Search

Phosphorylation by protein kinases has been established as a key factor in the regulation of cytoskeletal structure. However, little is known about the role of protein phosphatases in cytoskeletal regulation. To assess the possible functions of protein phosphatases in this respect, we studied the effects of the phosphatase inhibitors calyculin A, okadaic acid, and dinophysistoxin 1 (35-methylokadaic acid) on BHK-21 fibroblasts. Within minutes of incubation with these inhibitors, changes are seen in the structural organization of intermediate filaments, followed by a loss of microtubules, as assayed by immunofluorescence. These changes in cytoskeletal structure are accompanied by a rapid and selective increase in vimentin phosphorylation on interphase-specific sites, and they are fully reversible after removal of calyculin A. The results indicate that there is a rapid phosphate turnover on cytoskeletal intermediate filaments and further suggest that protein phosphatases are essential for the maintenance and structural integrity of two major cytoskeletal components.

The colchicine-binding protein in procine-brain tissue is a dimer of molecular weight 110,000 that is believed to be the subunit of neuronal microtubules. Conditions are established under which the dimers aggregate with reproducible kinetics. This aggregation reaction, which is monitored by development of turbidity, has the following characteristics: (a) Colchicine inhibits development of turbidity; (b) the reaction inhibited by colchicine is reversed by long-wave ultraviolet irradiation; (c) the aggregation is temperature-dependent; (d) the reaction is nucleotide triphosphate-specific, being stimulated by 1 mM GTP; (e) the reaction appears to be specific for microtubule subunits since in the presence of other added proteins and in curde cell extracts, only microtubule subunits aggregate. On the basis of these criteria, we conclude that we have established an in vitrosystem for the aggregation of microtubule subunits that shares some of the properties characteristic of the in vivoassembly of cytoplasmic and spindle microtubules.