柳 茂

Abstract Mitochondria are involved in various cellular processes, such as energy production, inflammatory responses, and cell death. Mitochondrial dysfunction is associated with many age-related diseases, including neurological disorders and heart failure. Mitochondrial quality is strictly maintained by mitochondrial dynamics linked to an adequate supply of phospholipids and other substances from the endoplasmic reticulum (ER). The outer mitochondrial membrane-localized E3 ubiquitin ligase MITOL/MARCHF5 is responsible for mitochondrial quality control through the regulation of mitochondrial dynamics, formation of mitochondria-ER contacts, and mitophagy. MITOL deficiency has been shown to impair mitochondrial function, cause an excessive inflammatory response, and increase vulnerability to stress, resulting in the exacerbation of the disease. In this study, we overview the ubiquitin-mediated regulation of mitochondrial function by MITOL and the relationship between MITOL and diseases.

Abnormal mitochondrial fragmentation by dynamin-related protein1 (Drp1) is associated with the progression of aging-associated heart diseases, including heart failure and myocardial infarction (MI). Here, we report a protective role of outer mitochondrial membrane (OMM)-localized E3 ubiquitin ligase MITOL/MARCH5 against cardiac senescence and MI, partly through Drp1 clearance by OMM-associated degradation (OMMAD). Persistent Drp1 accumulation in cardiomyocyte-specific MITOL conditional-knockout mice induced mitochondrial fragmentation and dysfunction, including reduced ATP production and increased ROS generation, ultimately leading to myocardial senescence and chronic heart failure. Furthermore, ischemic stress-induced acute downregulation of MITOL, which permitted mitochondrial accumulation of Drp1, resulted in mitochondrial fragmentation. Adeno-associated virus-mediated delivery of the MITOL gene to cardiomyocytes ameliorated cardiac dysfunction induced by MI. Our findings suggest that OMMAD activation by MITOL can be a therapeutic target for aging-associated heart diseases, including heart failure and MI.

The transfer of phospholipids from the endoplasmic reticulum to mitochondria via the mitochondria-endoplasmic reticulum (ER) contact site (MERCS) is essential for maintaining mitochondrial function and integrity. Here, we identified RMDN3/PTPIP51, possessing phosphatidic acid (PA)-transfer activity, as a neighboring protein of the mitochondrial E3 ubiquitin ligase MITOL/MARCH5 by proximity-dependent biotin labeling using APEX2. We found that MITOL interacts with and ubiquitinates RMDN3. Mutational analysis identified lysine residue 89 in RMDN3 as a site of ubiquitination by MITOL. Loss of MITOL or the substitution of lysine 89 to arginine in RMDN3 significantly reduced the PA-binding activity of RMDN3, suggesting that MITOL regulates the transport of PA to mitochondria by activating RMDN3. Our findings imply that ubiquitin signaling regulates phospholipid transport at the MERCS.

Amyloid-β (Aβ) plaques are strongly associated with the development of Alzheimer's disease (AD). However, it remains unclear how morphological differences in Aβ plaques determine the pathogenesis of Aβ. Here, we categorized Aβ plaques into four types based on the macroscopic features of the dense core, and found that the Aβ-plaque subtype containing a larger dense core showed the strongest association with neuritic dystrophy. Astrocytes dominantly accumulated toward these expanded/dense-core-containing Aβ plaques. Previously, we indicated that deletion of the mitochondrial ubiquitin ligase MITOL/MARCH5 triggers mitochondrial impairments and exacerbates cognitive decline in a mouse model with AD-related Aβ pathology. In this study, MITOL deficiency accelerated the formation of expanded/dense-core-containing Aβ plaques, which showed reduced contacts with astrocytes, but not microglia. Our findings suggest that expanded/dense-core-containing Aβ-plaque formation enhanced by the alteration of mitochondrial function robustly contributes to the exacerbation of Aβ neuropathology, at least in part, through the reduced contacts between Aβ plaques and astrocytes.

