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K Mikoshiba

Publications and source records attributed to K Mikoshiba.

At least 19 recordsLinked to original sources
PubMed2001

C2 domains from different Ca2+ signaling pathways display functional and mechanistic diversity.

The ubiquitous C2 domain is a conserved Ca2+ triggered membrane-docking module that targets numerous signaling proteins to membrane surfaces where they regulate diverse processes critical for cell signaling. In this study, we quantitatively compared the equilibrium and kinetic parameters of C2 domains isolated from three functionally distinct signaling proteins: cytosolic phospholipase A2-alpha (cPLA2-alpha), protein kinase C-beta (PKC-beta), and synaptotagmin-IA (Syt-IA). The results show that equilibrium C2 domain docking to mixed phosphatidylcholine and phosphatidylserine membranes occurs at micromolar Ca2+ concentrations for the cPLA2-alpha C2 domain, but requires 3- and 10-fold higher Ca2+ concentrations for the PKC-beta and Syt-IA C2 domains ([Ca2+](1/2) = 4.7, 16, 48 microM, respectively). The Ca2+ triggered membrane docking reaction proceeds in at least two steps: rapid Ca2+ binding followed by slow membrane association. The greater Ca2+ sensitivity of the cPLA2-alpha domain results from its higher intrinsic Ca2+ affinity in the first step compared to the other domains. Assembly and disassembly of the ternary complex in response to rapid Ca2+ addition and removal, respectively, require greater than 400 ms for the cPLA2-alpha domain, compared to 13 ms for the PKC-beta domain and only 6 ms for the Syt-IA domain. Docking of the cPLA2-alpha domain to zwitterionic lipids is triggered by the binding of two Ca2+ ions and is stabilized via hydrophobic interactions, whereas docking of either the PKC-beta or the Syt-IA domain to anionic lipids is triggered by at least three Ca2+ ions and is maintained by electrostatic interactions. Thus, despite their sequence and architectural similarity, C2 domains are functionally specialized modules exhibiting equilibrium and kinetic parameters optimized for distinct Ca2+ signaling applications. This specialization is provided by the carefully tuned structural and electrostatic parameters of their Ca2+ and membrane-binding loops, which yield distinct patterns of Ca2+ coordination and contrasting mechanisms of membrane docking.

Binding Sites↗
PubMed2001

Characterization of KIAA1427 protein as an atypical synaptotagmin (Syt XIII).

Synaptotagmin (Syt) belongs to a family of type-I membrane proteins and is a protein that consists of a short extracellular N-terminus, a single transmembrane domain, two C2 domains and a short C-terminus. Here, we cloned and characterized a mouse orthologue of human KIAA1427 protein as an atypical Syt (named Syt XIII). Subcellular fractionation and antibody-uptake experiments indicate that Syt XIII is indeed a type-I membrane protein, but, unlike other Syt isoforms, lacks an N-terminal extracellular domain. Syt XIII C2 domains show relatively little similarity to Syt I (less than 35% identity at the amino acid level), and lack key amino acids responsible for Ca2+ binding. Because of these substitutions, the Syt XIII C2 domains did not show Ca2+-dependent phospholipid-binding activity, and Syt XIII is thus classified as a Ca2+ -independent isoform. By contrast, the Syt XIII C-terminal domain is highly homologous with other Syt isoforms and can function as a common receptor for neurexin Ialpha in vitro. Since Syt XIII is expressed in various tissues outside the brain, Syt XIII may be involved in constitutive vesicle transport.

Amino Acid Sequence↗
PubMed2001

Synergistic contributions of cyclin-dependant kinase 5/p35 and Reelin/Dab1 to the positioning of cortical neurons in the developing mouse brain.

