Hiroyuki Kabayama

Mutation of the pleckstrin homology domain of Bruton's tyrosine kinase in immunodeficiency impaired inositol 1,3,4,5-tetrakisphosphate binding capacity.

Brutons tyrosine kinase (Btk), a cytoplasmic protein-tyrosine kinase, plays a pivotal role in B cell activation and development. Mutations in the pleckstrin homology (PH) domain of the Btk gene cause human X-linked agammaglobulinemia (XLA) and murine X-linked immunodeficiency (Xid). In this paper, we report that the PH domain of Btk functions as an inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate (IP6) binding domain (Kd of approximately 40 nM for IP4), and that all of the XLA (Phe replaced by Ser at position 25 (F25S), R28H, T33P, V64F, and V113D) and Xid mutations (R28C) found in the PH domain result in a dramatic reduction of IP4 binding activity. Furthermore, the rare alternative splicing variant, with 33 amino acids deleted in the PH domain, corresponding to exon 3 of the Btk gene, also impaired IP4 binding capacity. In contrast, a gain-of-function mutant called Btk*, which carries a E41K mutation in the PH domain, binds IP6 with two times higher affinity than the wild type. Our data suggest that B cell differentiation is closely correlated with the IP4 binding capacity of the PH domain of Btk.

Synaptotagmin IV is present at the Golgi and distal parts of neurites.

Abstract: Synaptotagmin IV (SytIV) is an immediate early gene induced by membrane depolarization in PC12 cells and in rat brain. However, little is known about the function of SytIV or the functional relationship between SytIV and SytI, because SytIV has yet to be localized. Here we show that SytIV was localized at the Golgi and distal part of neurites in nerve growth factor‐differentiated PC12 cells and cultured hippocampal neurons by immunocytochemistry using an isoform‐specific antibody (anti‐SytIV). These SytIV signals were not colocalized well with SytI signals. Upon membrane depolarization, SytIV signals were increased at both the Golgi and distal part of neurites within several hours in both types of cells. We further show that the increase of SytIV protein levels results from protein kinase A‐dependent gene up‐regulation. In hippocampal neurons, SytIV was developmentally regulated. These results suggest that SytIV may play a role at the Golgi and tips of neurites during development and synaptic plasticity.

Role of synaptotagmin, a Ca2+ and inositol polyphosphate binding protein, in neurotransmitter release and neurite outgrowth.

Synaptotagmin I (or II), a possible Ca(2+)-sensor of synaptic vesicles, has two functionally distinct C2 domains: the C2A domain binds Ca2+ and the C2B domain binds inositol high polyphosphates (IP4, IP5, and IP6). Ca(2+)-regulated exocytosis of secretory vesicles is proposed to be activated by Ca2+ binding to the C2A domain and inhibited by inositol polyphosphate binding to the C2B domain. Synaptotagmins now constitute a large family and are thought to be involved in both regulated and constitutive vesicular trafficking. They are classified from their distribution as neuronal (synaptotagmin I-V, X, and XI) and the ubiquitous type (synaptotagmin VI-IX). Among them, synaptotagmins III, V, VI and X are deficient in IP4 binding activity due to the amino acid substitutions in the C-terminal region of the C2B domain, suggesting that these isoforms can work for vesicular trafficking even in the presence of inositol high polyphosphates. Synaptotagmin I is also known to be present in neuronal growth cone vesicles. Antibody against the C2A domain (anti-C2A) that inhibits Ca(2+)-regulated exocytosis also blocked neurite outgrowth of the chick dorsal root ganglion (DRG) neuron, suggesting that Ca(2+)-dependent synaptotagmin activation is also crucial for neurite outgrowth.

