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Mitsuo Ikebe

Publications and source records attributed to Mitsuo Ikebe.

At least 37 records · Page 2Linked to original sources
PubMed2004

A one-headed class V myosin molecule develops multiple large (approximately 32-nm) steps successively.

Class V myosin (myosin-V) was first found as a processive motor that moves along an actin filament with large ( approximately 36-nm) successive steps and plays an important role in cargo transport in cells. Subsequently, several other myosins have also been found to move processively. Because myosin-V has two heads with ATP- and actin-binding sites, the mechanism of successive movement has been generally explained based on the two-headed structure. However, the fundamental problem of whether the two-headed structure is essential for the successive movement has not been solved. Here, we measure motility of engineered myosin-V having only one head by optical trapping nanometry. The results show that a single one-headed myosin-V undergoes multiple successive large (approximately 32-nm) steps, suggesting that a novel mechanism is operating for successive myosin movement.

Actin Cytoskeleton↗
PubMed2004

Protein kinase Cepsilon and the antiadrenergic action of adenosine in rat ventricular myocytes.

Adenosine-induced antiadrenergic effects in the heart are mediated by adenosine A(1) receptors (A(1)R). The role of PKCepsilon in the antiadrenergic action of adenosine was explored with adult rat ventricular myocytes in which PKCepsilon was overexpressed. Myocytes were transfected with a pEGFP-N1 vector in the presence or absence of a PKCepsilon construct and compared with normal myocytes. The extent of myocyte shortening elicited by electrical stimulation of quiescent normal and transfected myocytes was recorded with video imaging. PKCepsilon was found localized primarily in transverse tubules. The A(1)R agonist chlorocyclopentyladenosine (CCPA) at 1 microM rendered an enhanced localization of PKCepsilon in the t-tubular system. The beta-adrenergic agonist isoproterenol (Iso; 0.4 microM) elicited a 29-36% increase in myocyte shortening in all three groups. Although CCPA significantly reduced the Iso-produced increase in shortening in all three groups, the reduction caused by CCPA was greatest with PKCepsilon overexpression. The CCPA reduction of the Iso-elicited shortening was eliminated in the presence of a PKCepsilon inhibitory peptide. These results suggest that the translocation of PKCepsilon to the t-tubular system plays an important role in A(1)R-mediated antiadrenergic actions in the heart.

Adenosine↗
PubMed2004

Human deafness mutation of myosin VI (C442Y) accelerates the ADP dissociation rate.

The missense mutation of Cys(442) to Tyr of myosin VI causes progressive postlingual sensorineural deafness. Here we report the affects of the C442Y mutation on the kinetics of the actomyosin ATP hydrolysis mechanism and motor function of myosin VI. The largest changes in the kinetic mechanism of ATP hydrolysis produced by the C442Y mutation are about 10-fold increases in the rate of ADP dissociation from both myosin VI and actomyosin VI. The rates of ADP dissociation from acto-C442Y myosin VI-ADP and C442Y myosin VI-ADP are 20-40 times more rapid than the steady state rates and cannot be the rate-limiting steps of the hydrolysis mechanism in the presence or absence of actin. The 2-fold increase in the actin gliding velocity of C442Y compared with wild type (WT) may be explained at least in part by the more rapid rate of ADP dissociation. The C442Y myosin VI has a significant increase ( approximately 10-fold) in the steady state ATPase rate in the absence of actin relative to WT myosin VI. The steady state rate of actin-activated ATP hydrolysis is unchanged by the C442Y mutation at low (<10(-7) m) calcium but is calcium-sensitive with a 1.6-fold increase at high ( approximately 10(-4) m) calcium that does not occur with WT. The actin gliding velocity of the C442Y mutant decreases significantly at low surface density of myosin VI, suggesting that the mutation hampers the processive movement of myosin VI.

Actins↗
PubMed2004

ZIP kinase is responsible for the phosphorylation of myosin II and necessary for cell motility in mammalian fibroblasts.

Reorganization of actomyosin is an essential process for cell migration and myosin regulatory light chain (MLC20) phosphorylation plays a key role in this process. Here, we found that zipper-interacting protein (ZIP) kinase plays a predominant role in myosin II phosphorylation in mammalian fibroblasts. Using two phosphorylation site-specific antibodies, we demonstrated that a significant portion of the phosphorylated MLC20 is diphosphorylated and that the localization of mono- and diphosphorylated myosin is different from each other. The kinase responsible for the phosphorylation was ZIP kinase because (a) the kinase in the cell extracts phosphorylated Ser19 and Thr18 of MLC20 with similar potency; (b) immunodepletion of ZIP kinase from the cell extracts markedly diminished its myosin II kinase activity; and (c) disruption of ZIP kinase expression by RNA interference diminished myosin phosphorylation, and resulted in the defect of cell polarity and migration efficiency. These results suggest that ZIP kinase is critical for myosin phosphorylation and necessary for cell motile processes in mammalian fibroblasts.

