Biomedical subjects

Masaki Inagaki

Publications and source records attributed to Masaki Inagaki.

42 records · Page 3Linked to original sources

PubMed2002

Aurora-B phosphorylates Histone H3 at serine28 with regard to the mitotic chromosome condensation.

BACKGROUND: Histone H3 (H3) phosphorylation plays important roles in mitotic chromosome condensation. We reported that H3 phosphorylation occurs at Ser28, as well as at Ser10 during mitosis, at least in mammals. Aurora B was recently demonstrated to be responsible for Ser10 phosphorylation in S. cerevisiae, C. elegans, Drosophila and Xenopus egg extract. RESULTS: We compared the distribution of Aurora-B with that of H3 phosphorylation. Aurora-B was primarily localized in the heterochromatin of late G2 phase cells, where only Ser10 phosphorylation was observed. The treatment of such cells with calyculin A induced Ser28 phosphorylation in the Aurora-B-localized area. During prophase to metaphase, Aurora-B was distributed in condensing chromosomes where Ser10 and Ser28 were phosphorylated. Aurora-B can phosphorylate H3-Ser10 and -Ser28 in nucleosomes in vitro. Transfection of a dominant-negative mutant of Aurora-B resulted in a reduction of H3 phosphorylation, not only at Ser10 but also Ser28, during mitosis. CONCLUSIONS: With regard to mitotic chromosome condensation, Aurora-B directly phosphorylated H3, not only at Ser10 but also at Ser28. The level of Ser28 phosphorylation is diminished to undetectable levels by PP1 phosphatase prior to entry into mitosis.

Aurora Kinase B↗

PubMed2002

Phosphorylation and reorganization of vimentin by p21-activated kinase (PAK).

BACKGROUND: Intermediate filament (IF) is one of the three major cytoskeletal filaments. Vimentin is the most widely expressed IF protein component. The Rho family of small GTPases, such as Cdc42, Rac and Rho, are thought to control the organization of actin filaments as well as other cytoskeletal filaments. RESULTS: We determined if the vimentin filaments can be regulated by p21-activated kinase (PAK), one of targets downstream of Cdc42 or Rac. In vitro analyses revealed that vimentin served as an excellent substrate for PAK. This phosphorylated vimentin lost the potential to form 10 nm filaments. We identified Ser25, Ser38, Ser50, Ser65 and Ser72 in the amino-terminal head domain as the major phosphorylation sites on vimentin for PAK. The ectopic expression of constitutively active PAK in COS-7 cells induced vimentin phosphorylation. Fibre bundles or granulates of vimentin were frequent in these transfected cells. However, the kinase-inactive mutant induced neither vimentin phosphorylation nor filament reorganization. CONCLUSION: Our observations suggest that PAK may regulate the reorganization of vimentin filaments through direct vimentin phosphorylation.

Animals↗

PubMed2002

ERBIN associates with p0071, an armadillo protein, at cell-cell junctions of epithelial cells.

BACKGROUND: ERBIN, an ErbB2 receptor-interacting protein, belongs to a recently described family of proteins termed the LAP [leucine-rich repeats and PSD-95/dLg-A/ZO-1 (PDZ) domains] family which has essential roles in establishment of cell polarity. RESULTS: To identify new ERBIN-binding proteins, we screened a yeast two-hybrid library, using the carboxyl-terminal fragment of ERBIN containing PDZ domain as the bait, and we isolated p0071 (also called plakophilin-4) as an ERBIN-interacting protein. p0071 is a member of the p120 catenin family, which are defined as proteins with 10 armadillo repeats, and localizes along the cell-cell border. The ERBIN PDZ domain binds the COOH-terminus of p0071 containing the PDZ domain-binding sequence. Endogenous ERBIN was co-immunoprecipitated with p0071. In fully polarized Madin-Darby canine kidney (MDCK) cells, ERBIN co-localized largely with beta-catenin and partly with desmoplakin along the lateral plasma membrane domain. At these cell-cell contact regions, ERBIN co-localizes with p0071. Over-expression of the dominant active forms of Cdc42, Rac1 or RhoA, Rho family small GTPases, resulted in a marked accumulation of ERBIN at the cell-cell contacts of MDCK and HeLa cells. CONCLUSION: These results show that ERBIN interacts in vivo with p0071 and that it may be involved in the organization of adherens junctions and the desmosomes of epithelia. In addition, we demonstrated that the subcellular localization of ERBIN might be regulated by Rho family small GTPases.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Densin-180, a synaptic protein, links to PSD-95 through its direct interaction with MAGUIN-1.