Mouse models of various neuropsychiatric disorders, such as schizophrenia, often display an immature dentate gyrus, characterized by increased numbers of immature neurons and neuronal progenitors and a dearth of mature neurons. We previously demonstrated that the CRMP5-associated GTPase (CRAG), a short splice variant of Centaurin-γ3/AGAP3, is highly expressed in the dentate gyrus. CRAG promotes cell survival and antioxidant defense by inducing the activation of serum response factors at promyelocytic leukemia protein bodies, which are nuclear stress-responsive domains, during neuronal development. However, the physiological role of CRAG in neuronal development remains unknown. Here, we analyzed the role of CRAG using dorsal forebrain-specific CRAG/Centaurin-γ3 knockout mice. The mice revealed maturational abnormality of the hippocampal granule cells, including increased doublecortin-positive immature neurons and decreased calbindin-positive mature neurons, a typical phenotype of immature dentate gyri. Furthermore, the mice displayed hyperactivity in the open-field test, a common measure of exploratory behavior, suggesting that these mice may serve as a novel model for neuropsychiatric disorder associated with hyperactivity. Thus, we conclude that CRAG is required for the maturation of neurons in the dentate gyrus, raising the possibility that its deficiency might promote the development of psychiatric disorders in humans.

Mitochondrial pathophysiology is implicated in the development of Alzheimer's disease (AD). An integrative database of gene dysregulation suggests that the mitochondrial ubiquitin ligase MITOL/MARCH5, a fine-tuner of mitochondrial dynamics and functions, is downregulated in patients with AD. Here, we report that the perturbation of mitochondrial dynamics by MITOL deletion triggers mitochondrial impairments and exacerbates cognitive decline in a mouse model with AD-related Aβ pathology. Notably, MITOL deletion in the brain enhanced the seeding effect of Aβ fibrils, but not the spontaneous formation of Aβ fibrils and plaques, leading to excessive secondary generation of toxic and dispersible Aβ oligomers. Consistent with this, MITOL-deficient mice with Aβ etiology exhibited worsening cognitive decline depending on Aβ oligomers rather than Aβ plaques themselves. Our findings suggest that alteration in mitochondrial morphology might be a key factor in AD due to directing the production of Aβ form, oligomers or plaques, responsible for disease development.

Parkin promotes cell survival by removing damaged mitochondria via mitophagy. However, although some studies have suggested that Parkin induces cell death, the regulatory mechanism underlying the dual role of Parkin remains unknown. Herein, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) regulates Parkin-mediated cell death through the FKBP38-dependent dynamic translocation from the mitochondria to the ER during mitophagy. Mechanistically, MITOL mediates ubiquitination of Parkin at lysine 220 residue, which promotes its proteasomal degradation, and thereby fine-tunes mitophagy by controlling the quantity of Parkin. Deletion of MITOL leads to accumulation of the phosphorylated active form of Parkin in the ER, resulting in FKBP38 degradation and enhanced cell death. Thus, we have shown that MITOL blocks Parkin-induced cell death, at least partially, by protecting FKBP38 from Parkin. Our findings unveil the regulation of the dual function of Parkin and provide a novel perspective on the pathogenesis of PD.

In mitochondrial disorders, short stature and growth failure are common symptoms, but their underlying mechanism remains unknown. In this study, we examined the cause of growth failure of mice induced by nestin promoter-driven knockout of the mitochondrial ubiquitin ligase MITOL (MARCH5), a key regulator of mitochondrial function. MITOL-knockout mice have congenital hypoplasia of the anterior pituitary caused by decreased expression of pituitary transcript factor 1 (Pit1). Consistently, both mRNA levels of growth hormone (GH) and prolactin levels were markedly decreased in the anterior pituitary of mutant mice. Growth failure of mutant mice was partly rescued by hypodermic injection of recombinant GH. To clarify whether this abnormality was induced by the primary effect of MITOL knockdown in the anterior pituitary or a secondary effect of other lesions, we performed lentiviral-mediated knockdown of MITOL on cultured rat pituitary GH3 cells, which secrete GH. GH production was severely compromised in MITOL-knockdown GH3 cells. In conclusion, MITOL plays a critical role in the development of the anterior pituitary; therefore, mice with MITOL dysfunction exhibited pituitary dwarfism caused by anterior pituitary hypoplasia. Our findings suggest that mitochondrial dysfunction is commonly involved in the unknown pathogenesis of pituitary dwarfism.