Animals↗
PubMed2004

Ca2+-induced activation of ATPase activity of myosin Va is accompanied with a large conformational change.

We succeeded in expressing the recombinant full-length myosin Va (M5Full) and studied its regulation mechanism. The actin-activated ATPase activity of M5Full was significantly activated by Ca(2+), whereas the truncated myosin Va without C-terminal globular domain is not regulated by Ca(2+) and constitutively active. Sedimentation analysis showed that the sedimentation coefficient of M5Full undergoes a Ca(2+)-induced conformational transition from 14S to 11S. Electron microscopy revealed that at low ionic strength, M5Full showed an extended conformation in high Ca(2+) while it formed a folded shape in the presence of EGTA, in which the tail domain was folded back towards the head-neck region. Furthermore, we found that the motor domain of myosin Va folds back to the neck domain in Ca(2+) while the head-neck domain is more extended in EGTA. It is thought that the association of the motor domain to the neck inhibits the binding of the tail to the neck thus destabilizing a folded conformation in Ca(2+). This conformational transition is closely correlated to the actin-activated ATPase activity. These results suggest that the tail and neck domain play a role in the Ca(2+) dependent regulation of myosin Va.

Actins↗
PubMed2004

The myosin cardiac loop participates functionally in the actomyosin interaction.

The motor protein myosin in association with actin transduces chemical free energy in ATP into work in the form of actin translation against an opposing force. Mediating the actomyosin interaction in myosin is an actin binding site distributed among several peptides on the myosin surface including surface loops contributing to affinity and actin regulation of myosin ATPase. A structured surface loop on beta-cardiac myosin, the cardiac or C-loop, was recently demonstrated to affect myosin ATPase and was indirectly implicated in the actomyosin interaction. The C-loop is a conserved feature of all myosin isoforms with crystal structures, suggesting that it is an essential part of the core energy transduction machinery. It is shown here that proteolytic digestion of the C-loop in beta-cardiac myosin eliminates actin-activated myosin ATPase and reduces actomyosin affinity in rigor more than 100-fold. Studies of C-loop function in smooth muscle myosin were also undertaken using site-directed mutagenesis. Mutagenesis of a single charged residue in the C-loop of smooth muscle myosin alters actomyosin affinity and doubles myosin in vitro motility and actin-activated ATPase velocities, thereby involving a charged region of the loop in the actomyosin interaction. It appears likely that the C-loop is an essential electrostatic binding site for actin involved in modulation of actomyosin affinity and regulation of actomyosin ATPase velocity.

Actomyosin↗
PubMed2004

Dynamic changes in expression of myosin phosphatase in a model of portal hypertension.

Myosin phosphatase is a target for signaling pathways that modulate calcium sensitivity of force production in smooth muscle. Myosin phosphatase targeting subunit 1 (MYPT1) isoforms are generated by cassette-type alternative splicing of exons in the central and 3' portion of the transcript. Exclusion of the 3' alternative exon, coding for the leucine zipper (LZ)-positive MYPT1 isoform, is associated with the ability to desensitize to calcium (relax) in response to NO/cGMP-dependent signaling. We examined expression of MYPT1 isoforms and smooth muscle phenotype in normal rat vessels and in a prehepatic model of portal hypertension characterized by arteriolar dilation. The large capacitance vessels, aorta, pulmonary artery, and inferior vena cava expressed predominantly the 3' exon-out/LZ-positive MYPT1 isoform. The first-order mesenteric resistance artery (MA1) and portal vein (PV) expressed severalfold higher levels of MYPT1 with predominance of the 3' exon-included/LZ-negative isoform. There was minor variation in the presence of the MYPT1 central alternative exons. Myosin heavy and light chain splice variants in part cosegregated with MYPT1 isoforms. In response to portal hypertension induced by PV ligature, abundance of MYPT1 in PV and MA1 was significantly reduced and switched to the LZ-positive isoform. These changes were evident within 1 day of PV ligature and were maintained for up to 10 days before reverting to control values at day 14. Alteration of MYPT1 expression was part of a complex change in protein expression that can be generalized as a modulation from a phasic (fast) to a tonic (slow) contractile phenotype. Implications of vascular smooth muscle phenotypic diversity and reversible phenotypic modulation in portal hypertension with regards to regulation of blood flow are discussed.

