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

Sekimata, Masayuki; Tsujimura, Kunio; Tanaka, Jin; Takeuchi, Yukiko; Inagaki, Naoyuki; Inagaki, Masaki

doi:10.1083/jcb.132.4.635

Cited by 43 publications

(41 citation statements)

References 39 publications

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“…3A, lane 5) (Liao and Omary, 1996). The increase in the association coincident with the timing of S and G2/M phases of the cell cycle, also correlated with IFs hyperphosphorylation (Liao and Omary, 1996;Sekimata et al, 1996). This lends support to our observations with…”

Section: Association Of 14-3-3␥ and Gfap In Astrocytessupporting

confidence: 86%

“…Hyperphosphorylation of IFs is directly correlated with their disassembly into non-filamentous soluble structures (Kawajiri et al, 2003). Under physiological conditions, it is known that GFAP becomes hyperphosphorylated during S and G2/M phases of the cell cycle (Sekimata et al, 1996). To elucidate whether the change in GFAP phosphorylation during the cell cycle would affect its interaction with 14-3-3␥, we synchronized astrocytes in primary cultures at G1/S stages of the cell cycle by aphidicolin treatment (Liao and Omary, 1996), and carried out coimmunoprecipitation assays.…”

Section: Association Of 14-3-3␥ and Gfap In Astrocytesmentioning

confidence: 99%

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14-3-3γ affects dynamics and integrity of glial filaments by binding to phosphorylated GFAP

Guo

Jiang

et al. 2006

Journal of Cell Science

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Recent findings indicated a protective role of GFAP in ischemic brain, injured spinal cord, and in neurodegenerative disease. We previously demonstrated that 14-3-3␥ ␥, once thought to be neuronal specific, was upregulated by ischemia in astrocytes and may play a specific protective role in astrocytes. Here we report that 14-3-3␥ ␥ associates with both soluble and filamentous GFAP in a phosphorylation-and cell-cycle-dependent manner in primary cultured astrocytes. The amount of association increases during G2/M phase due to more phosphorylated GFAP. Moreover, this interaction is independent of vimentin, another type III intermediate filament protein in astrocytes which forms glial filaments with GFAP. A series of domain deletion mutants and substitution mutations at phosphorylation sites (from serine to alanine) on GFAP demonstrated that serine 8 in the head domain is essential for the direct association of GFAP to 14-3-3␥ ␥. Overexpression of 14-3-3␥ ␥ destroyed the integrity and affected the movement of GFAP intermediate filaments. This data demonstrates that 14-3-3␥ ␥ contributes to the regulation of dynamics of GFAP filaments, which may contribute to the stability of the cytoskeleton and the mechanisms of central nervous system neurodegenerative disease.Supplementary material available online at

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Section: Association Of 14-3-3␥ and Gfap In Astrocytessupporting

confidence: 86%

Section: Association Of 14-3-3␥ and Gfap In Astrocytesmentioning

confidence: 99%

14-3-3γ affects dynamics and integrity of glial filaments by binding to phosphorylated GFAP

Guo

Jiang

et al. 2006

Journal of Cell Science

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“…Less is known about the sites and kinases involved in GFAP phosphorylation in intact cell systems, but a start has been made. Japanese workers used monoclonal antibodies recognizing specific phosphorylation sites and immunofluorescence to demonstrate that one threonine and three serines in the head region of GFAP are phosphorylated in vivo at different stages of the cell cycle (56)(57)(58). With regard to the kinases responsible for these in vivo phosphorylations, present evidence points to the direct or indirect participation of PKA, CaMK II, the cdc-2 kinase and an unknown kinase (CF kinase) (58).…”

Section: General and Functional Aspects Of Gfap Phosphorylationmentioning

confidence: 99%

“…Phosphorylation of disassembled subunits inhibits their assembly into filaments (47,61), with subunit exchange in the case of GFAP being suppressed in proportion to the extent of phosphorylation (62). Moreover, IF protein phosphorylation plays a crucial role in dividing cells, where evidence from cell cultures has shown that the disassembly of the cytoskeleton preceding mitosis in some cells is regulated by a site-specific increase in the phosphorylation state of vimentin and GFAP (57,(63)(64)(65). In interphase cells changes in the phosphorylation state of IF proteins may have profound effects on the structure of the cytoskeleton, as shown by studies in which hyperphosphorylation of vimentin induced by protein phosphatase inhibitors (66,67) or the antitumor drug fostriecin (68) was associated with intermediate filament reorganization.…”

Section: General and Functional Aspects Of Gfap Phosphorylationmentioning

confidence: 99%

Control of the phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in the immature rat hippocampus by glutamate and calcium ions: possible key factor in astrocytic plasticity

