The Experts below are selected from a list of 2030358 Experts worldwide ranked by ideXlab platform
Eva-maria Mandelkow - One of the best experts on this subject based on the ideXlab platform.
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MARKK, a Ste20-like kinase, activates the polarity-inducing kinase MARK/PAR-1
The EMBO journal, 2003Co-Authors: Thomas Timm, Eva-maria Mandelkow, Jacek Biernat, Jian Jiao, Joël VandekerckhoveAbstract:MARK, a kinase family related to PAR-1 involved in establishing cell polarity, phosphorylates microtubule-associated proteins (tau/MAP2/MAP4) at KXGS motifs, causes detachment from microtubules, and their disassembly. The sites are prominent in tau from Alzheimer's disease brains. We studied the activation of MARK and identified the upstream kinase, MARKK, a member of the Ste20 kinase family. It phosphorylates MARK within the activation loop (T208 in MARK2). A fraction of MARK in brain tissue is doubly phosphorylated (at T208/S212), reminiscent of the activation of MAP kinase; however, the phosphorylation of the second site in MARK (S212) is inhibitory. In cells the activity of MARKK enhances microtubule dynamics through the activation of MARK and leads to phosphorylation and detachment of tau or equivalent MAPs from microtubules. Overexpression of MARK eventually leads to microtubule breakdown and cell death, but in neuronal cells the primary effect is to allow the development of neurites during differentiation.
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Phosphorylation of MAP2c and MAP4 by MARK kinases leads to the destabilization of microtubules in cells.
Cytoskeleton, 1999Co-Authors: Andreas Ebneth, Gerard Drewes, Eva-maria MandelkowAbstract:Max-Planck Unit for Structural Molecular Biology, Hamburg, Germany Microtubules serve as transport tracks in molecular mechanisms governing cellular shape and polarity. Rapid transitions between stable and dynamic microtubules are regulated by several factors, including microtubule-associated proteins (MAPs). We have shown that MAP/microtubule affinity regulating kinases (MARK) can phosphorylate the microtubule-associated-proteins MAP4, MAP2c, and tau on their microtubule-binding domain in vitro. This leads to their detachment from microtubules (MT) and an increased dynamic instability of MT. Here we show that MARK protein kinases phosphorylate MAP2 and MAP4 on their microtubulebinding domain in transfected CHO cells. In CHO cells expressing MARK1 or MARK2 under control of an inducible promoter, MARK2 phosphorylates an endogenous MAP4-related protein. Prolonged expression of MARK2 results in microtubule-disruption, detachment of cells from the substratum, and cell death. Concomitant with microtubule disruption, we also observed a breakdown of the vimentin network, whereas actin fibers remained unaffected. Thus, MARK seems to play an important role in controlling cytoskeletal dynamics. Cell Motil. Cytoskeleton 44:209‐224, 1999. r 1999 Wiley-Liss, Inc.
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MARK, a novel family of protein kinases that phosphorylate microtubule- associated proteins and trigger microtubule disruption
Cell, 1997Co-Authors: Gerard Drewes, Ute Preuss, Andreas Ebneth, Eva-maria MandelkowAbstract:MARK phosphorylates the microtubule-associated proteins tau, MAP2, and MAP4 on their microtubule-binding domain, causing their dissociation from microtubules and increased microtubule dynamics. We describe the molecular cloning, distribution, activation mechanism, and overexpression of two MARK proteins from rat that arise from distinct genes. They encode Ser/Thr kinases of 88 and 81 kDa, respectively, and show similarity to the yeast kin1+ and C. elegans par-1 genes that are involved in the establishment of cell polarity. Expression of both isoforms is ubiquitous, and homologous genes are present in humans. Catalytic activity depends on phosphorylation of two residues in subdomain VIII. Overexpression of MARK in cells leads to hyperphosphorylation of MAPs on KXGS motifs and to disruption of the microtubule array, resulting in morphological changes and cell death.