Mitochondria are highly dynamic organelles that constantly fuse, divide, and move, and their function is regulated and maintained by their morphologic changes. Mitochondrial disease (MD) comprises a group of disorders involving mitochondrial dysfunction. However, it is not clear whether changes in mitochondrial morphology are related to MD. In this study, we examined mitochondrial morphology in fibroblasts from patients with MD (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and Leigh syndrome). We observed that MD fibroblasts exhibited significant mitochondrial fragmentation by upregulation of Drp1, which is responsible for mitochondrial fission. Interestingly, the inhibition of mitochondrial fragmentation by Drp1 knockdown enhanced cellular toxicity and led to cell death in MD fibroblasts. These results suggest that mitochondrial fission plays a critical role in the attenuation of mitochondrial damage in MD fibroblasts.

CRMP-5-associated GTPase (CRAG), a short splicing variant of centaurin-γ3/AGAP3, is predominantly expressed in the developing brain. We previously demonstrated that CRAG, but not centaurin-γ3, translocates to the nucleus and activates the serum response factor (SRF)-c-Fos pathway in cultured neuronal cells. However, the physiological relevance of CRAG in vivo is unknown. Here, we found that CRAG/centaurin-γ3-knockout mice showed intensively suppressed kainic acid-induced c-fos expression in the hippocampus. Analyses of molecular mechanisms underlying CRAG-mediated SRF activation revealed that CRAG has an essential role in GTPase activity, interacts with ELK1 (a co-activator of SRF), and activates SRF in an ELK1-dependent manner. Furthermore, CRAG and ELK1 interact with promyelocytic leukaemia bodies through SUMO-interacting motifs, which is required for SRF activation. These results suggest that CRAG plays a critical role in ELK1-dependent SRF-c-fos activation at promyelocytic leukaemia bodies in the developing brain.

Little is known about the molecular mechanisms of cognitive deficits in psychiatric disorders. CAMDI is a psychiatric disorder-related factor, the deficiency of which in mice results in delayed neuronal migration and psychiatrically abnormal behaviors. Here, we found that CAMDI-deficient mice exhibited impaired recognition memory and spatial reference memory. Knockdown of CAMDI in hippocampal neurons increased the amount of internalized alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor (AMPAR) and attenuated the chemical long-term potentiation (LTP)-dependent cell surface expression of AMPAR. KIBRA was identified as a novel CAMDI-binding protein that retains AMPAR in the cytosol after internalization. KIBRA inhibited CAMDI-dependent Rab11 activation, thereby attenuating AMPAR cell surface expression. These results suggest that CAMDI regulates AMPAR cell surface expression during LTP. CAMDI dysfunction may partly explain the mechanism underlying cognitive deficits in psychiatric diseases.

The DISC1-interacting protein CAMDI has been suggested to promote radial migration through centrosome regulation. However, its physiological relevance is unclear. Here, we report the generation and characterization of CAMDI-deficient mice. CAMDI-deficient mice exhibit delayed radial migration with aberrant neural circuit formation and psychiatric behaviors including hyperactivity, repetitive behavior, and social abnormality typically observed in autism spectrum disorder patients. Analyses of direct targets of CAMDI identify HDAC6 whose -tubulin deacetylase activity is inhibited by CAMDI at the centrosome. CAMDI deficiency increases HDAC6 activity, leading to unstable centrosomes with reduced -tubulin and acetylated -tubulin levels. Most importantly, psychiatric behaviors as well as delayed migration are significantly rescued by treatment with Tubastatin A, a specific inhibitor of HDAC6. Our findings indicate that HDAC6 hyperactivation by CAMDI deletion causes psychiatric behaviors, at least in part, through delayed radial migration due to impaired centrosomes.

