Regulation of a major microtubule-associated protein by MPF and MAP kinase.

Abstract

We have now identified and purified from Xenopus eggs a major microtubuleassociated protein, p220, that may be a target protein for these two M phase-activated kinases. p220, when purified from interphase cells, potently bound to microtubules and stimulated tubulin polymerization, whereas p220 purifled from M phase cells showed little or no such activities. Cell staining with a monoclonal anti-p220 antibody revealed that p220 is localized on cytoplasmic microtubule networks during interphase, while it is distributed rather diffusely throughout the cell during M phase. We have further found that p220 is phosphorylated specifically in M phase. Moreover, p220 purified from interphase cells served as a good substrate for MAP kinase and MPF in vitro, and two-dimensional phosphopeptide mapping pattern of the p220 phosphorylated in vitro was very similar to that of p220 phosphorylated at M phase in vivo. These results suggest that the drastic change in p220 activity during the transition from interphase to M phase may be induced by its phosphorylation in M phase probably catalyzed by MAP kinase and MPF.

M-phase-specific phosphorylation and structural rearrangement of the cytoplasmic cross-linking protein plectin involve p34cdc2 kinase.

“…Second, it could effect the assembly state of the filament itself by phosphorylation of its core subunit proteins. Unlike the microtubule and microfilament systems, where the phosphorylation of filament-associated proteins has been suggested as a major mechanism for structural rearrangements (Mak et al, 1991;Tombes et al, 1991;Yashimiro et al, 1991;Shiina et al, 1992;Verde et al, 1992;Hosoya et al, 1993), the regulation of IF assembly and its structural organization has mostly been reported to involve phosphorylation of the core subunits (for review see Skalli et al, 1992b). Only a few studies have also addressed the fate of IF-associated proteins during mitosis.…”

“…In eukaryotic cells entry into mitosis is characterized by a profound reorganization of the cytoskeleton, such as the formation of the mitotic spindle (McIntosh and Koonce, 1989), the reorganization of microfilaments to the contractile ring , the structural rearrangement of the cytoplasmic intermediate filament (IF) network to cage-like bundles or aggregates (for a review see Georgatos, 1993), and the breakdown of the nuclear envelope, accompanied by a disassembly of the nuclear IF structure, the nuclear lamina (Gerace and Burke, 1988). p34cdc2 kinase (Verde et al, 1990(Verde et al, , 1992 probably due to a phosphorylation-dependent dissociation of microtubule-associated protein (mammalian MAP 4, MAP 1B, and Xenopus p220) from the microtubule surface (Tombes et al, 1991;Shiina et al, 1992). p34cdc2-dependent phosphorylation of the actin-binding protein caldesmon releases the protein from actin filaments and is likely to cause microfilament disassembly (Yashimiro et al, 1990(Yashimiro et al, ,1991Mak et al, 1991;Hosoya et al, 1993), whereas phosphorylation of myosin II regulatory light chain decreases actomyosin ATPase activity and seems to inhibit cytokinesis until completion of mitosis Yamakita et al, 1994).…”

A Novel RNA-Binding Protein in Neuronal RNA Granules: Regulatory Machinery for Local Translation

“…Cultured cells were immunostained as described previously (Shiina et al, 1992). Rat brains were infused with Tissue-Tek (Sakura Finetek, Tokyo, Japan), frozen in liquid nitrogen, cut into cryosections, and mounted on silan-coated glass coverslips.…”

Phosphorylation of Microtubule-associated Proteins MAP2 and MAP4 by the Protein Kinase p110mark

“…MAPs isolated from tissue or cells are phosphoproteins (Sloboda et al, 1975;Vallee, 1980;Burns et al, 1984;Tsuyama et al, 1986;Brugg and Matus, 1991;Watanabe et al, 1993), MAPs are good substrates for many protein kinases in vitro (Theurkauf and Vallee, 1983;Lindwall and Cole, 1984;Mori et al, 1991;Drewes et al, 1992), and phosphorylation interferes with their microtubule stabilizing capacity (Brugg and Matus, 1991;Shiina et al, 1992;Drechsel et al, 1992;Biernat et al, 1993;Brandt et al, 1994;Ookata et al, 1995;Trinczek et al, 1995). In the case of tau protein, phosphorylation has been extensively studied, because aberrantly phosphorylated tau is involved in the neurofibrillar pathology of Alzheimer's disease (reviewed by Goedert (1993), Mandelkow and Mandelkow (1993), and Trojanowski and Lee (1994)).…”