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Phosphorylation of Microtubule-associated Proteins MAP2 and MAP4 by the Protein Kinase p110mark: PHOSPHORYLATION SITES AND REGULATION OF MICROTUBULE DYNAMICS (∗)
The Journal of biological chemistry, 1996Co-Authors: Susanne Illenberger, Gerard Drewes, Eva-maria Mandelkow, Jacek Biernat, B. Trinczek, Helmut E. Meyer, Joanna B. OlmstedAbstract:The phosphorylation of microtubule-associated proteins (MAPs) is thought to be a key factor in the regulation of microtubule stability. We have shown recently that a novel protein kinase, termed p110 microtubule-affinity regulating kinase ("MARK"), phosphorylates microtubule-associated protein tau at the KXGS motifs in the region of internal repeats and causes the detachment of tau from microtubules (Drewes, G., Trinczek, B., Illenberger, S., Biernat, J., Schmitt-Ulms, G., Meyer, H.E., Mandelkow, E.-M., and Mandelkow, E. (1995) J. Biol. Chem. 270, 7679-7688). Here we show that p110mark phosphorylates analogous KXGS sites in the microtubule binding domains of the neuronal MAP2 and the ubiquitous MAP4. Phosphorylation in vitro leads to the dissociation of MAP2 and MAP4 from microtubules and to a pronounced increase in dynamic instability. Thus, the phosphorylation of the repeated motifs in the microtubule binding domains of MAPs by p110mark might provide a mechanism for the regulation of microtubule dynamics in cells.
Susumu Kotani - One of the best experts on this subject based on the ideXlab platform.
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Microtubule-associated protein (MAP) 4 interacts with microtubules in an intrinsically disordered manner
Bioscience biotechnology and biochemistry, 2014Co-Authors: Yurika Hashi, Gota Kawai, Susumu KotaniAbstract:We previously used nuclear magnetic resonance (NMR) to analyze the structure of a synthetic tricosapeptide corresponding to an active site of microtubule-associated protein 4 (MAP4). To further the structural analysis, we have constructed a minimal active domain fragment of MAP4, encompassing the entire active site, and obtained its NMR spectra. The secondary structure prediction using partially assigned NMR data suggested that the fragment is largely unfolded. Two other independent techniques also demonstrated its unfolded nature, indicating that MAP4 belongs to the class of intrinsically disordered proteins (IDPs). The NMR spectra of the fragment-microtubule mixture revealed that the fragment binds to the microtubule using multiple binding sites, apparently contradicting our previous quantitative studies. Given that MAP4 is intrinsically disordered, we propose a mechanism in which any one of the binding sites is active at a time, which is one of the typical interaction mechanisms proposed for IDPs.
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Microtubule-associated protein 4 binds to actin filaments and modulates their properties.
Journal of biochemistry, 2011Co-Authors: Kazuyuki Matsushima, Kiyotaka Tokuraku, Mohammad Rubayet Hasan, Susumu KotaniAbstract:We previously reported that an isoform of microtubule-associated protein 4 (MAP4) is localized to the distal area of developing neurites, where microtubules are relatively scarce, raising the possibility that MAP4 interacts with another major cytoskeletal component, actin filaments. In the present study, we examined the in vitro interaction between MAP4 and actin filaments, using bacterially expressed MAP4 and its truncated fragments. Sedimentation assays revealed that MAP4 and its microtubule-binding domain fragments bind to actin filaments under physiological conditions. The apparent dissociation constant and the binding stoichiometry of the fragments to actin were about 0.1 µm and 1 : 3 (MAP4/actin), respectively. Molecular dissection studies revealed that the actin-binding site on MAP4 is situated at the C-terminal part of the proline-rich region, where the microtubule-binding site is also located. Electron microscopy revealed that the MAP4-bound actin filaments become straighter and longer and that the number of actin bundles increases with greater concentrations of added MAP4 fragment, indicating that MAP4 binding alters the properties of the actin filaments. A multiple sequence alignment of the proline-rich regions of MAP4 and tau revealed two putative actin-binding consensus sequences.
