Phosphorylation sites linked to glial filament disassembly in vitro locate in a non-alpha-helical head domain.

Self Cite

Abstract. We Z 11:2895-2902). This observation implies a possibility that there is a protein kinase specifically activated at metaphaseanaphase transition. To further analyze the cell cycledependent GFAP phosphorylation, we prepared monoclonal antibodies KT13 and KT34 which recognize the phosphorylation of GFAP at Serl3 and Ser34, respectively. Immunocytochemical studies with KT13 and KT34 revealed that the GFAP phosphorylation in the cleavage furrow during late mitotic phase occurred not only in glioma cells but also in human SW-13 and mouse Ltk-cells in which GFAP was ectopically expressed, thus the phosphorylation can be monitored in a wide range of cell types. Furthermore, we detected kinase activity which phosphorylates GFAP at Serl3 and Ser34 in the lysates of late mitotic cells but not in those of interphase cells or early mitotic cells. These results suggest that there exists a protein kinase which is specifically activated at the transition of metaphase to anaphase not only in GFAP-expressing cells but also in cells without GFAP.

Section: Discussionmentioning

confidence: 99%

Section: Purification Of Proteinsmentioning

confidence: 99%

Section: Production and Characterization Of Monoclonal Antibodies Kt13 And Kt34mentioning

confidence: 99%

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Detection of protein kinase activity specifically activated at metaphase-anaphase transition.

Sekimata

Tsujimura

Tanaka

et al. 1996

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“…We reported that intermediate filaments (IFs) are pertinent in vitro PKC substrates (Inagaki et al, 1987a(Inagaki et al, , 1988(Inagaki et al, , 1990Gonda et al, 1990;Yano et al, 1991b). During mitosis, IFs undergo dynamic change in organization accompanied with phosphorylation.…”

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confidence: 99%

Mitosis-specific phosphorylation of vimentin by protein kinase C coupled with reorganization of intracellular membranes.

Takai

Ogawara

Tomono

et al. 1996

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Abstract. Using two types of anti-phosphopeptide antibodies which specifically recognize vimentin phosphorylated by protein kinase C (PKC) at two distinct PKC sites, we found that PKC acted as a mitotic vimentin kinase. Temporal change of vimentin phosphorylation by PKC differed from changes by cdc2 kinase. The mitosis-specific vimentin phosphorylation by PKC was dramatically enhanced by treatment with a PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), while no phosphorylation of vimentin by PKC was observed in interphase cells treated with TPA. By contrast, the disruption of subcellular compartmentalization of interphase cells led to vimentin phosphorylation by PKC. Cytoplasmic and nuclear membranes are fragmented and dispersed in the cytoplasm and some bind to vimentin during mitosis. Thus, targeting of activated PKC, coupled with the reorganization of intracellular membranes which contain phospholipids essential for activation, leads to the mitosis-specific phosphorylation of vimentin. We propose that during mitosis, PKC may phosphorylate an additional subset of proteins not phosphorylated in interphase.

“…First, IF proteins are commonly phosphorylated (Cabral and Gottesman, 1979;Gard and l.,azaddes, 1982;Steinert et al, 1982) and their level of phosphorylation increases during M-phase (Evans and Fink, 1982;Celis et al, 1983;Evans, 1984Evans, , 1989Klymkowsky et al, 1991). Second, the in vitro phosphorylation of IF proteins blocks the ability of the protein to polymerize or induces the disassembly of preassembled IFs (Inagaki et al, 1987(Inagaki et al, , 1988(Inagaki et al, , 1990Geisler and Weber, 1988;Evans, 1988;Ando et al, 1989;Geisler et al, 1989;Kitamura et al, 1989;Chou et al, 1990;Yano et al, 1991). Finally, induction of protein kinases by drugs, growth factors (Coca-Prados, 1985;Baribanlt et al, 1989;Escribano and Rozengurt, 1988;Huang et al, 1988;Ciesielski-Treska et al, 1991), or the direct injection of active kinase (Lamb et al, 1989) induces IF reorganization.…”

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confidence: 99%

Host cell factors controlling vimentin organization in the Xenopus oocyte.

Dent

Cary

Bachant

et al. 1992

Abstract. To study vimentin filament organization in vivo we injected Xenopus oocytes, which have no significant vimentin system of their own, with in vitro-synthesized RNAs encoding Xenopus vimentins.Exogenous vimentins were localized primarily to the cytoplasmic surface of the nucleus and to the subplasma membrane "cortex." In the cortex of the animal hemisphere, wild-type vimentin forms punctate structures and short filaments. In contrast, long anastomosing vimentin filaments are formed in the vegetal hemisphere cortex. This asymmetry in the organization of exogenous vimentin is similar to that of the endogenous keratin system (Klymkowsky, M. W., L. A. vimentin filaments remain intact and vimentin is hyperphosphorylated. To examine the role of MPF kinase in the M-phase reorganization of vimentin we deleted the conserved proline of vimentin's single MPF-kinase site; this mutation had no apparent effect on the prophase or M-phase behavior of vimentin. In contrast, deletion of amino acids 19-68 or 18-61 of the NH2-terminal "head" domain produced proteins that formed extended filaments in the animal hemisphere of the prophase oocyte. We suggest that the animal hemisphere cortex of the prophase oocyte contains a factor that actively suppresses the formation of extended vimentin filaments through a direct interaction with vimentin's head domain. During maturation this "suppressor of extended filaments" appears to be inactivated, leading to the formation of an extended vimentin filament system.