Decreased phosphorylation of four 20-kDa proteins precedes staurosporine-induced disruption of the actin/myosin cytoskeleton in rat astrocytes

The changes in protein phosphorylation and cytoskeletal structure preceding the dramatic morphological changes in staurosporine-treated rat astrocytes were examined, and the dependence of these effects on protein kinase C (PKC) was studied. Fluorescence and photoelectron microscopy revealed that a 20-min exposure to the kinase inhibitor staurosporine at 100 nM substantially decreased the thickness and linear appearance of actin microfilament bundles (stress fibers) prior to major changes 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 within the cytoplasm was also dramatically altered by staurosporine, progressing from a linear punctate pattern coincident with the linear pattern of filamentous actin to a diffuse pattern in cells in which microfilament dissolution was taking place. Two-dimensional gel analysis of astrocyte phosphoproteins demonstrated 50-80% reduction of ³²P incorporation into four 20-kDa spots, one of which was recognized by an antibody to MLC, following a 15-min treatment with 100 nM staurosporine. Depletion 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 protein phosphorylation. Two other protein kinase inhibitors which affect astrocyte morphology, H-7 and the MLC kinase inhibitor ML-9, were also observed to disrupt microfilament bundles with accompanying decreases in ³²P 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 from actin microfilament bundles, the disruption of actin patterns, and the subsequent morphological alterations. These data also suggest that staurosporine and H-7 may exert their effects via a pathway involving inhibition of MLC kinase.