Cyclin-dependent kinase (Cdk) 5 is a unique member of the Cdk family, because Cdk5 kinase activity is detected only in the nervous tissue. Two neuron-specific activating subunits of Cdk5, p35 and p39, have been identified. Overlapping expression pattern of these isoforms in the embryonic mouse brain and the significant residual Cdk5 kinase activity in brain homogenate of the p35-/- mice indicate the redundant functions of the Cdk5 activators in vivo. Severe neuronal migration defects in p35-/-Cdk5 +/- mice further support the idea that the redundant expression of the Cdk5 activators may cause a milder phenotype in p35-/- mice compared with Cdk5-/- mice. Mutant mice lacking either Cdk5 or p35 exhibit certain similarities with Reelin/Dab1-mutant mice in the disorganization of cortical laminar structure in the brain. To elucidate the relationship between Cdk5/p35 and Reelin/Dab1 signaling, we generated mouse lines that have combined defects of these genes. The addition of heterozygosity of either Dab1 or Reelin mutation to p35-/- causes the extensive migration defects of cortical neurons in the cerebellum. In the double-null mice of p35 and either Dab1 or Reelin, additional migration defects occur in the Purkinje cells in the cerebellum and in the pyramidal neurons in the hippocampus. These additional defects in neuronal migration in mice lacking both Cdk5/p35 and Reelin/Dab1 indicate that Cdk5/p35 may contribute synergistically to the positioning of the cortical neurons in the developing mouse brain.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Crucial role of type 1, but not type 3, inositol 1,4,5-trisphosphate (IP(3)) receptors in IP(3)-induced Ca(2+) release, capacitative Ca(2+) entry, and proliferation of A7r5 vascular smooth muscle cells.

Stimulation of G protein- or tyrosine kinase-coupled receptors regulates cell proliferation through intracellular Ca(2+) ([Ca(2+)](i)) signaling. In A7r5 cells, we confirmed that inositol 1,4,5-trisphosphate (IP(3)) mediates vasopressin (VP)-evoked Ca(2+) release from intracellular stores and showed that types 1 (IP(3)R(1)) and 3 (IP(3)R(3)) IP(3) receptors were expressed. Using antisera selective for IP(3)R(1) or IP(3)R(3) and another that interacted equally well with both subtypes, together with membranes from SF:9 cells expressing only single IP(3)R subtypes to calibrate immunoblotting, we established that A7r5 cells express 81% IP(3)R(1) and 19% IP(3)R(3). To elucidate the contributions of IP(3)R(1) and IP(3)R(3) to Ca(2+) signaling and proliferation, stable clones expressing promoter-inducible antisense cDNA fragments (-90 to +9) corresponding to the two IP(3)R subtypes were selected. Mild inhibition of IP(3)R(1) (71+/-8% of control level) slightly attenuated the IP(3)-evoked Ca(2+) release (IICR) induced by VP but significantly decreased the subsequent capacitative Ca(2+) entry (CCE) and proliferation. Moderate inhibition (34+/-6%) strongly decreased both IICR and CCE and further blocked proliferation. Complete inhibition almost abolished IICR and CCE and arrested proliferation entirely. Complete inhibition of IP(3)R(3) expression slightly attenuated IICR without affecting CCE or proliferation. In cells microinjected with a low dose of heparin, VP-induced CCE was more susceptible than IICR to mild inhibition of both IP(3)R(1) and IP(3)R(3). A high dose of heparin had a similar effect to complete inhibition of IP(3)R(1) expression: it blocked VP-evoked IICR entirely and CCE by 90%. We conclude that IP(3)R(1), but not IP(3)R(3), is crucial for IICR, CCE, and proliferation of vascular smooth muscle cells.

Animals↗
PubMed2001

Physical and functional interactions between Zic and Gli proteins.

Zic and Gli family proteins are transcription factors that share similar zinc finger domains. Recent studies indicate that Zic and Gli collaborate in neural and skeletal development. We provide evidence that the Zic and Gli proteins physically and functionally interact through their zinc finger domains. Moreover, Gli proteins were translocated to cell nuclei by coexpressed Zic proteins, and both proteins regulated each other's transcriptional activity. Our result suggests that the physical interaction between Zic and Gli is the molecular basis of their antagonistic or synergistic features in developmental contexts and that Zic proteins are potential modulators of the hedgehog-mediated signaling pathway.