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Structural and Functional Features
2011Co-Authors: Kiyotaka Tokuraku, Kazuyuki Matsushima, Hiroyuki Nakagawa, Susumu KotaniAbstract:In contrast with neural microtubule-associated proteins (MAPs) such as MAP1, MAP2, and tau, MAP4 is identified as a ubiquitous MAP. MAP4 stimu- lates tubulin polymerization and stabilizes polymerized-microtubules as do neural MAPs. Because MAP2, MAP4, and tau are structurally similar, the three MAPs are considered to constitute a superfamily. The architecture of the superfamily proteins consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain, and the microtubule-binding domain is further divided into three subdomains: the Pro-rich region ,t herepeat region, and the tail region. Recent studies have revealed the functions of these domains/subdomains in the MAP4 molecule: the projection domain keeps individual microtubules separated by suppressing the bundle-forming ability of the microtubule-binding domain, the Pro-rich region promotes the nucleation of microtubules, the repeat region promotes their elongation, and the tail region may contribute to the proper folding of the molecule. Isoforms of MAP4 are produced from the single MAP4 gene by alterna- tive RNA splicing, thereby five isoforms, with a deletion in the Pro-rich region or the repeat region, are expressed. The expression of these isoforms depends on the tissue type and the developmental stage, suggesting that the function of MAP4 is elabo- rately regulated by alternative splicing. In this chapter, we will address the structural and functional futures of MAP4 focusing on the functions of domains/subdomains and add some insights into the roles of the protein in neurons. Keywords MAP1 · MAP2 · MAP3 · MAP4 · MAP5 · Tau · Microtubule
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Microtubule-Associated Protein 4
Advances in Neurobiology, 2010Co-Authors: Kiyotaka Tokuraku, Kazuyuki Matsushima, Hiroyuki Nakagawa, Susumu KotaniAbstract:In contrast with neural microtubule-associated proteins (MAPs) such as MAP1, MAP2, and tau, MAP4 is identified as a ubiquitous MAP. MAP4 stimulates tubulin polymerization and stabilizes polymerized-microtubules as do neural MAPs. Because MAP2, MAP4, and tau are structurally similar, the three MAPs are considered to constitute a superfamily. The architecture of the superfamily proteins consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain, and the microtubule-binding domain is further divided into three subdomains: the Pro-rich region, the repeat region, and the tail region. Recent studies have revealed the functions of these domains/subdomains in the MAP4 molecule: the projection domain keeps individual microtubules separated by suppressing the bundle-forming ability of the microtubule-binding domain, the Pro-rich region promotes the nucleation of microtubules, the repeat region promotes their elongation, and the tail region may contribute to the proper folding of the molecule. Isoforms of MAP4 are produced from the single MAP4 gene by alternative RNA splicing, thereby five isoforms, with a deletion in the Pro-rich region or the repeat region, are expressed. The expression of these isoforms depends on the tissue type and the developmental stage, suggesting that the function of MAP4 is elaborately regulated by alternative splicing. In this chapter, we will address the structural and functional futures of MAP4 focusing on the functions of domains/subdomains and add some insights into the roles of the protein in neurons.
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Distinct neuronal localization of microtubule-associated protein 4 in the mammalian brain.
Neuroscience letters, 2010Co-Authors: Kiyotaka Tokuraku, Kazuyuki Matsushima, Satoshi Okuyama, Tsuneya Ikezu, Susumu KotaniAbstract:Although recent studies have suggested the role of microtubule-associated protein (MAP) 4 in some neuron-specific events, there are no reports that directly observed its neuronal localization. Here we show the detailed expression of MAP4 in the mammalian brain. Immunoblotting revealed the presence of MAP4 in all neuronal tissues. The site-specific localization of MAP4 was observed in sagittal brain sections: MAP4 was rich in brain-specific cells, cerebellum Purkinje cells and hippocampus pyramidal cells. When primary cultures of cortical neurons were immunostained, MAP4 was detected in the cell bodies and processes with patchy staining pattern. These results suggested that MAP4 play some roles in the central nervous system, such as the dynamic cytoskeletal reorganization and regulation of the microtubule-dependent long-range transport.
Kiyotaka Tokuraku - One of the best experts on this subject based on the ideXlab platform.
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Structural insight into microtubule stabilization and kinesin inhibition by Tau family MAPs.