Tax1-binding protein 1 (TAX1BP1) is a ubiquitin-binding protein that restricts nuclear factor-kappa B (NF-kappa B) activation and facilitates the termination of aberrant inflammation. However, its roles in B-cell activation and differentiation are poorly understood. To evaluate the function of TAX1BP1 in B cells, we established TAX1BP1-deficient DT40 B cells that are hyper-responsive to CD40-induced extracellular signal-regulated kinase (ERK) activation signaling, exhibit prolonged and exaggerated ERK phosphorylation and show enhanced B lymphocyte-induced maturation protein 1 (Blimp-1; a transcription factor inducing plasma cell differentiation) expression that is ERK-dependent. Furthermore, TAX1BP1-deficient cells exhibit significantly decreased surface IgM expression and increased IgM secretion. Moreover, TAX1BP1-deficient mice display reduced germinal center formation and antigen-specific antibody production. These findings show that TAX1BP1 restricts ERK activation and Blimp-1 expression and regulates germinal center formation.

The mitochondrial ubiquitin ligase MITOL regulates mitochondria! dynamics. We report here that MITOL regulates mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) domain formation through nnitofusin2 (Mfn2). MITOL interacts with and ubiquitinates mitochondrial Mfn2, but not ER-associated Mfn2. Mutation analysis identified a specific interaction between MITOL C-terminal domain and Mfn2 HR1 domain. MITOL mediated lysine-63-linked polyubiquitin chain addition to Mfn2, but not its proteasomal degradation. MITOL knockdown inhibited Mfn2 complex formation and caused Mfn2 mislocalization and MAM dysfunction. Sucrose-density gradient centrifugation and blue native PAGE retardation assay demonstrated that MITOL is required for GTP-dependent Mfn2 oligomerization. MITOL knockdown reduced Mfn2 GTP binding, resulting in reduced GTP hydrolysis. We identified K192 in the GTPase domain of Mfn2 as a major ubiquitination site for MITOL. A K192R mutation blocked oligomerization even in the presence of GTP. Taken together, these results suggested that MITOL regulates ER tethering to mitochondria by activating Mfn2 via K192 ubiquitination.

Nitric oxide (NO) is implicated in neuronal cell survival. However, excessive NO production mediates neuronal cell death, in part via mitochondrial dysfunction. Here, we report that the mitochondrial ubiquitin ligase, MITOL, protects neuronal cells from mitochondrial damage caused by accumulation of S-nitrosylated microtubule-associated protein 1B-light chain 1 (LC1). S-nitrosylation of LC1 induces a conformational change that serves both to activate LC1 and to promote its ubiquination by MITOL, indicating that microtubule stabilization by LC1 is regulated through its interaction with MITOL. Excessive NO production can inhibit MITOL, and MITOL inhibition resulted in accumulation of S-nitrosylated LC1 following stimulation of NO production by calcimycin and N-methyl-D-aspartate. LC1 accumulation under these conditions resulted in mitochondrial dysfunction and neuronal cell death. Thus, the balance between LC1 activation by S-nitrosylation and down-regulation by MITOL is critical for neuronal cell survival. Our findings may contribute significantly to an understanding of the mechanisms of neurological diseases caused by nitrosative stress-mediated mitochondrial dysfunction.

We previously demonstrated that CRAM (CRMP5)-associated GTPase (CRAG), a short splicing variant of centaurin-gamma 3/AGAP3, facilitated degradation of expanded polyglutamine protein (polyQ) via the nuclear ubiquitin-proteasome pathway. Taking advantage of this feature, we also showed that lentivirus-mediated CRAG expression in the Purkinje cells of mice expressing polyQ resulted in clearance of the polyQ aggregates and rescue from ataxia. However, the molecular basis of the function of CRAG in cell survival against polyQ remains unclear. Here we report that CRAG, but not centaurin-gamma 3, induces transcriptional activation of c-Fos-dependent activator protein-1 (AP-1) via serum response factor (SRF). Mutation analysis indicated that the nuclear localization signal and both the N- and C-terminal regions of CRAG are critical for SRF-dependent c-Fos activation. CRAG knockdown by siRNA or expression of a dominant negative mutant of CRAG significantly attenuated the c-Fos activation triggered by either polyQ or the proteasome inhibitor MG132. Importantly, c-Fos expression partially rescued the enhanced cytotoxicity of CRAG knockdown in polyQ-expressing or MG132-treated cells. Finally, we suggest the possible involvement of CRAG in the sulfiredoxin-mediated antioxidant pathway via AP-1. Taken together, these results demonstrated that CRAG enhances the cell survival signal against the accumulation of unfolded proteins, including polyQ, through not only proteasome activation, but also the activation of c-Fos-dependent AP-1.