3T3 Cells↗
PubMed2001

Synaptotagmin-like protein 1-3: a novel family of C-terminal-type tandem C2 proteins.

Synaptotagmins (Syt), rabphilin-3A, and Doc2 belong to a family of carboxyl terminal type (C-type) tandem C2 proteins and are thought to be involved in vesicular trafficking. We have cloned and characterized a novel family of C-type tandem C2 proteins, designated Slp1-3 (synaptotagmin-like protein 1-3). The Slp1-3 C2 domains show high homology to granuphilin-a C2 domains, but the amino-terminal domain of Slp1-3 does not contain any known protein motifs or a transmembrane domain. A subcellular fractionation study indicated that Slp1-3 proteins are peripheral membrane proteins. Phospholipid binding experiments indicated that Slp3 is a Ca(2+)-dependent isoform, but Slp1 and Slp2 are Ca(2+)-independent isoforms, because only the Slp3 C2A domain showed Ca(2+)-dependent phospholipid binding activity. The C-terminus of Slp1-3 also bound neurexin Ialpha in vitro, in the same manner as Syt family proteins, which may be important for the membrane association of Slp1-3. In addition, Slp family proteins are differentially distributed in different mouse tissues and at different developmental stages.

Amino Acid Sequence↗
PubMed2001

Induction of brain-derived neurotrophic factor by convulsant drugs in the rat brain: involvement of region-specific voltage-dependent calcium channels.

A high level of hippocampal brain-derived neurotrophic factor (BDNF) in normally aged as compared with young rats suggests that it is important to maintain a considerable level of hippocampal BDNF during aging in order to keep normal hippocampal functions. To elucidate possible mechanisms of endogenous BDNF increase, changes in levels of BDNF were studied in the rat brain following systemic administration of various convulsant agents; excitotoxic glutamate agonists, NMDA, kainic acid and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA); GABA receptor antagonists, picrotoxin, pentylenetetrazole (PTZ) and lindane (gamma-hexachlorocyclohexane); and L-type voltage-dependent calcium channel agonist, BAY-K 8644. Kainic acid and AMPA, but not NMDA, caused remarkable increases in BDNF protein in the rat hippocampus and entorhinal cortex. Picrotoxin, PTZ and lindane stimulated BDNF production in the entorhinal cortex and also in the hippocampus of rats showing very severe convulsions. On the other hand, BAY-K 8644 treatment increased BDNF levels in the neocortex and entorhinal cortex. Maximal levels of BDNF protein were observed at 12--24 h, 8--16 h and 6 h following administration of kainic acid, PTZ and BAY-K 8644, respectively. Kainic acid stimulated BDNF synthesis in presynaptic hippocampal granule neurons, but not in postsynaptic neurons with its receptors, while PTZ and BAY-K 8644 produced the same effects in postsynaptic neurons in the entorhinal cortex (in granule neurons in the hippocampus) and in the whole cortex, respectively. Nifedipine inhibited almost completely BAY-K 8644, but not PTZ, effects. omega-Conotoxin GVIA and DCG-IV partially blocked kainic acid-induced enhancement of BDNF, indicating involvement of L-type and N-type voltage-dependent calcium channels, respectively. In addition, BDNF levels in the hippocampus of mice deficient in D-myo-inositol-1,4,5-triphosphate receptor gene were scarcely different from those in the same region of controls, suggesting little involvement of intracellular calcium increase through this receptor. BAY-K 8644, but not kainic acid or PTZ, stimulated the phosphorylation of cyclic AMP responsive element binding protein. Our results indicate convulsant-dependent stimulation of BDNF production and involvement of region-specific voltage-dependent calcium channels.

Animals↗
PubMed2001

A proposed mechanism for the potentiation of cAMP-mediated acid secretion by carbachol.