The Journal of cell biology, 2018Co-Authors: Hideki Shigematsu, Kiyotaka Tokuraku, Chihiro Doki, Tsuyoshi Imasaki, Takuya Sumi, Mari Aoki, Tomomi Uchikubo-kamo, Ayako Sakamoto, Mikako Shirouzu, Ryo NittaAbstract:The Tau family microtubule-associated proteins (MAPs) promote microtubule stabilization and regulate microtubule-based motility. They share the C-terminal microtubule-binding domain, which includes three to five tubulin-binding repeats. Different numbers of repeats formed by alternative splicing have distinct effects on the activities of these proteins, and the distribution of these variants regulates fundamental physiological phenomena in cells. In this study, using cryo-EM, we visualized the MAP4 microtubule complex with the molecular motor kinesin-1. MAP4 bound to the C-terminal domains of tubulins along the protofilaments stabilizes the longitudinal contacts of the microtubule. The strongest bond of MAP4 was found around the intertubulin–dimer interface such that MAP4 coexists on the microtubule with kinesin-1 bound to the intratubulin–dimer interface as well. MAP4, consisting of five repeats, further folds and accumulates above the intertubulin–dimer interface, interfering with kinesin-1 movement. Therefore, these cryo-EM studies reveal new insight into the structural basis of microtubule stabilization and inhibition of kinesin motility by the Tau family MAPs.
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Microtubule-associated protein 4 binds to actin filaments and modulates their properties.
Journal of biochemistry, 2011Co-Authors: Kazuyuki Matsushima, Kiyotaka Tokuraku, Mohammad Rubayet Hasan, Susumu KotaniAbstract:We previously reported that an isoform of microtubule-associated protein 4 (MAP4) is localized to the distal area of developing neurites, where microtubules are relatively scarce, raising the possibility that MAP4 interacts with another major cytoskeletal component, actin filaments. In the present study, we examined the in vitro interaction between MAP4 and actin filaments, using bacterially expressed MAP4 and its truncated fragments. Sedimentation assays revealed that MAP4 and its microtubule-binding domain fragments bind to actin filaments under physiological conditions. The apparent dissociation constant and the binding stoichiometry of the fragments to actin were about 0.1 µm and 1 : 3 (MAP4/actin), respectively. Molecular dissection studies revealed that the actin-binding site on MAP4 is situated at the C-terminal part of the proline-rich region, where the microtubule-binding site is also located. Electron microscopy revealed that the MAP4-bound actin filaments become straighter and longer and that the number of actin bundles increases with greater concentrations of added MAP4 fragment, indicating that MAP4 binding alters the properties of the actin filaments. A multiple sequence alignment of the proline-rich regions of MAP4 and tau revealed two putative actin-binding consensus sequences.
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Structural and Functional Features
2011Co-Authors: Kiyotaka Tokuraku, Kazuyuki Matsushima, Hiroyuki Nakagawa, Susumu KotaniAbstract:In contrast with neural microtubule-associated proteins (MAPs) such as MAP1, MAP2, and tau, MAP4 is identified as a ubiquitous MAP. MAP4 stimu- lates tubulin polymerization and stabilizes polymerized-microtubules as do neural MAPs. Because MAP2, MAP4, and tau are structurally similar, the three MAPs are considered to constitute a superfamily. The architecture of the superfamily proteins consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain, and the microtubule-binding domain is further divided into three subdomains: the Pro-rich region ,t herepeat region, and the tail region. Recent studies have revealed the functions of these domains/subdomains in the MAP4 molecule: the projection domain keeps individual microtubules separated by suppressing the bundle-forming ability of the microtubule-binding domain, the Pro-rich region promotes the nucleation of microtubules, the repeat region promotes their elongation, and the tail region may contribute to the proper folding of the molecule. Isoforms of MAP4 are produced from the single MAP4 gene by alterna- tive RNA splicing, thereby five isoforms, with a deletion in the Pro-rich region or the repeat region, are expressed. The expression of these isoforms depends on the tissue type and the developmental stage, suggesting that the function of MAP4 is elabo- rately regulated by alternative splicing. In this chapter, we will address the structural and functional futures of MAP4 focusing on the functions of domains/subdomains and add some insights into the roles of the protein in neurons. Keywords MAP1 · MAP2 · MAP3 · MAP4 · MAP5 · Tau · Microtubule