Animals↗
PubMed2004

Unconstrained steps of myosin VI appear longest among known molecular motors.

Myosin VI is a two-headed molecular motor that moves along an actin filament in the direction opposite to most other myosins. Previously, a single myosin VI molecule has been shown to proceed with steps that are large compared to its neck size: either it walks by somehow extending its neck or one head slides along actin for a long distance before the other head lands. To inquire into these and other possible mechanism of motility, we suspended an actin filament between two plastic beads, and let a single myosin VI molecule carrying a bead duplex move along the actin. This configuration, unlike previous studies, allows unconstrained rotation of myosin VI around the right-handed double helix of actin. Myosin VI moved almost straight or as a right-handed spiral with a pitch of several micrometers, indicating that the molecule walks with strides slightly longer than the actin helical repeat of 36 nm. The large steps without much rotation suggest kinesin-type walking with extended and flexible necks, but how to move forward with flexible necks, even under a backward load, is not clear. As an answer, we propose that a conformational change in the lifted head would facilitate landing on a forward, rather than backward, site. This mechanism may underlie stepping of all two-headed molecular motors including kinesin and myosin V.

Actins↗
PubMed2003

F-actin and myosin II binding domains in supervillin.

Detergent-resistant membranes contain signaling and integral membrane proteins that organize cholesterol-rich domains called lipid rafts. A subset of these detergent-resistant membranes (DRM-H) exhibits a higher buoyant density ( approximately 1.16 g/ml) because of association with membrane skeleton proteins, including actin, myosin II, myosin 1G, fodrin, and an actin- and membrane-binding protein called supervillin (Nebl, T., Pestonjamasp, K. N., Leszyk, J. D., Crowley, J. L., Oh, S. W., and Luna, E. J. (2002) J. Biol. Chem. 277, 43399-43409). To characterize interactions among DRM-H cytoskeletal proteins, we investigated the binding partners of the novel supervillin N terminus, specifically amino acids 1-830. We find that the supervillin N terminus binds directly to myosin II, as well as to F-actin. Three F-actin-binding sites were mapped to sequences within amino acids approximately 280-342, approximately 344-422, and approximately 700-830. Sequences with combinations of these sites promote F-actin cross-linking and/or bundling. Supervillin amino acids 1-174 specifically interact with the S2 domain in chicken gizzard myosin and nonmuscle myosin IIA (MYH-9) but exhibit little binding to skeletal muscle myosin II. Direct or indirect binding to filamin also was observed. Overexpression of supervillin amino acids 1-174 in COS7 cells disrupted the localization of myosin IIB without obviously affecting actin filaments. Taken together, these results suggest that supervillin may mediate actin and myosin II filament organization at cholesterol-rich membrane domains.

Actins↗
PubMed2003

Functional diversity between Rho-kinase- and MLCK-mediated cytoskeletal actions in a myofibroblast-like hepatic stellate cell line.

Using a rat myofibroblast-like hepatic stellate cell line, we studied the actomyosin-based cytoskeletal actions mediated by Rho-kinase and/or myosin light chain kinase (MLCK). Calmodulin/MLCK inhibitors W-7 and ML-7 attenuated cell migration dose-relatedly at concentrations from 10(-6) to 10(-4)M and collagen gel-contraction by the cells at 10(-4)M, respectively. Rho-kinase inhibitors Y-27632 and HA1077 attenuated the gel-contraction at concentrations from 10(-6) to 10(-4) M, respectively. These Rho-kinase inhibitors attenuated cell migration at 10(-7)M but enhanced the migration at 10(-4)M, respectively. They altered cell morphology showing prominent peripheral actin bundles and sparse central stress fibers, in comparison with the calmodulin/MLCK inhibitors. Both ML-7 and Y-27632 attenuated phosphorylation of myosin regulatory light chain and cell attachment to extracellular substrate. ML-7 attenuated the activation of GTP-binding protein Rac, while Y-27632 did not. These findings suggest that the actomyosin-based cytoskeletal actions can be functionally diverse depending on the Rho-kinase-mediated pathway and the MLCK-mediated pathway.

Animals↗
PubMed2003

Two functional heads are required for full activation of smooth muscle myosin.