BACKGROUND: Densin-180, a brain-specific protein highly concentrated at the postsynaptic density (PSD), belongs to the LAP [leucine-rich repeats and PSD-95/Dlg-A/ZO-1 (PDZ) domains] family of proteins, some of which play fundamental roles in the establishment of cell polarity. RESULTS: To identify new Densin-180-interacting proteins, we screened a yeast two-hybrid library using the COOH-terminal fragment of Densin-180 containing the PDZ domain as bait, and we isolated MAGUIN-1 as a Densin-180-binding protein. MAGUIN-1, a mammalian homologue of Drosophila connector enhancer of KSR (CNK), is known to interact with PSD-95 and has a short isoform, MAGUIN-2. The Densin-180 PDZ domain bound to the COOH-terminal PDZ domain-binding motif of MAGUIN-1. Densin-180 co-immunoprecipitated with MAGUIN-1 as well as with PSD-95 from the rat brain. In dissociated hippocampal neurones Densin-180 co-localized with MAGUINs and PSD-95, mainly at neuritic spines. In transfected cells, Densin-180 formed a ternary complex with MAGUIN-1 and PSD-95, whereas no association was detected between Densin-180 and PSD-95 in the absence of MAGUIN-1. MAGUIN-1 formed a dimer or multimer via the COOH-terminal leucine-rich region which is present in MAGUIN-1 but not in -2. Among the PDZ domains of PSD-95, the first was sufficient for interaction with MAGUIN-1. CONCLUSION: These results suggest that the potential to dimerize or multimerize allows MAGUIN-1 to bind simultaneously to both Densin-180 and PSD-95, leading to the ternary complex assembly of these proteins at the postsynaptic membrane.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Vimentin intermediate filament reorganization by Cdc42: involvement of PAK and p70 S6 kinase.

Rho family GTPases play a major role in actin cytoskeleton reorganization. Recent studies have shown that the activation of Rho family GTPases also induces collapse of the vimentin intermediate filament (IF) network in fibroblasts. Here, we report that Cdc42V12 induces the reorganization of vimentin IFs in Hela cells, and such reorganization is independent of actin and microtubule status. We analyzed the involvement of three serine/threonine kinase effectors, MRCK, PAK and p70 S6K in the Cdc42-induced vimentin reorganization. Surprisingly, the ROK-related MRCK is not involved in this IF reorganization. We detected phosphorylation of vimentin Ser72, a site phosphorylated by PAK, after Cdc42 activation. PAK inhibition partially blocked Cdc42-induced vimentin IF collapse suggesting the involvement of other effectors. We report that p70 S6 kinase (S6K)1 participates in this IF rearrangement since the inhibitor rapamycin or a dominant inhibitory S6K could reduce the Cdc42V12 or bradykinin-induced vimentin collapse. Further, inhibition of PAK and S6K in combination very effectively prevents Cdc42-induced vimentin IF collapse. Conversely, only in combination active PAK and S6K could induce a vimentin IF rearrangement that mimics the Cdc42 effect. Thus, Cdc42-induced vimentin reorganization involves PAK and, in a novel cytoskeletal role, p70 S6K.

3T3 Cells↗

PubMed2001

Densin-180 interacts with delta-catenin/neural plakophilin-related armadillo repeat protein at synapses.

Densin-180, a protein purified from the postsynaptic density fraction of the rat forebrain, is the founding member of a newly described family of proteins termed the LAP (leucine-rich repeats and PSD-95/Dlg-A/ZO-1 (PDZ) domains) family that plays essential roles in establishment of cell polarity. To identify Densin-180-binding proteins, we screened a yeast two-hybrid library using the carboxyl-terminal fragment of Densin-180 containing PDZ domain as bait, and we isolated delta-catenin/neural plakophilin-related armadillo repeat protein (NPRAP) as a Densin-180-interacting protein. delta-catenin/NPRAP, a member of the armadillo repeat family, is a nervous system-specific adherens junction protein originally discovered as an interactor with presenilin-1, a protein involved in Alzheimer's disease. Densin-180 PDZ domain binds the COOH terminus of delta-catenin/NPRAP containing the PDZ domain-binding sequence. Endogenous Densin-180 was co-immunoprecipitated with delta-catenin/NPRAP and N-cadherin. Although Densin-180 was reported to be a transmembrane protein, Densin-180 was not accessible to surface biotinylation in dissociated hippocampal neurons; hence Densin-180 may be a cytosolic protein. Densin-180 co-localized with delta-catenin/NPRAP at synapses in delta-catenin/NPRAP and may be involved in organization of the synaptic cell-cell junction through interaction with the delta-catenin/NPRAP-N-cadherin complex.

Animals↗