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Microtubule-Associated Protein 4
Advances in Neurobiology, 2010Co-Authors: Kiyotaka Tokuraku, Kazuyuki Matsushima, Hiroyuki Nakagawa, Susumu KotaniAbstract:In contrast with neural microtubule-associated proteins (MAPs) such as MAP1, MAP2, and tau, MAP4 is identified as a ubiquitous MAP. MAP4 stimulates tubulin polymerization and stabilizes polymerized-microtubules as do neural MAPs. Because MAP2, MAP4, and tau are structurally similar, the three MAPs are considered to constitute a superfamily. The architecture of the superfamily proteins consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain, and the microtubule-binding domain is further divided into three subdomains: the Pro-rich region, the repeat region, and the tail region. Recent studies have revealed the functions of these domains/subdomains in the MAP4 molecule: the projection domain keeps individual microtubules separated by suppressing the bundle-forming ability of the microtubule-binding domain, the Pro-rich region promotes the nucleation of microtubules, the repeat region promotes their elongation, and the tail region may contribute to the proper folding of the molecule. Isoforms of MAP4 are produced from the single MAP4 gene by alternative RNA splicing, thereby five isoforms, with a deletion in the Pro-rich region or the repeat region, are expressed. The expression of these isoforms depends on the tissue type and the developmental stage, suggesting that the function of MAP4 is elaborately regulated by alternative splicing. In this chapter, we will address the structural and functional futures of MAP4 focusing on the functions of domains/subdomains and add some insights into the roles of the protein in neurons.
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Distinct neuronal localization of microtubule-associated protein 4 in the mammalian brain.
Neuroscience letters, 2010Co-Authors: Kiyotaka Tokuraku, Kazuyuki Matsushima, Satoshi Okuyama, Tsuneya Ikezu, Susumu KotaniAbstract:Although recent studies have suggested the role of microtubule-associated protein (MAP) 4 in some neuron-specific events, there are no reports that directly observed its neuronal localization. Here we show the detailed expression of MAP4 in the mammalian brain. Immunoblotting revealed the presence of MAP4 in all neuronal tissues. The site-specific localization of MAP4 was observed in sagittal brain sections: MAP4 was rich in brain-specific cells, cerebellum Purkinje cells and hippocampus pyramidal cells. When primary cultures of cortical neurons were immunostained, MAP4 was detected in the cell bodies and processes with patchy staining pattern. These results suggested that MAP4 play some roles in the central nervous system, such as the dynamic cytoskeletal reorganization and regulation of the microtubule-dependent long-range transport.
Hiroyuki Nakagawa - One of the best experts on this subject based on the ideXlab platform.
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Microtubule elongation along actin filaments induced by microtubule-associated protein 4 contributes to the formation of cellular protrusions
Journal of biochemistry, 2020Co-Authors: Chihiro Doki, Kohei Nishida, Shoma Saito, Miyuki Shiga, Hikari Ogara, Ayumu Kuramoto, Masahiro Kuragano, Motohiro Nozumi, Michihiro Igarashi, Hiroyuki NakagawaAbstract:Actin-microtubule crosstalk is implicated in the formation of cellular protrusions, but the mechanism remains unclear. In this study, we examined the regulation of cell protrusion involving a ubiquitously expressed microtubule-associated protein (MAP) 4, and its superfamily proteins, neuronal MAP2 and tau. Fluorescence microscopy revealed that these MAPs bound to F-actin and microtubules simultaneously, and formed F-actin/microtubule hybrid bundles. The hybrid bundle-forming activity was in the order of MAP2 > MAP4 ≫ tau. Interestingly, the microtubule assembly-promoting activity of MAP4 and MAP2, but not of tau, was upregulated by their interaction with F-actin. When MAP4 was overexpressed in NG108-15 cells, the number of cell processes and maximum process length of each cell increased significantly by 28% and 30%, respectively. Super-resolution microscopy revealed that 95% of microtubules in cell processes colocalized with F-actin, and MAP4 was always found in their vicinity. These results suggest that microtubule elongation along F-actin induced by MAP4 contributes to the formation of cellular protrusions. Since MAP4, MAP2 and tau had different crosstalk activity between F-actin and microtubules, it is likely that the functional differentiation of these MAPs is a driving force for neural evolution, causing significant changes in cell morphology.
