Intermediate filaments are a major component of the cytoskeleton of eukaryotic cells. Although there appear to be at least five distinct classes of these filaments, cells of mesenchymal origin and most cells in culture contain the intermediate filament composed of the subunit protein vimentin. Vimentin exists in a nonphosphorylated as well as in a phosphorylated form, and there is increased phosphorylation of this protein when the filament undergoes marked redistribution in various cells. The role of phosphorylation on assembly-disassembly and organization of the vimentin filament has remained obscure. We report here a stable and purified system allowing biochemical examination of vimentin filament assembly and disassembly. Using this in vitro system, we carried out stoichiometrical phosphorylations, using purified protein kinases. We obtained evidence for site-specific, phosphorylation-dependent disassembly of the vimentin filament.

Glial fibrillary acidic protein (GFAP) is a component of glial filaments specific to astroglia. We now report the spatial and temporal distributions of four phosphorylated sites in the GFAP molecule during mitosis of astroglial cells, determined by antibodies which can distinguish phosphorylated epitopes from non‐phosphorylated‐epitopes. Immunofluorescence microscopy showed that the Ser8 residues in the entire cytoplasmic glial filament system are initially phosphorylated when the cells enter mitosis. In cytokinesis, the phosphoSer8 residues become dephosphorylated, whereas Thr7, Ser13 and Ser34 in glial filaments at the cleavage furrow become the preferred sites of phosphorylation. The cdc2 kinase purified from mitotic cells can phosphorylate GFAP at Ser8 but not at Thr7, Ser13 or Ser34, in vitro. These results suggest that cdc2 kinase acts as a glial filament kinase only at the G2‐M phase transition while other glial filament kinases are probably activated at the cleavage furrow before final separation of the daughter cells.

Keratins, constituent proteins of intermediate filaments of epithelial cells, are phosphoproteins containing phosphoserine and phosphothreonine. We examined the in vitro phosphorylation of keratin filaments by CAMPdependent protein kinase, protein kinase C and Ca2 +/calmodulin-dependent protein kinase 11. When rat liver keratin filaments reconstituted by type I keratin 18 (molecular mass 47 kDa; acidic type) and type I1 keratin 8 (molecular mass 55 kDa; basic type) in a 1 : 1 ratio were used as substrates, all the protein kinases phosphorylated both of the constituent proteins to a significant rate and extent, and disassembly of the keratin filament structure occurred. Kinetic analysis suggested that all these protein kinases preferentially phosphorylate keratin 8, compared to keratin 18. The amino acid residues of keratins 8 and 18 phosphorylated by CAMP-dependent protein kinase or protein kinase C were almost exclusively serine, while those phosphorylated by Ca2 c calmoddin-dependent protein kinase I1 were serine and threonine. Peptide mapping analysis indicated that these protein kinases phosphorylate keratins 8 and 18 in a different manner. These observations gave the way for in vivo studies of the role of phosphorylation in the reorganization of keratin filaments.

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native NF was nearly the same as that of purified NF-L. However, phosphorylation did not cause the native NFs to disassemble into oligomers, as was the case for purified NF-L. Instead, partial fragmentation was detected in sedimentation experiments and by electron microscopic observations. This is probably not due to the presence of the three NF subunits in NF or to differences in phosphorylation sites because reassembled NF containing all three NF subunits were disassembled into oligomeric forms by phosphorylation with A-kinase and the phosphorylation by A-kinase occurred at the head domain of NF-L whether NF were native or reassembled. Disassembling intermediates of reassembled NF containing all three NF subunits were somewhat different from disassembling intermediates of NF-L. Thinning and loosening of filaments was frequently observed preceding complete disassembly. From the fact that the thinning was also observed in the native filaments phosphorylated by A-kinase, it is reasonable to propose the native NF is fragmented through a process of thinning that is stimulated by phosphorylation in the head domain of the NF subunits.

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ABSTRACT.A monoclonal antibody was raised against the highest molecular weight protein associated with microtubules (MAP-1). Its specific binding to MAP-1 was determined by immunoblotting of the gel electrophoretogram of microtubule proteins prepared from porcine brain. The antibody reacted only with MAP-1, not with MAP-2, tau or tubulin. Indirect Immunofluorescent staining by this antibody showed bright intranuclear spots, the centrosome and the faint meshwork of the cytoplasm in several types of cultured mammalian cells; HeLa, PtK2, human skin fibroblasts, mouse melanoma cells, Chinese hamster ovary cells. The nulcear spots in the interphase cells, were replaced by diffuse enhanced fluorescence throughout the cell except for chromosomes during mitosis. They reappeared in late telophase, first in the cytoplasm, late in the nucleus. The punctate pattern of nuclear immunofluorescence was not affected by microtubule-depolymerizing agents. The result that it persisted on residual cell structures after extraction with a high salt concentration buffer containing Triton X-100 followed by digestion with DNase I and RNase A suggests that the antigen is associated with the nuclear skeleton.