The motor activity of smooth muscle myosin II is regulated by the regulatory light chain phosphorylation, but it is not understood how phosphorylation activates motor activity. To address this question, we produced asymmetric heavy meromyosin (HMM), which is composed of a wild-type (WT) heavy chain and a mutant heavy chain having no motor activity (i.e. S236T or G457A). The actin-activated ATPase activities (Vmax) of asymmetric HMMs were only 21.8 and 8.4% of the wild-type HMM for S236A/WT HMM and G456A/WT HMM, respectively. If the two heads of HMM are independent for their ATPase activities, asymmetric HMM should show 50% of the activity of wild-type HMM; however, the activity of asymmetric HMM was much lower than the expected value. The results suggest that the activity of the wild-type head is attenuated by the presence of inactive head. Consistently, the actin-gliding velocity of the asymmetric HMM (i.e. S236T/WT or G457A/WT) was less than one-fifth of the wild-type HMM. The present study supports an idea that the two heads of smooth muscle myosin II interact with each other and the presence of two active heads is required for full activation.

Actins↗
PubMed2003

Determination of human myosin III as a motor protein having a protein kinase activity.

The class III myosin is the most divergent member of the myosin superfamily, having a domain with homology to a protein kinase. However, the function of class III myosin at a molecular level is not known at all, and it has been questioned whether it is actually an actin-based motor molecule. Here, we showed that human myosin III has an ATPase activity that is significantly activated by actin (20-fold) with Kactin of 112 microm and Vmax of 0.34 s-1, indicating the mechanoenzymatic activity of myosin III. Furthermore, we found that human myosin III has actin translocating activity (0.11 +/- 0.05 microm/s) using an in vitro actin gliding assay, and it moves toward the plus end of actin filaments. Myosin III containing calmodulin as the light chain subunit showed a protein kinase activity and underwent autophosphorylation. The autophosphorylation was the intramolecular process, and the sites were at the C-terminal end of the motor domain. Autophosphorylation significantly activated the kinase activity, although it did not affect the ATPase activity. The present study is the first report that clearly demonstrates that the class III myosin is an actin-based motor protein having a protein kinase activity.

Actins↗
PubMed2003

Agonist-induced changes in the phosphorylation of the myosin- binding subunit of myosin light chain phosphatase and CPI17, two regulatory factors of myosin light chain phosphatase, in smooth muscle.

The inhibition of myosin light chain phosphatase (MLCP) enhances smooth muscle contraction at a constant [Ca2+]. There are two components, myosin-binding subunit of MLCP (MBS) and CPI17, thought to be responsible for the inhibition of MLCP by external stimuli. The phosphorylation of MBS at Thr-641 and of CPI17 at Thr-38 inhibits the MLCP activity in vitro. Here we determined the changes in the phosphorylation of MBS and CPI17 after agonist stimulation in intact as well as permeabilized smooth muscle strips using phosphorylation-site-specific antibodies as probes. The CPI17 phosphorylation transiently increased after agonist stimulation in both alpha-toxin skinned and intact fibres. The time course of the increase in CPI17 phosphorylation after stimulation correlated with the increase in myosin regulatory light chain (MLC) phosphorylation. The increase in CPI17 phosphorylation was significantly diminished by Y27632, a Rho kinase inhibitor, and GF109203x, a protein kinase C inhibitor, suggesting that both the protein kinase C and Rho kinase pathways influence the change in CPI17 phosphorylation. On the other hand, a significant level of MBS phosphorylation at Thr-641, an inhibitory site, was observed in the resting state for both skinned and intact fibres and the agonist stimulation did not significantly alter the MBS phosphorylation level at Thr-641. While the removal of the agonist markedly decreased MLC phosphorylation and induced relaxation, the phosphorylation of MBS was unchanged, while CPI17 phosphorylation markedly diminished. These results strongly suggest that the phosphorylation of CPI17 plays a more significant role in the agonist-induced increase in myosin phosphorylation and contraction of smooth muscle than MBS phosphorylation in the Ca2+-independent activation mechanism of smooth muscle contraction.

Amino Acid Sequence↗
PubMed2002

Characterization of the motor activity of mammalian myosin VIIA.