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Structural and Functional Features
2011Co-Authors: Kiyotaka Tokuraku, Kazuyuki Matsushima, Hiroyuki Nakagawa, Susumu KotaniAbstract:In contrast with neural microtubule-associated proteins (MAPs) such as MAP1, MAP2, and tau, MAP4 is identified as a ubiquitous MAP. MAP4 stimu- lates tubulin polymerization and stabilizes polymerized-microtubules as do neural MAPs. Because MAP2, MAP4, and tau are structurally similar, the three MAPs are considered to constitute a superfamily. The architecture of the superfamily proteins consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain, and the microtubule-binding domain is further divided into three subdomains: the Pro-rich region ,t herepeat region, and the tail region. Recent studies have revealed the functions of these domains/subdomains in the MAP4 molecule: the projection domain keeps individual microtubules separated by suppressing the bundle-forming ability of the microtubule-binding domain, the Pro-rich region promotes the nucleation of microtubules, the repeat region promotes their elongation, and the tail region may contribute to the proper folding of the molecule. Isoforms of MAP4 are produced from the single MAP4 gene by alterna- tive RNA splicing, thereby five isoforms, with a deletion in the Pro-rich region or the repeat region, are expressed. The expression of these isoforms depends on the tissue type and the developmental stage, suggesting that the function of MAP4 is elabo- rately regulated by alternative splicing. In this chapter, we will address the structural and functional futures of MAP4 focusing on the functions of domains/subdomains and add some insights into the roles of the protein in neurons. Keywords MAP1 · MAP2 · MAP3 · MAP4 · MAP5 · Tau · Microtubule
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Microtubule-Associated Protein 4
Advances in Neurobiology, 2010Co-Authors: Kiyotaka Tokuraku, Kazuyuki Matsushima, Hiroyuki Nakagawa, Susumu KotaniAbstract:In contrast with neural microtubule-associated proteins (MAPs) such as MAP1, MAP2, and tau, MAP4 is identified as a ubiquitous MAP. MAP4 stimulates tubulin polymerization and stabilizes polymerized-microtubules as do neural MAPs. Because MAP2, MAP4, and tau are structurally similar, the three MAPs are considered to constitute a superfamily. The architecture of the superfamily proteins consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain, and the microtubule-binding domain is further divided into three subdomains: the Pro-rich region, the repeat region, and the tail region. Recent studies have revealed the functions of these domains/subdomains in the MAP4 molecule: the projection domain keeps individual microtubules separated by suppressing the bundle-forming ability of the microtubule-binding domain, the Pro-rich region promotes the nucleation of microtubules, the repeat region promotes their elongation, and the tail region may contribute to the proper folding of the molecule. Isoforms of MAP4 are produced from the single MAP4 gene by alternative RNA splicing, thereby five isoforms, with a deletion in the Pro-rich region or the repeat region, are expressed. The expression of these isoforms depends on the tissue type and the developmental stage, suggesting that the function of MAP4 is elaborately regulated by alternative splicing. In this chapter, we will address the structural and functional futures of MAP4 focusing on the functions of domains/subdomains and add some insights into the roles of the protein in neurons.
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Differences in the regulation of microtubule stability by the pro-rich region variants of microtubule-associated protein 4.
FEBS letters, 2006Co-Authors: Mohammad Rubayet Hasan, Susumu Kotani, Kazuyuki Matsushima, Mingyue Jin, Shigeaki Miyamoto, Hiroyuki NakagawaAbstract:We have recently reported a neural variant of microtubule-associated protein 4 with a short pro-rich region (MAP4-SP). Here, we show that the neural MAP4 has reduced microtubule-stabilizing activity, compared to the ubiquitous MAP4 with a long pro-rich region (MAP4-LP), both in vitro and in vivo. Fluorescence recovery after photobleaching analyses revealed that the interaction of MAP4-SP with the microtubules is very rapid, with a half-time of fluorescence recovery of 7 ± 2.36 s, compared to 19.5 ± 3.03 s in case of MAP4-LP. The dynamic interaction of MAP4-SP with microtubules in neural cells may contribute to the dynamic behaviors of extending neurites.
