DECREASED PHOSPHORYLATION OF 4 20-KDA PROTEINS PRECEDES STAUROSPORINE-INDUCED DISRUPTION OF THE ACTIN MYOSIN CYTOSKELETON IN RAT ASTROCYTES/

The changes in protein phosphorylation and cytoskeletal structure prec eding the dramatic morphological changes in staurosporine-treated rat astrocytes were examined, and the dependence of these effects on prote in kinase C (PKC) was studied. Fluorescence and photoelectron microsco py revealed that a 20-min exposure to the kinase inhibitor staurospori ne at 100 nM substantially decreased the thickness and linear appearan ce of actin microfilament bundles (stress fibers) prior to major chang es in cell shape, while 60 min of staurosporine depleted virtually all microfilament bundles and caused arborization and contraction of the cell body. The distribution of myosin light chain (MLC) labeling withi n the cytoplasm was also dramatically altered by staurosporine, progre ssing from a linear punctate pattern coincident with the linear patter n of filamentous actin to a diffuse pattern in cells in which microfil ament dissolution was taking place. Two-dimensional gel analysis of as trocyte phosphoproteins demonstrated 50-80% reduction of P-32 incorpor ation into four 20-kDa spots, one of which was recognized by an antibo dy to MLC, following a 15-min treatment with 100 nM staurosporine. Dep letion of functional PKC from astrocytes by a 24-h exposure to phorbol myristate acetate prior to staurosporine exposure did not reduce the extent of the cytoskeletal alterations or alter the decrease in protei n phosphorylation. Two other protein kinase inhibitors which affect as trocyte morphology, H-7 and the MLC kinase inhibitor ML-9, were also o bserved to disrupt microfilament bundles with accompanying decreases i n P-32 incorporation into these same phosphoproteins, whereas the more selective PKC inhibitor Ro 31-8220 did not do either. The early onset of decreased phosphorylation of the 20-kDa proteins supports a direct relationship between the rapid dissociation of myosin light chain fro m actin microfilament bundles, the disruption of actin patterns, and t he subsequent morphological alterations. These data also suggest that staurosporine and H-7 may exert their effects via a pathway involving inhibition of MLC kinase. (C) 1994 Academic Press, Inc.