Acid secretion in isolated rabbit gastric glands was monitored by the accumulation of [(14)C]aminopyrine. Stimulation of the glands with carbachol synergistically augmented the response to dibutyryl cAMP. The augmentation persisted even after carbachol was washed out and was resistant to chelated extracellular Ca(2+) and to inhibitors of either protein kinase C or calmodulin kinase II. Cytochalasin D at 10 microM preferentially blocked the secretory effect of carbachol and its synergism with cAMP, whereas it had no effect on histamine- or cAMP-stimulated acid secretion within 15 min. Cytochalasin D inhibited the carbachol-stimulated intracellular Ca(2+) concentration ([Ca(2+)](i)) increase due to release from the Ca(2+) store. Treatment of the glands with cytochalasin D redistributed type 3 inositol 1,4,5-trisphosphate receptor (the major subtype in the parietal cell) from the fraction containing membranes of large size to the microsomal fraction, suggesting a dissociation of the store from the plasma membrane. These findings suggest that intracellular Ca(2+) release by cholinergic stimulation is critical for determining synergism with cAMP in parietal cell activation and that functional coupling between the Ca(2+) store and the receptor is maintained by actin microfilaments.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

Role of the conserved WHXL motif in the C terminus of synaptotagmin in synaptic vesicle docking.

Synaptotagmin (Syt) I, an abundant synaptic vesicle protein, consists of one transmembrane region, two C2 domains, and a short C terminus. This protein is essential for both synaptic vesicle exocytosis and endocytosis via its C2 domains. Although the short C terminus is highly conserved among the Syt family and across species, little is known about the exact role of the conserved C terminus of Syt I. In this paper, we report a function of the Syt I C terminus in synaptic vesicle docking at the active zones. Presynaptic injection of a peptide corresponding to the C-terminal 21 amino acids of Syt I (named Syt-C) into the squid giant synapse blocked synaptic transmission without affecting the presynaptic action potential or the presynaptic Ca(2+) currents. The same procedure repeated with a mutant C-terminal peptide (Syt-CM) had no effect on synaptic transmission. Repetitive presynaptic stimulation with Syt-C produced a rapid decrease in the amplitude of the postsynaptic potentials as the synaptic block progressed, indicating that the peptide interferes with the docking step rather than the fusion step of synaptic vesicles. Electron microscopy of the synapses injected with the Syt-C peptide showed a marked decrease in the number of docked synaptic vesicles at the active zones, as compared with controls. These results indicate that Syt I is a multifunctional protein that is involved in at least three steps of synaptic vesicle cycle: docking, fusion, and reuptake of synaptic vesicles.

Action Potentials↗
PubMed2000

Molecular cloning and expression profile of Xenopus calcineurin A subunit(1).

We have cloned a cDNA encoding a catalytic subunit of calcineurin (CnA) expressed in Xenopus oocytes. The deduced amino acid sequence indicates 96.3% and 96.8% identities with the mouse and human CnAalpha isoforms, respectively. Xenopus CnA (XCnA) RNA and protein are expressed as maternal and throughout development. Recombinant XCnA protein interacted with calmodulin in the presence of Ca(2+). Deletion of calmodulin binding domain and auto-inhibitory domain revealed calcium independent phosphatase activity, thereby showing that XCnA is likely to be modulated by both calmodulin and calcium.

Amino Acid Sequence↗
PubMed2000

Expression of synaptotagmin I or II promotes neurite outgrowth in PC12 cells.

Synaptotagmin I (Syt I), a possible Ca(2+) sensor for neurotransmitter release, was suggested to be involved in neurite outgrowth of chick dorsal root ganglion (DRG) neurons, based on introduction of the antibody against the C2A domain into cells via mechanical lesions. Recently, however, the functional block antibody against the C2A domain was shown to block axonal repair processes, which raised a question as to whether Syt I is indeed involved in neurite outgrowth. In this study, we expressed Syt I or II in PC12 cells and found that these expression did indeed promote neurite outgrowth, as compared to control cells. We further showed that expression of the phospholipid binding activity-deficient mutant of Syt II (Delta180-183) had little effect on the neurite outgrowth of PC12 cells. These results indicate the Ca(2+)/phospholipid binding site of Syt I or II to be essential for neurite outgrowth.