Seven human Sir2 homologues (sirtuin) have been identified to date. In this study, we clarified the mechanism of subcellular localization of two SIRT5 isoforms (i.e., SIRT5iso1 and SIRT5iso2) encoded by the human SIRT5 gene and whose C-termini slightly differ from each other. Although both isoforms contain cleavable mitochondrial targeting signals at their N-termini, we found that the cleaved SIRT5iso2 was localized mainly in mitochondria, whereas the cleaved SIRT5iso1 was localized in both mitochondria and cytoplasm. SIRT5 delta C, which is composed of only the common domain, showed the same mitochondrial localization as that of SIRT5iso2. These results suggest that the cytoplasmic localization of cleaved SIRT5iso1 is dependent on the SIRT5iso1-specific C-terminus. Further analysis showed that the C-terminus of SIRT5iso2, which is rich in hydrophobic amino acid residues, functions as a mitochondrial membrane insertion signal. In addition, a de novo protein synthesis inhibition experiment using cycloheximide showed that the SIRT5iso1-specific C-terminus is necessary for maintaining the stability of SIRT5iso1. Moreover, genome sequence analysis from each organism examined indicated that SIRT5iso2 is a primate-specific isoform. Taken together, these results indicate that human SIRT5 potentially controls various primate-specific functions via two isoforms with different intracellular localizations or stabilities.

Expansion of a polyglutamine tract in ataxin-3 (polyQ) causes Machado-Joseph disease, a late-onset neurodegenerative disorder characterized by ubiquitin-positive aggregate formation. Several lines of evidence demonstrate that polyQ also accumulates in mitochondria and causes mitochondria! dysfunction. To uncover the mechanism of mitochondrial quality-control via the ubiquitin-proteasome pathway, we investigated whether MITOL, a novel mitochondrial ubiquitin ligase localized in the mitochondrial outer membrane, is involved in the degradation of pathogenic ataxin-3 in mitochondria. In this study, we used N-terminal-truncated pathogenic ataxin-3 with a 71-glutamine repeat (Delta NAT-3Q71) and found that MITOL promoted Delta NAT-3Q71 degradation via the ubiquitin-proteasome pathway and attenuated mitochondrial accumulation of Delta NAT-3Q71. Conversely, MITOL knockdown induced an accumulation of detergent-insoluble Delta NAT-3Q71 with large aggregate formation, resulting in cytochrome c release and subsequent cell death. Thus, MITOL plays a protective role against polyQ toxicity, and thereby may be a potential target for therapy in polyQ diseases. Our findings indicate a protein quality-control mechanism at the mitochondrial outer membrane via a MITOL-mediated ubiquitin-proteasome pathway. (C) 2010 Elsevier B.V. and Mitochorndria Research Society. All rights reserved.

Centrosomes play a crucial role in the directed migration of developing neurons. However, the underlying mechanism is poorly understood. This study has identified a novel disrupted in schizophrenia 1 (DISC1)-interacting protein, named CAMDI after coiled-coil protein associated with myosin II and DISC1, which translocates to the centrosome in a DISC1-dependent manner. Knockdown of CAMDI by shRNA revealed severely impaired radial migration with disoriented centrosomes. A yeast two-hybrid screen identified myosin II as a binding protein of CAMDI. CAMDI interacts preferentially with phosphomyosin II and induces an accumulation of phosphomyosin II at the centrosome in a DISC1-dependent manner. Interestingly, one single nucleotide polymorphism of the CAMDI gene (R828W) is identified, and its gene product was found to reduce the binding ability to phosphomyosin II. Furthermore, mice with overexpression of R828W in neurons exhibit an impaired radial migration. Our findings indicate that CAMDI is required for radial migration probably through DISC1 and myosin II-mediated centrosome positioning during neuronal development.