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Identification of a neural cell specific variant of microtubule-associated protein 4.
Cell structure and function, 2005Co-Authors: Kazuyuki Matsushima, Kiyotaka Tokuraku, Hiroyuki Nakagawa, Mohammad Rubayet Hasan, Masafumi Aosaki, Susumu KotaniAbstract:The microtubule-binding domain of MAP4, a ubiquitous microtubule-associated protein, contains a region rich in proline and basic residues (proline-rich region). We searched the bovine adrenal gland for MAP4 isoforms, and identified a novel variant lacking 72 consecutive amino acid residues within the proline-rich region, as compared with the full-length MAP4. The amino acid sequence of the missing region was highly conserved (about 85% identity/similarity) among the corresponding regions of bovine, human, mouse, and rat MAP4, which suggested the functional significance of this region. A comparison of the genomic sequence with the cDNA sequence revealed that the missing region is encoded by a single exon. A MAP4 variant cDNA homologous to the bovine form was also detected in rat cells, suggesting that the new variant can be generated by alternative splicing, not only in bovine but also in other mammalian species. The mRNA expression of the novel isoform was restricted to the brain and the adrenal medulla, suggesting that this isoform is specific to a certain cell type. Using a bacterially expressed fragment corresponding to the microtubule-binding domain of the novel isoform, we analyzed its in vitro characteristics. The fragment induced microtubule assembly and bound to preformed microtubules, but the activities were slightly lower than those of the conventional MAP4 fragment, which carries the full-length proline-rich region. The microtubules assembled in the presence of the fragment failed to be bundled. Instead, a constant spacing between neighboring microtubules was observed.
Kazuyuki Matsushima - One of the best experts on this subject based on the ideXlab platform.
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Microtubule-associated protein 4 binds to actin filaments and modulates their properties.
Journal of biochemistry, 2011Co-Authors: Kazuyuki Matsushima, Kiyotaka Tokuraku, Mohammad Rubayet Hasan, Susumu KotaniAbstract:We previously reported that an isoform of microtubule-associated protein 4 (MAP4) is localized to the distal area of developing neurites, where microtubules are relatively scarce, raising the possibility that MAP4 interacts with another major cytoskeletal component, actin filaments. In the present study, we examined the in vitro interaction between MAP4 and actin filaments, using bacterially expressed MAP4 and its truncated fragments. Sedimentation assays revealed that MAP4 and its microtubule-binding domain fragments bind to actin filaments under physiological conditions. The apparent dissociation constant and the binding stoichiometry of the fragments to actin were about 0.1 µm and 1 : 3 (MAP4/actin), respectively. Molecular dissection studies revealed that the actin-binding site on MAP4 is situated at the C-terminal part of the proline-rich region, where the microtubule-binding site is also located. Electron microscopy revealed that the MAP4-bound actin filaments become straighter and longer and that the number of actin bundles increases with greater concentrations of added MAP4 fragment, indicating that MAP4 binding alters the properties of the actin filaments. A multiple sequence alignment of the proline-rich regions of MAP4 and tau revealed two putative actin-binding consensus sequences.
