PHOSPHORYLATION OF INTERMEDIATE FILAMENTS DURING MITOSIS

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Intermediate filaments are a major component of the cytoskeleton of animal cells. Preliminary evidence indicates that intermediate filaments of vimentin, desmin, and cytokeratin types undergo an increased phosphorylation in cultured cells during mitosis when filament organization is altered. Experiments with vimentin-type filaments show that the change in phosphorylation in mitotic cells is serine site specific. The proposed experiments will use cultured mouse (L-929), hamster (BHK21) and human (A-431) cells that have been double thymidine blocked to produce non-mitotic cells, and cells mitotically selected after release from the thymidine block. Phosphorylated intermediate filament proteins will be isolated from these mitotic and non-mitotic cells and specific fragments or domains produced by chemical and limited enzymatic cleavage. These domain fragments will be peptide mapped to determine the functional area of the protein that is specifically modified at mitosis. Experiments will be conducted with intact cells and in vitro to determine if a unique vimentin protein kinase activity is expressed in mitotic cells. In vivo and in vitro phosphorylation experiments will also attempt to determine if the mitosis-specific phosphorylation is a cAMP-independent process. Experiments will be performed on vimentin filaments isolated from mitotic and non-mitotic cells and phosphorylated in vitro with purified protein kinase to determine if there is a change in serine site availability in filaments during mitosis. Finally, quantitative peptide mapping experiments will be carried out to determine the relative occupancy of phosphate at serine sites on vimentin isolated from mitotic and non-mitotic cells. The goal of these studies is to determine if a specific modification of intermediate filament phosphorylation represents a regulatory mechanism in cytoskeletal organization important in the control of cell proliferation. The long term objective is to understand if this mechanism is involved in the maintenance of the transformed or neoplastic phenotype.