We have previously identified a novel mitochondrial ubiquitin ligase, MITOL, which is localized in the mitochondrial outer membrane and is involved in the control of mitochondrial dynamics. In this study, we examined whether MITOL eliminates misfolded proteins localized to mitochondria. Mutant superoxide dismutase1 (mSOD1), one of misfolded proteins, has been shown to localize in mitochondria and induce mitochondrial dysfunction, possibly involving in the onset and progression of amyotrophic lateral sclerosis. We found that in the mitochondria, MITOL interacted with and ubiquitinated mSOD1 but not wild-type SOD1. In vitro ubiquitination assay revealed that MITOL directly ubiquitinates mSOD1. Cycloheximide-chase assay in the Neuro2a cells indicated that MITOL overexpression promoted mSOD1 degradation and suppressed both the mitochondrial accumulation of mSOD1 and mSOD1-induced reactive oxygen species (ROS) generation. Conversely, the overexpression of MITOL CS mutant and MITOL knockdown by specific siRNAs resulted in increased accumulation of mSOD1 in mitochondria, which enhanced mSOD1-induced ROS generation and cell death. Thus, our findings indicate that MITOL plays a protective role against mitochondrial dysfunction caused by the mitochondrial accumulation of mSOD1 via the ubiquitin-proteasome pathway.

In this study, we have identified a novel mitochondrial ubiquitin ligase, designated MITOL, which is localized in the mitochondrial outer membrane. MITOL possesses a Plant Homeo-Domain (PHD) motif responsible for E3 ubiquitin ligase activity and predicted four-transmembrane domains. MITOL displayed a rapid degradation by auto-ubiquitination activity in a PHD-dependent manner. HeLa cells stably expressing a MITOL mutant lacking ubiquitin ligase activity or MITOL-deficient cells by small interfering RNA showed an aberrant mitochondrial morphology such as fragmentation, suggesting the enhancement of mitochondrial fission by MITOL dysfunction. Indeed, a dominant-negative expression of Drp1 mutant blocked mitochondrial fragmentation induced by MITOL depletion. We found that MITOL associated with and ubiquitinated mitochondrial fission protein hFis1 and Drp1. Pulse-chase experiment showed that MITOL overexpression increased turnover of these fission proteins. In addition, overexpression phenotype of hFis1 could be reverted by MITOL co-overexpression. Our finding indicates that MITOL plays a critical role in mitochondrial dynamics through the control of mitochondrial fission proteins.

Polyglutamine diseases are inherited neurodegenerative diseases caused by the expanded polyglutamine proteins (polyQs). We have identified a novel guanosine triphosphatase (GTPase) named CRAG that contains a nuclear localization signal (NLS) sequence and forms nuclear inclusions in response to stress. After ultraviolet irradiation, CRAG interacted with and induced an enlarged ring-like structure of promyelocytic leukemia protein (PML) body in a GTPase-dependent manner. Reactive oxygen species (ROS) generated by polyQ accumulation triggered the association of CRAG with polyQ and the nuclear translocation of the CRAG-polyQ complex. Furthermore, CRAG promoted the degradation of polyQ at PML/CRAG bodies through the ubiquitin-proteasome pathway. CRAG knockdown by small interfering RNA in neuronal cells consistently blocked the nuclear translocation of polyQ and enhanced polyQ-mediated cell death. We propose that CRAG is a modulator of PML function and dynamics in ROS signaling and is protectively involved in the pathogenesis of polyglutamine diseases.

Collapsin response mediator proteins (CRMPs) have been implicated in signaling of axonal guidance, including semaphorins. We have previously identified a unique member of this gene family, CRMP-associated molecule CRAM (CRMP-5), which is phylogenetically divergent from the other four CRMPs. In this study, we have examined the distribution and function of CRAM in developing neurons. Immunohistochemical analysis showed accumulation of CRAM in the filopodia of growth cones. Experiments using cytochalasin D indicated that filopodial localization of CRAM was independent of filamentous actin. Overexpression of CRAM in neuronal cells significantly promoted filopodial growth and led to the formation of supernumerary growth cones, which acquired resistance to semaphorin-3A stimulation. Finally, knockdown of CRAM by using RNA interference blocked filopodial formation and revealed an aberrant morphology of growth cones. We propose that CRAM regulates filopodial dynamics and growth cone development, thereby restricting the response of growth cone to repulsive guidance cues.