Myosin VIIA was cloned from rat kidney, and the construct (M7IQ5) containing the motor domain, IQ domain, and the coiled-coil domain as well as the full-length myosin VIIA (M7full) was expressed. The M7IQ5 contained five calmodulins. Based upon native gel electrophoresis and gel filtration, it was found that M7IQ5 was single-headed, whereas M7full was two-headed, suggesting that the tail domain contributes to form the two-headed structure. M7IQ5 had Mg(2+)-ATPase activity that was markedly activated by actin with K(actin) of 33 microm and V(max) of 0.53 s(-1) head(-1). Myosin VIIA required an extremely high ATP concentration for ATPase activity, ATP-induced dissociation from actin, and in vitro actin-translocating activity. ADP markedly inhibited the actin-activated ATPase activity. ADP also significantly inhibited the ATP-induced dissociation of myosin VIIA from actin. Consistently, ADP decreased K(actin) of the actin-activated ATPase. ADP decreased the actin gliding velocity, although ADP did not stop the actin gliding even at high concentration. These results suggest that myosin VIIA has slow ATP binding or low affinity for ATP and relatively high affinity for ADP. The directionality of myosin VIIA was determined by using the polarity-marked dual fluorescence-labeled actin filaments. It was found that myosin VIIA is a plus-directed motor.

Actins↗
PubMed2002

Phosphorylation of CPI17 and myosin binding subunit of type 1 protein phosphatase by p21-activated kinase.

CPI17 and myosin binding subunit of type 1 protein phosphatase (MBS) are the regulators of myosin light chain phosphatase (MLCP). The function of both regulators is controlled by phosphorylation. The phosphorylation of CPI17 at Thr38 significantly enhances the inhibitory activity of CPI17 and the phosphorylation at Thr641 of MBS decreases the MLCP activity. Here, we found that p21-activated protein kinase (PAK) phosphorylates both CPI17 at Thr38 and MBS at Thr641. For CPI17, PAK specifically phosphorylated at Thr38, since the mutation of Thr38 to Ala completely abolished the phosphorylation. On the other hand, PAK phosphorylated Thr641 but not Thr799 of MBS, the site phosphorylated by Rho kinase. Because PAK phosphorylates MBS more than 1 mol/mol, it is anticipated that PAK also phosphorylates other sites in addition to Thr641. CPI17 phosphorylation induced by PAK significantly enhanced the inhibitory activity of CPI17. On the other hand, the phosphorylation of MBS by PAK also decreased the MLCP activity. These results raise the possibility that the PAK pathway plays a role in MLCP regulation.

Amino Acid Sequence↗
PubMed2002

M20, the small subunit of PP1M, binds to microtubules.

Myosin light chain phosphatase (PP1M) is composed of three subunits, i.e., M20, MBS, and a catalytic subunit. Whereas MBS is assigned as a myosin binding subunit, the function of M20 is unknown. In the present study, we found that M20 binds to microtubules. The binding activity was revealed by cosedimentation of M20 with microtubules and binding of tubulin to M20 affinity resin. Green fluorescent protein (GFP)-tagged M20 (M20-GFP) was expressed in chicken primary smooth muscle cells and COS-7 cells and was used as a probe for studying the association between M20 and microtubules in living cells. M20-GFP was localized on filamentous structures in both cell types. Colocalization analysis revealed that M20-GFP colocalized with tubulin. Treatment with nocodazole, but not cytochalasin B, abolished the filamentous structure of M20-GFP. These results indicate that M20-GFP associates with microtubules in cells. Microinjection of rhodamine-tubulin into the M20-expressing cells revealed that incorporation of rhodamine-tubulin into microtubules was significantly facilitated by microtubule-associated M20. Consistent with this result, M20 enhanced the rate of tubulin polymerization in vitro and produced elongated microtubules. These results suggest that M20 has a microtubule binding activity and plays a role in regulating microtubule dynamics.

Animals↗
PubMed2002

DOC-2/DAB2 is the binding partner of myosin VI.

Myosin VI is a molecular motor that moves processively along actin filaments and is believed to play a role in cargo movement in cells. Here we found that DOC-2/DAB2, a signaling molecule inhibiting the Ras cascade, binds to myosin VI at the globular tail domain. DOC-2/DAB2 binds stoichiometrically to myosin VI with one molecule per one myosin VI heavy chain. The C-terminal 122 amino acid residues of DOC-2/DAB2, containing the Grb2 binding site, is identified to be critical for the binding to myosin VI. Actin gliding assay revealed that the binding of DOC-2/DAB2 to myosin VI can support the actin filament gliding by myosin VI, suggesting that it can function as a myosin VI anchoring molecule. The C-terminal domain but not the N-terminal domain of DOC-2/DAB2 functions as a myosin VI anchoring site. The present findings suggest that myosin VI plays a role in transporting DOC-2/DAB2, a Ras cascade signaling molecule, thus involved in Ras signaling pathways.

Actin Cytoskeleton↗