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Structural and Functional Features
2011Co-Authors: Kiyotaka Tokuraku, Kazuyuki Matsushima, Hiroyuki Nakagawa, Susumu KotaniAbstract:In contrast with neural microtubule-associated proteins (MAPs) such as MAP1, MAP2, and tau, MAP4 is identified as a ubiquitous MAP. MAP4 stimu- lates tubulin polymerization and stabilizes polymerized-microtubules as do neural MAPs. Because MAP2, MAP4, and tau are structurally similar, the three MAPs are considered to constitute a superfamily. The architecture of the superfamily proteins consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain, and the microtubule-binding domain is further divided into three subdomains: the Pro-rich region ,t herepeat region, and the tail region. Recent studies have revealed the functions of these domains/subdomains in the MAP4 molecule: the projection domain keeps individual microtubules separated by suppressing the bundle-forming ability of the microtubule-binding domain, the Pro-rich region promotes the nucleation of microtubules, the repeat region promotes their elongation, and the tail region may contribute to the proper folding of the molecule. Isoforms of MAP4 are produced from the single MAP4 gene by alterna- tive RNA splicing, thereby five isoforms, with a deletion in the Pro-rich region or the repeat region, are expressed. The expression of these isoforms depends on the tissue type and the developmental stage, suggesting that the function of MAP4 is elabo- rately regulated by alternative splicing. In this chapter, we will address the structural and functional futures of MAP4 focusing on the functions of domains/subdomains and add some insights into the roles of the protein in neurons. Keywords MAP1 · MAP2 · MAP3 · MAP4 · MAP5 · Tau · Microtubule
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Microtubule-Associated Protein 4
Advances in Neurobiology, 2010Co-Authors: Kiyotaka Tokuraku, Kazuyuki Matsushima, Hiroyuki Nakagawa, Susumu KotaniAbstract:In contrast with neural microtubule-associated proteins (MAPs) such as MAP1, MAP2, and tau, MAP4 is identified as a ubiquitous MAP. MAP4 stimulates tubulin polymerization and stabilizes polymerized-microtubules as do neural MAPs. Because MAP2, MAP4, and tau are structurally similar, the three MAPs are considered to constitute a superfamily. The architecture of the superfamily proteins consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain, and the microtubule-binding domain is further divided into three subdomains: the Pro-rich region, the repeat region, and the tail region. Recent studies have revealed the functions of these domains/subdomains in the MAP4 molecule: the projection domain keeps individual microtubules separated by suppressing the bundle-forming ability of the microtubule-binding domain, the Pro-rich region promotes the nucleation of microtubules, the repeat region promotes their elongation, and the tail region may contribute to the proper folding of the molecule. Isoforms of MAP4 are produced from the single MAP4 gene by alternative RNA splicing, thereby five isoforms, with a deletion in the Pro-rich region or the repeat region, are expressed. The expression of these isoforms depends on the tissue type and the developmental stage, suggesting that the function of MAP4 is elaborately regulated by alternative splicing. In this chapter, we will address the structural and functional futures of MAP4 focusing on the functions of domains/subdomains and add some insights into the roles of the protein in neurons.
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Distinct neuronal localization of microtubule-associated protein 4 in the mammalian brain.
Neuroscience letters, 2010Co-Authors: Kiyotaka Tokuraku, Kazuyuki Matsushima, Satoshi Okuyama, Tsuneya Ikezu, Susumu KotaniAbstract:Although recent studies have suggested the role of microtubule-associated protein (MAP) 4 in some neuron-specific events, there are no reports that directly observed its neuronal localization. Here we show the detailed expression of MAP4 in the mammalian brain. Immunoblotting revealed the presence of MAP4 in all neuronal tissues. The site-specific localization of MAP4 was observed in sagittal brain sections: MAP4 was rich in brain-specific cells, cerebellum Purkinje cells and hippocampus pyramidal cells. When primary cultures of cortical neurons were immunostained, MAP4 was detected in the cell bodies and processes with patchy staining pattern. These results suggested that MAP4 play some roles in the central nervous system, such as the dynamic cytoskeletal reorganization and regulation of the microtubule-dependent long-range transport.
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Differences in the regulation of microtubule stability by the pro-rich region variants of microtubule-associated protein 4.
FEBS letters, 2006Co-Authors: Mohammad Rubayet Hasan, Susumu Kotani, Kazuyuki Matsushima, Mingyue Jin, Shigeaki Miyamoto, Hiroyuki NakagawaAbstract:We have recently reported a neural variant of microtubule-associated protein 4 with a short pro-rich region (MAP4-SP). Here, we show that the neural MAP4 has reduced microtubule-stabilizing activity, compared to the ubiquitous MAP4 with a long pro-rich region (MAP4-LP), both in vitro and in vivo. Fluorescence recovery after photobleaching analyses revealed that the interaction of MAP4-SP with the microtubules is very rapid, with a half-time of fluorescence recovery of 7 ± 2.36 s, compared to 19.5 ± 3.03 s in case of MAP4-LP. The dynamic interaction of MAP4-SP with microtubules in neural cells may contribute to the dynamic behaviors of extending neurites.