We have previously demonstrated that Fes/Fps (Fes) tyrosine kinase is involved in Semaphorin3A-mediated signaling. Here we report a role for Fes tyrosine kinase in microtubule dynamics. A fibrous formation of Fes was observed in a kinase-dependent manner, which associated with microtubules and functionally correlated with microtubule bundling. Microtubule regeneration assays revealed that Fes aggregates colocalized with gamma-tubulin at microtubule nucleation sites in a Fes/CIP4 homology (FCH) domain-dependent manner and that expression of FCH domain-deleted Fes mutants blocked normal centrosome formation. In support of these observations, mouse embryonic fibroblasts derived from Fes-deficient mice displayed an aberrant structure of nucleation and centrosome with unbundling and disoriented filaments of microtubules. Our findings suggest that Fes plays a critical role in microtubule dynamics including microtubule nucleation and bundling through its FCH domain.

Background: Collapsin response mediator proteins (CRMPs) and CRAM belong to the unc-33 gene family which is implicated in axon guidance and outgrowth during neural development. However, their exact roles remain largely unknown. To understand the molecular basis of CRMP/CRAM function, we have undertaken to identify CRMP/CRAM interacting proteins. Results: We, have identified a novel mitochondrial septin (M-septin) as one of the CRMP/CRAM interacting proteins from the developing rat brain. M-septin is a major, alternatively spliced variant of the H5 gene in developing mouse brain and its expression is up-regulated during the neuronal differentiation of embryonal carcinoma P19 cells. In COS-7 cells, M-septin is specifically localized to mitochondria whereas H5 is diffusely distributed to the perinuclear cytoplasm and plasma membranes. In contrast to H5, M-septin induces the mitochondrial translocation of CRAM but not CRMP2. Finally, M-Septin is found to be transiently translocated to mitochondria before the induction of the neurites and then dissociates from the mitochondria after neurite extension in P19 cells. Conclusions: Our results suggest that M-septin has a role which is distinct from H5, and together with CRMP/CRAM, may play an important role in the neuronal-differentiation and axon guidance through the control of mitochondrial function.

Collapsin response mediator proteins (CRMPs)/TOAD64/Ulips/DRPs and CRAM have emerged as strong candidates for a role in semaphorin signaling. In this study we identified Fes/Fps (Fes) tyrosine kinase in the CRMP-CRAM complex and investigated whether Fes was involved in semaphorin3A (Sema3A) signaling. In COS-7 cells, the interaction between Fes and plexinA1 (PlexA1) and the tyrosine phosphorylation of PlexA1 by Fes were observed; however, these events were significantly attenuated by co-expression of neuropilin-1 (NP-1). Even with NP-1 co-expression, Sema3A was able to enhance the association of Fes with PlexA1 and Fes-mediated tyrosine phosphorylation of PlexA1, CRAM and CRMP2. Co-expression of Fes with PlexA1 exhibited COS-7 cell contraction activity, indicating that Fes can convert inactive PlexA1 to its active form, whereas combination of Fes/NP-1/PlexA1 or Fes kinase-negative mutants/PlexA1 did not alter cell morphology. Finally, Sema3A-induced growth cone collapse of dorsal root ganglion neurons was suppressed by expression of Fes kinase-negative mutants. Taken together, our findings suggest that Fes links Sema3A signals to CRMP-CRAM, and that NP-1 negatively regulates PlexA1 activation by Fes in resting condition.

Mice deficient in the Syk tyrosine kinase showed severe petechiae in utero and died shortly after birth. The mechanism of this bleeding, however, remains unknown. Here it is shown that this bleeding is caused by morphologic defects of Syk-deficient endothelial cells during embryogenesis. Immunoblot and reverse transcriptase-polymerase chain reaction Northern blot analysis indicated that Syk is expressed in several endothelial cell lines. Immunocytochemical analysis also confirmed that Syk is expressed in the normal embryonic endothelial cells and is absent in Syk-deficient mice. Furthermore, electron microscopic analysis of Syk-deficient mice revealed an abnormal morphogenesis and a decreased number of endothelial cells. The results indicate a critical role for Syk in endothelial cell function and in maintaining vascular integrity in vivo. (C) 2001 by The American Society of Hematology.

