 immunoreactivity in ERK3 immunoprecipitates. When fibroblasts were maintained on a compliant (8-kPa) substrate, and hence quiescent, the lower mobility forms of MK5 immunoreactivity were less abundant relative to myofibroblasts. Furthermore, in whole heart lysates from micesacrificed 8 weeks after constriction of the transverse aorta (TAC), Phos-tag PAGE revealed banding patterns consistent with increased MK5 phosphorylation relative to sham hearts. Taken together, these observations suggest: 1) p38α/β are the primarymediators of MK5 phosphorylation at Thr182 in cardiac fibroblasts, 2) ERK3 may be responsible for targeting activated MK5 to specific cytosolic sites, 3) Thr182 is not the only site at which MK5 is phosphorylated in vivo , and 4) MK5 phosphorylation increases with fibroblast activation.