Functional involvement of synaptotagmin I/II C2A domain in neurite outgrowth of chick dorsal root ganglion neuron

Abstract Synaptotagmin I or II (Syt I/II) is involved in Ca 2+ -regulated exocytosis of secretory vesicles, probably serving as a Ca 2+ -sensor via its C2A domain. Synaptotagmin is also known to be expressed in neuronal growth cone vesicles, but its functional involvement in neurite outgrowth remains largely unknown. In this study, we examined the function of Syt I/II in neurite outgrowth in cultured chick dorsal root ganglion neurons using an anti-synaptotagmin I and II C2A domain (anti-STI/II-C2A) antibody that inhibits Ca 2+ -regulated exocytosis. Immunoblots confirmed the high specificity of the anti-STI/II-C2A antibody and showed the expression of synaptotagmin I or II in chick dorsal root ganglion neurons. Immunocytochemistry revealed that synaptotagmin I or II is enriched at the growth cone region of chick dorsal root ganglion neurons, in both lamellipodia and filopodia. Whole or Fab-fragment of the anti-STI/II-C2A antibody loaded into dorsal root ganglion neurons by trituration significantly inhibited neurite outgrowth, whereas preimmune IgG had no effect. These results showed that the C2A domain of synaptotagmin I or II plays a crucial role in neurite outgrowth.

Syntaxin 6 regulates nerve growth factor-dependent neurite outgrowth.

Neurite outgrowth is crucial for neural circuit formation. Intracellular membrane trafficking is involved in the cell surface expansion that is necessary for neurite outgrowth. It is known that syntaxin 6 is predominantly located in the Golgi region in undifferentiated PC12 cells and that it regulates trans-Golgi network trafficking and the secretory pathway via its coiled-coil domains. However, whether it also regulates neurite outgrowth remains unknown. In this paper, we found that syntaxin 6 was located both in the Golgi apparatus and the distal tips of the neurites of nerve growth factor (NGF)-treated PC12 cells. We also showed that the overexpression of the first coiled-coil domain of syntaxin 6 inhibited NGF-dependent neurite outgrowth. However, the coiled-coil domain-disrupting mutant had little effect on neurite outgrowth. These results suggest that the first coiled-coil domain of syntaxin 6 plays a crucial role in NGF-dependent neurite outgrowth.

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 Ca2+‐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 Ca2+‐dependent self‐oligomerization activity mediated by the C2B domain but allows partial interaction with wild‐type proteins in a Ca2+‐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 Ca2+‐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.

Regulation of Irp gene expression by H-NS and Lrp proteins in Escherichia coli : dominant negative mutations in Irp

Lrp (leucine-responsive regulatory protein) is a global transcription factor of Escherichia coli and regulates, negatively or positively, many genes including lysU, which encodes lysyl-tRNA synthetase. Dominant negative mutations that derepress lysU expression were isolated in this study. These mutations affected a predicted DNA-binding domain of Lrp and mutants were defective both in activation of ilvIH expression and in repression of lysU expression. Consistent with the previous notion that lrp is autoregulated, lrp expression was derepressed by these mutations and repressed by multi-copy plasmids carrying lrp+. Moreover, we found by gene fusion and Northern blot hybridization that the “histonelike” protein, H-NS, bound specifically to a promoter segment of lrp in vitro, and the level of lrp expression increased in the hns null mutant. These results indicated that the lrp gene is not only feedback regulated by Lrp but is also controlled by H-NS protein.

Ca2+ induces macropinocytosis via F-actin depolymerization during growth cone collapse

Growth cone collapse occurs in repulsive axon guidance and is accompanied by a reduction in the surface area of the plasma membrane of growth cones. However, the mechanism of this reduction is unclear. Here, we show that during growth cone collapse, caffeine-induced Ca(2+) release from ryanodine-sensitive Ca(2+) stores triggers the formation of large vacuoles in growth cones by macropinocytosis, a clathrin-independent endocytosis for the massive retrieval of the cellular plasma membrane, and subsequent retrograde membrane transport. We observed a significant correlation of the area of caffeine-induced macropinosomes with growth cone collapse. We also detected macropinocytosis induced by semaphorin 3A, a typical repulsive cue, and correlation between the area of semaphorin 3A-induced macropinocytic vacuoles and growth cone collapse. Moreover, jasplakinolide, an inhibitor of F-actin depolymerization, blocked caffeine-induced macropinocytosis. We propose that the coordinated regulation of actin cytoskeletal reorganization and macropinocytosis-mediated retrograde membrane trafficking may contribute to Ca(2+)-induced axon growth inhibition.