Animals↗
PubMed2000

Calcium stores regulate the polarity and input specificity of synaptic modification.

Activity-induced synaptic modification is essential for the development and plasticity of the nervous system. Repetitive correlated activation of pre- and postsynaptic neurons can induce persistent enhancement or decrement of synaptic efficacy, commonly referred to as long-term potentiation or depression (LTP or LTD). An important unresolved issue is whether and to what extent LTP and LTD are restricted to the activated synapses. Here we show that, in the CA1 region of the hippocampus, reduction of postsynaptic calcium influx by partial blockade of NMDA (N-methyl-D-aspartate) receptors results in a conversion of LTP to LTD and a loss of input specificity normally associated with LTP, with LTD appearing at heterosynaptic inputs. The induction of LTD at homo- and heterosynaptic sites requires functional ryanodine receptors and inositol triphosphate (InsP3) receptors, respectively. Functional blockade or genetic deletion of type 1 InsP3 receptors led to a conversion of LTD to LTP and elimination of heterosynaptic LTD, whereas blocking ryanodine receptors eliminated only homosynaptic LTD. Thus, postsynaptic Ca2+, deriving from Ca2+ influx and differential release of Ca2+ from internal stores through ryanodine and InsP3 receptors, regulates both the polarity and input specificity of activity-induced synaptic modification.

Animals↗
PubMed2000

Molecular properties of Zic proteins as transcriptional regulators and their relationship to GLI proteins.

Zic family genes encode zinc finger proteins, which play important roles in vertebrate development. The zinc finger domains are highly conserved between Zic proteins and show a notable homology to those of Gli family proteins. In this study, we investigated the functional properties of Zic proteins and their relationship to the GLI proteins. We first established an optimal binding sequence for Zic1, Zic2, and Zic3 proteins by electrophoretic mobility shift assay-based target selection and mutational analysis. The selected sequence was almost identical to the GLI binding sequence. However, the binding affinity was lower than that of GLI. Consistent results were obtained in reporter assays, in which transcriptional activation by Zic proteins was less dependent on the GLI binding sequence than GLI1. Moreover, Zic proteins activated a wide range of promoters irrespective of the presence of a GLI binding sequence. When Zic and GLI proteins were cotransfected into cultured cells, Zic proteins enhanced or suppressed sequence-dependent, GLI-mediated transactivation depending on cell type. Taken together, these results suggest that Zic proteins may act as transcriptional coactivators and that their function may be modulated by the GLI proteins and possibly by other cell type-specific cofactors.

Base Sequence↗
PubMed2000

Drosophila AD3 mutation of synaptotagmin impairs calcium-dependent self-oligomerization activity.

Genetic analysis of a Drosophila synaptotagmin (Syt) I mutant (AD3) has revealed that Tyr-334 within the C2B domain is essential for efficient Ca(2+)-dependent neurotransmitter release. However, little is known as to why a missense mutation (Tyr-334-Asn) disrupts the function of the C2B domain at the molecular level. Here, we present evidence that a Tyr-312 to Asn substitution in mouse Syt II, which corresponds to the Drosophila AD3 mutation, completely impairs Ca(2+)-dependent self-oligomerization activity mediated by the C2B domain but allows partial interaction with wild-type proteins in a Ca(2+)-dependent manner. This observation is consistent with the fact that the AD3 allele is homozygous lethal but complements another mutant phenotype. We also showed that the Ca(2+)-dependent C2B self-oligomerization is inhibited by inositol 1,3,4, 5-tetrakisphosphate, a potent inhibitor of neurotransmitter release. All of these findings strongly support the idea that self-oligomerization of Syt I or II is essential for neurotransmitter release in vivo.

Amino Acid Sequence↗
PubMed2000

Galphas family G proteins activate IP(3)-Ca(2+) signaling via gbetagamma and transduce ventralizing signals in Xenopus.