Rodnight¹,

Gonçalves²,

Wofchuk³

et al. 1997

Braz J Med Biol Res

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The present review describes recent research on the regulation by glutamate and Ca 2+ of the phosphorylation state of the intermediate filament protein of the astrocytic cytoskeleton, glial fibrillary acidic protein (GFAP), in immature hippocampal slices. The results of this research are discussed against a background of modern knowledge of the functional importance of astrocytes in the brain and of the structure and dynamic properties of intermediate filament proteins. Astrocytes are now recognized as partners with neurons in many aspects of brain function with important roles in neural plasticity. Site-specific phosphorylation of intermediate filament proteins, including GFAP, has been shown to regulate the dynamic equilibrium between the polymerized and depolymerized state of the filaments and to play a fundamental role in mitosis. Glutamate was found to increase the phosphorylation state of GFAP in hippocampal slices from rats in the post-natal age range of 12-16 days in a reaction that was dependent on external Ca 2+ . The lack of external Ca 2+ in the absence of glutamate also increased GFAP phosphorylation to the same extent. These effects of glutamate and Ca 2+ were absent in adult hippocampal slices, where the phosphorylation of GFAP was completely Ca 2+ -dependent. Studies using specific agonists of glutamate receptors showed that the glutamate response was mediated by a G protein-linked group II metabotropic glutamate receptor (mGluR). Since group II mGluRs do not act by liberating Ca 2+ from internal stores, it is proposed that activation of the receptor by glutamate inhibits Ca 2+ entry into the astrocytes and consequently down-regulates a Ca 2+ -dependent dephosphorylation cascade regulating the phosphorylation state of GFAP. The functional significance of these results may be related to the narrow developmental window when the glutamate response is present. In the rat brain this window corresponds to the period of massive synaptogenesis during which astrocytes are known to proliferate. Possibly, glutamate liberated from developing synapses during this period may signal an increase in the phosphorylation state of GFAP and a consequent increase in the number of mitotic astrocytes.

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“…These six amino acid residues are highly conserved among species, implying the biological importance of GFAP phosphorylation [7]. Enzymes including protein kinase A (PKA), protein kinase C (PKC), calcium/calmodulin dependent protein kinase II (CaMKII), Rho kinase, cleavage furrow (CF) kinase and Cdc2 kinase modulate the phosphorylation of one or more of the GFAP residues [8][9][10][11][12]. Phosphorylation in the N-terminal region (head domain) affects GFAP assembly and in the C-terminal region (tail domain), phosphorylation of Ser-389 affects interactions between GFAP and other intermediate filaments or proteins [10].…”

Section: Introductionmentioning

confidence: 99%

Phosphorylation of GFAP is Associated with Injury in the Neonatal Pig Hypoxic-Ischemic Brain

Sullivan

Miller

et al. 2012

Neurochem Res

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Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in the astrocytecytoskeleton that plays an important role in the structure and function of the cell. GFAP can be phosphorylated at six serine (Ser) or threonine (Thr) residues but little is known about the role of GFAP phosphorylation in physiological and pathophysiological states. We have generated antibodies against two phosphorylated GFAP (pGFAP) proteins: p8GFAP, where GFAP is phosphorylated at Ser-8 and p13GFAP, where GFAP is phosphorylated at Ser-13. We examined p8GFAP and p13GFAP expression in the control neonatal pig brain and at 24h and 72h after an hypoxic-ischemic (HI) insult. Immunohistochemistry demonstrated pGFAP expression in astrocytes with an atypical cytoskeletal morphology, even in control brains. Semi-quantitative western blotting revealed that p8GFAP expression was significantly increased at 24h post-insult in HI animals with seizures in frontal, parietal, temporal and occipital cortices. At 72h post-insult, p8GFAP and p13GFAP expression were significantly increased in HI animals with seizures in brain regions that are vulnerable to cellular damage (cortex and basal ganglia), but no changes were observed in brain regions that are relatively spared following an HI insult (brain stem and cerebellum). Increased pGFAP expression was associated with poor neurological outcomes such as abnormal encephalography (EEG) and neurobehaviour, and increased histological brain damage.Phosphorylation of GFAP may play an important role in astrocyte remodelling during development and disease and could potentially contribute to the plasticity of the central nervous system. 3

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

Cited by 43 publications

References 39 publications

14-3-3γ affects dynamics and integrity of glial filaments by binding to phosphorylated GFAP

14-3-3γ affects dynamics and integrity of glial filaments by binding to phosphorylated GFAP

Control of the phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in the immature rat hippocampus by glutamate and calcium ions: possible key factor in astrocytic plasticity

Phosphorylation of GFAP is Associated with Injury in the Neonatal Pig Hypoxic-Ischemic Brain

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