Syntaxin 1B Suppresses Macropinocytosis and Semaphorin 3A-Induced Growth Cone Collapse

Growth cone collapse is a crucial process for repulsive axon guidance and is accompanied by a reduction in growth cone surface area. This process of reduction may be regulated by endocytosis; however, its molecular mechanism is unclear. Macropinocytosis is a clathrin-independent form of endocytosis in which large areas of plasma membrane can be engulfed. We have reported previously that macropinocytosis is induced in growth cones of chick dorsal root ganglion neurons by semaphorin 3A (Sema3A), a repulsive axon guidance cue, and that Sema3A-induced reduction in growth cone surface area and macropinocytic vacuole area were correlated, suggesting a positive role for macropinocytosis in Sema3A-induced growth cone collapse. In the present study, we found that syntaxin 1B (Syx1B), a membrane trafficking protein, is a negative regulator of macropinocytosis, and its expression is downregulated by Sema3A signaling. Macropinocytosis inhibitor ethylisopropylamiloride or Syx1B overexpression suppressed Sema3A-induced macropinocytosis and growth cone collapse. These results indicate that Syx1B couples macropinocytosis-mediated massive internalization of the plasma membrane to Sema3A-induced growth cone collapse.

Parkin promotes proteasomal degradation of synaptotagmin IV by accelerating polyubiquitination

Abstract Parkin is an E3 ubiquitin ligase whose mutations cause autosomal recessive juvenile Parkinsons disease (PD). Unlike the human phenotype, parkin knockout (KO) mice show no apparent dopamine neuron degeneration, although they demonstrate reduced expression and activity of striatal mitochondrial proteins believed to be necessary for neuronal survival. Instead, parkin‐KO mice show reduced striatal evoked dopamine release, abnormal synaptic plasticity, and non‐motor symptoms, all of which appear to mimic the preclinical features of Parkinsons disease. Extensive studies have screened candidate synaptic proteins responsible for reduced evoked dopamine release, and synaptotagmin XI (Syt XI), an isoform of Syt family regulating membrane trafficking, has been identified as a substrate of parkin in humans. However, its expression level is unaltered in the striatum of parkin‐KO mice. Thus, the target(s) of parkin and the molecular mechanisms underlying the impaired dopamine release in parkin‐KO mice remain unknown. In this study, we focused on Syt IV because of its highly homology to Syt XI, and because they share an evolutionarily conserved lack of Ca2 +‐binding capacity; thus, Syt IV plays an inhibitory role in Ca2 +‐dependent neurotransmitter release in PC12 cells and neurons in various brain regions. We found that a proteasome inhibitor increased Syt IV protein, but not Syt XI protein, in neuron‐like, differentiated PC12 cells, and that parkin interacted with and polyubiquitinated Syt IV, thereby accelerating its protein turnover. Parkin overexpression selectively degraded Syt IV protein, but not Syt I protein (indispensable for Ca2 +‐dependent exocytosis), thus enhancing depolarization‐dependent exocytosis. Furthermore, in parkin‐KO mice, the level of striatal Syt IV protein was increased. Our data indicate a crucial role for parkin in the proteasomal degradation of Syt IV, and provide a potential mechanism of parkin‐regulated, evoked neurotransmitter release. HighlightsSynaptotagmin IV (Syt IV), an evoked exocytosis regulator, is degraded by parkin.The E3 ubiqutin ligase parkin polyubiquitinates Syt IV and accelerates its turnover.Syt IV is increased in the striatum of parkin‐KO mice.Parkin overexpression enhances evoked exocytosis by degradation of Syt IV.