Protein kinase C‐induced redistribution of the cytoskeleton and phosphorylation of vimentin in cultured brain macrophages

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Abstract
The phorbol ester 12‐O‐tetradecanoyl‐acetate (TPA) induced prominent and transient changes in the organization of the cytoskeleton in cultured amoeboid microglial cells including redistribution of actin toward the center of the cells and in the subplasmalemmal region, appearance of fine actin filaments, retraction of micro tubules (MT), and rearrangement of intermediate filaments (IF) containing vimentin. The possible implication of protein kinase C (PKC) in mediating the effects of TPA was suggested by a parallel shift of PKC activity from the soluble to membrane fractions and phosphorylation of several microglial proteins.The rearrangement of IF closely correlated with increased vimentin phosphorylation, detected by pulse labeling of intact cells. Two monoclonal antivimentin antibodies, B3 and V9, showed different staining patterns. Immunoreactivity with the antibody B3 was more restricted and could be abolished by treatment of fixed, permeabilized cells with alkaline phosphatase, thus suggesting that the antibody reacts with a phosphorylated epitope. Using this antibody, rearrangement of IF involving vimentin phosphorylation was detected within 15 to 60 min of treatment with 50 nM TPA and consisted in the appearance of intense perinuclear fluorescent label. This perinuclear fluorescence persisted up to 24 hr after TPA removal and gradually diminished during the following 2 to 3 days. Immunochemical analysis of nonionic detergent‐soluble and ‐insoluble extracts from untreated and TPA‐treated cells revealed no differences in vimentin solubility suggesting that TPA induced vimentin phosphorylation does not result in notable vimentin filament disassembly. However the extent of vimentin degradation was more prominent in TPA‐treated cultures indicating a higher sensitivity of vimentin to proteolytic degradation.The data show that PKC‐mediated phosphorylation of vimentin results in precise spatial and temporal rearrangement of IF which are not associated with altered vimentin solubility, but possibly changes the mechanical properties and interactions of vimentin filaments.