Syk is a protein-tyrosine kinase that is widely expressed in haematopoietic cells and involved in coupling activated immunoreceptors to downstream signaling. On the other hand, Syk-deficient mice showed severe petechiae in utero and died shortly after birth. Recently we have shown the expression of Syk in endothelial cells and morphological defects of these cells in embryonic Syk-deficient mice. Here we report that both proliferation and migration of human umbilical vein endothelial cells were severely impaired by adenovirus-mediated expression of Syk dominant negative mutants. Furthermore, a close relationship between Syk kinase activity and extracellular signal-regulated kinase activation was suggested. Our results indicate that Syk plays a critical role in endothelial cell functions, including morphogenesis, cell growth, migration, and survival, and contributes to maintaining vascular integrity in vivo. (C) 2001 Academic Press.

Four members of collapsin response mediator proteins (CRMPs) are thought to be involved in the semaphorin-induced growth cone collapse during neural development. Here we report the identification of a novel CRMPS associated protein, designated CRAM for CRMP3-associated molecule, that belongs to the unc-33 gene family. The deduced amino acid sequence reveals that the CRAM gene encodes a protein of 563 amino acids, shows 57% identity with dihydropyrimidinase, and shows 50-51% identity with CRMPs. CRAM appears to form a large complex composed of CRMPS and other unidentified proteins in vivo. Indeed, CRAM physically associates with CRMPS when co-expressed in COS-7 cells. The expression of CRAM is brain-specific, is high in fetal and neonatal rat brain, and decreases to very low levels in adult brain. Moreover, CRAM expression is up-regulated during neuronal differentiation of embryonal carcinoma P19 and PC12 cells. Finally, immunoprecipitation analysis of rat brain extracts shows that CRAM is co-immunoprecipitated with proteins that contain protein-tyrosine kinase activity, Taken together, our results suggest that CRAM, which interacts with CRMP3 and protein-tyrosine kinase(s), is a new member of an emerging family of molecules that potentially mediate signals involved in the guidance and outgrowth of axons.

CD45 is a tyrosine phosphatase that is required for normal B cell receptor (BCR)-mediated signaling. It has been shown that Src-family tyrosine kinases such as Lyn could be a potential substrate for CD45. In vitro studies indicate that activities of Src family tyrosine kinases are regulated by tyrosine phosphorylation; C-terminal phosphorylation is inhibitory, and autophosphorylation is stimulatory. We report here that both autophosphorylation and C-terminal negative regulatory tyrosines of Lyn were hyperphosphorylated in CD45-deficient DT40 B cells, In this mutant cell, BCR-induced protein-tyrosine phosphorylation and calcium mobilization were severely compromised, as seen in Lyn-deficient cells. Consistent with this observation, Lyn activation upon receptor ligation was profoundly decreased in CD45-deficient cells. Taken together, our results suggest that dephosphorylation of tyrosine residues at both autophosphorylation and negative regulatory sites is mediated by CD45 in vivo, and that dephosphorylation of C-terminal tyrosine is a prerequisite for participation of Lyn in BCR signaling.

We have previously reported that a non-receptor-type protein-tyrosine kinase p72(syk), exists in both membrane and cytosolic fractions in porcine platelets and is activated after thrombin stimulation. To facilitate the understanding of the function of p72(syk), we have investigated the topological features, kinase activities and the interaction with another signal-transducing molecule, namely phosphatidylinositol 3-kinase, during platelet activation. Membrane and cytosolic fractions were separated from thrombin-treated porcine platelets, and the amount of p72(syk) was quantified by the immunoblot technique or the kinase activity of each fraction was determined by an immunoprecipitation kinase assay. After stimulation by thrombin, cytosolic p72(syk) rapidly translocated to the membrane fraction within 10 s and there was also a significant increase in the amount of p72(syk) in the cytoskeletal fraction. The autophosphorylation activity of membrane-associated p72(syk) significantly increased approximately tenfold and reached a maximum at 10 s; the activity subsequently decreased to almost the basal level within 120 s. For similar time courses, association of p72(syk) with phosphatidylinositol 3-kinase and tyrosine phosphorylation of p72(syk) were observed. These results suggest that translocation, activation, and association of p72(syk) with transducing molecules such as phosphatidylinositol 3-kinase, events which occur during platelet activation, may participate in early signal-transduction events.