Organization of intermediate filament, a major component of cytoskeleton, is regulated by
protein phosphorylation/dephosphorylation, which is a dynamic process governed by a balance between the activities of involved
protein kinases and
phosphatases. Blocking dephosphorylation by
protein phosphatase inhibitors such as
okadaic acid (OA) leads to an apparent activation of
protein kinase(s) and to genuine activation of
phosphatase-regulated
protein kinase(s). Treatment of 9L rat
brain tumor cells with OA results in a drastically increased phosphorylation of
vimentin, an intermediate filament
protein. In-gel renaturing assays and in vitro
kinase assays using
vimentin as the exogenous substrate indicate that certain
protein kinase(s) is activated in OA-treated cells. With specific
protein kinase inhibitors, we show the possible involvement of the cdc2
kinase- and
p38 mitogen-activated protein kinase (p38MAPK)-mediated pathways in this process. Subsequent in vitro assays demonstrate that
vimentin may serve as an excellent substrate for MAPK-activated
protein kinase-2 (MAPKAPK-2), the downstream effector of p38MAPK, and that
MAPKAPK-2 is activated with OA treatment. Comparative analysis of tryptic
phosphopeptide maps also indicates that corresponding
phosphopeptides emerged in
vimentin from OA-treated cells and were phosphorylated by
MAPKAPK-2. Taken together, the results clearly demonstrate that
MAPKAPK-2 may function as a
vimentin kinase in vitro and in vivo. These findings shed new light on the possible involvement of the p38MAPK signaling cascade, via
MAPKAPK-2, in the maintenance of integrity and possible physiological regulation of intermediate filaments.