During early embryonic development, IP(3)-Ca(2+) signaling transduces ventral signaling at the time of dorsoventral axis formation. To identify molecules functioning upstream in this signal pathway, we examined effects of a panel of inhibitory antibodies against Galphaq/11, Galphas/olf, or Galphai/o/t/z. While all these antibodies showed direct inhibition of their targets, their effects on redirection of the ventral mesoderm to a dorsal fate varied. Anti-Galphas/olf antibody showed strong induction of dorsal fate, anti-Galphai/o/t/z antibody did so weakly, and anti-Galphaq/11 antibody was without effect. Injection of betaARK, a Gbetagamma inhibitor, mimicked the dorsalizing effect of anti-Galphas/olf antibody, whereas injection of adenylyl cyclase inhibitors at a concentration which inhibited Galphas-coupled cAMP increase did not do so. The activation of Galphas-coupled receptor gave rise to Ca(2+) transients. All these results suggest that activation of the Galphas-coupled receptor relays dorsoventral signal to Gbetagamma, which then stimulates PLCbeta and then the IP(3)-Ca(2+) system. This signaling pathway may play a crucial role in transducing ventral signals.

Amino Acid Sequence↗
PubMed2000

Doc2gamma, a third isoform of double C2 protein, lacking calcium-dependent phospholipid binding activity.

The Doc2 (double C2) family consists of two isoforms (Doc2alpha and Doc2beta) characterized by an N-terminal Munc13-1 interacting domain (Mid) and two C2 domains that interact with Ca(2+) and phospholipid at the C-terminus. This Ca(2+)-binding property is thought to be important to the regulation of neurotransmitter release. In this paper, we report a third isoform of mouse Doc2, named Doc2gamma. Doc2gamma also contains a putative Mid domain and two C2 domains, and it is 45.6 and 43.2% identical to mouse Doc2alpha and Doc2beta, respectively, at the amino acid level. In contrast to the other Doc2 isoforms, the C2 domains of Doc2gamma impair Ca(2+)-dependent phospholipid binding activity. The highest expression of Doc2gamma mRNA was found in the heart, but occurs ubiquitously, the same as Doc2beta. These findings indicate that Doc2gamma may also function as an effector for Munc13-1 and that it may be involved in the regulation of vesicular trafficking.

Amino Acid Sequence↗
PubMed2000

Distinct self-oligomerization activities of synaptotagmin family. Unique calcium-dependent oligomerization properties of synaptotagmin VII.

Synaptotagmins constitute a large protein family, characterized by one transmembrane region and two C2 domains, and can be classified into several subclasses based on phylogenetic relationships and biochemical activities (Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427). Synaptotagmin I (Syt I), a possible Ca(2+) sensor for neurotransmitter release, showed both Ca(2+)-dependent (via the C2 domain) and -independent (via the NH(2)-terminal domain) self-oligomerization, which are thought to be important for synaptic vesicle exocytosis. However, little is known about the relationship between these two interactions and the Ca(2+)-dependent oligomerization properties of other synaptotagmin isoforms. In this study, we first examined the Ca(2+)-dependent self-oligomerization properties of synaptotagmin family by co-expression of T7- and FLAG-tagged Syts (full-length or cytoplasmic domain) in COS-7 cells. We found that Syt VII is a unique class of synaptotagmins that only showed robust Ca(2+)-dependent self-oligomerization at the cytoplasmic domain with EC(50) values of about 150 micrometer Ca(2+). In addition, Syt VII preferentially interacted with the previously described subclass of Syts (V, VI, and X) in a Ca(2+)-dependent manner. Co-expression of full-length and cytoplasmic portion of Syts VII (or II) indicate that Syt VII cytoplasmic domain oligomerizes in a Ca(2+)-dependent manner without being tethered at the NH(2)-terminal domain, whereas Ca(2+)-dependent self-oligomerization at the cytoplasmic domain of other isoforms (e.g. Syt II) occurs only when the two molecules are tethered at the NH(2)-terminal domain.

Animals↗