Publications by authors named "Taketoshi Kajimoto"

25 Publications

  • Page 1 of 1

Involvement of Receptor-Mediated S1P Signaling in EGF-Induced Macropinocytosis in COS7 Cells.

Kobe J Med Sci 2020 Nov 17;66(3):E94-E101. Epub 2020 Nov 17.

Division of Biochemistry, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.

Macropinocytosis is a highly conserved cellular process of endocytosis by which extracellular fluid and nutrients are taken up into cells through large, heterogeneous vesicles known as macropinosomes. Growth factors such as epidermal growth factor (EGF) can induce macropinocytosis in many types of cells, although precise mechanism underlying EGF-induced macropinocytosis remains unclear. In the present studies we have shown the involvement of S1P signaling in EGF-induced macropinocytosis in COS7 cells. First, EGF-induced macropinocytosis was strongly impaired in sphingosine kinase isozymes, SphK1 or SphK2-depleted cells, which was completely rescued by the expression of the corresponding wild-type isozyme but not the catalytically inactive one, suggesting the involvement of sphingosine 1-phosphate (S1P) in this phenomenon. Next, we observed that EGF-induced macropinocytosis was strongly inhibited in S1P type 1 receptor (S1P1R)-knockdown cells, implying involvement of S1P1R in this event. Furthermore, we could successfully demonstrate EGF-induced trans-activation of S1P1R using one-molecular fluorescence resonance energy transfer (FRET) technique. Moreover, for EGF-induced Rac1 activation, a step essential to F-actin formation and subsequent macropinocytosis, S1P signaling is required for its full activation, as judged by FRET analysis. These findings indicate that growth factors such as EGF utilize receptor-mediated S1P signaling for the regulation of macropinocytosis to fulfil vital cell activity.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7837661PMC
November 2020

Apical-basal polarity inhibits epithelial-mesenchymal transition and tumour metastasis by PAR-complex-mediated SNAI1 degradation.

Nat Cell Biol 2019 03 25;21(3):359-371. Epub 2019 Feb 25.

Department of Pharmacology, Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA.

Loss of apical-basal polarity and activation of epithelial-mesenchymal transition (EMT) both contribute to carcinoma progression and metastasis. Here, we report that apical-basal polarity inhibits EMT to suppress metastatic dissemination. Using mouse and human epithelial three-dimensional organoid cultures, we show that the PAR-atypical protein kinase C (aPKC) polarity complex inhibits EMT and invasion by promoting degradation of the SNAIL family protein SNAI1. Under intact apical-basal polarity, aPKC kinases phosphorylate S249 of SNAI1, which leads to protein degradation. Loss of apical-basal polarity prevents aPKC-mediated SNAI1 phosphorylation and stabilizes the SNAI1 protein to promote EMT and invasion. In human breast tumour xenografts, inhibition of the PAR-complex-mediated SNAI1 degradation mechanism promotes tumour invasion and metastasis. Analyses of human breast tissue samples reveal negative correlations between PAR3 and SNAI1 protein levels. Our results demonstrate that apical-basal polarity functions as a critical checkpoint of EMT to precisely control epithelial-mesenchymal plasticity during tumour metastasis.
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http://dx.doi.org/10.1038/s41556-019-0291-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546105PMC
March 2019

Activation of atypical protein kinase C by sphingosine 1-phosphate revealed by an aPKC-specific activity reporter.

Sci Signal 2019 01 1;12(562). Epub 2019 Jan 1.

Department of Pharmacology, University of California at San Diego, La Jolla, CA 92037, USA.

Atypical protein kinase C (aPKC) isozymes are unique in the PKC superfamily in that they are not regulated by the lipid second messenger diacylglycerol, which has led to speculation about whether a different second messenger acutely controls their function. Here, using a genetically encoded reporter that we designed, aPKC-specific C kinase activity reporter (aCKAR), we found that the lipid mediator sphingosine 1-phosphate (S1P) promoted the cellular activity of aPKC. Intracellular S1P directly bound to the purified kinase domain of aPKC and relieved autoinhibitory constraints, thereby activating the kinase. In silico studies identified potential binding sites on the kinase domain, one of which was validated biochemically. In HeLa cells, S1P-dependent activation of aPKC suppressed apoptosis. Together, our findings identify a previously undescribed molecular mechanism of aPKC regulation, a molecular target for S1P in cell survival regulation, and a tool to further explore the biochemical and biological functions of aPKC.
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http://dx.doi.org/10.1126/scisignal.aat6662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657501PMC
January 2019

Essential Role of Sphingosine Kinase 2 in the Regulation of Cargo Contents in the Exosomes from K562 Cells.

Kobe J Med Sci 2018 May 25;63(4):E123-E129. Epub 2018 May 25.

Division of Biochemistry, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.

Sphingosine 1-phosphate (S1P) is a bioactive phosphorylated product of sphingosine catalyzed by sphingosine kinase (SphK) and implicated in diverse cellular functions including vesicular trafficking. In the present study we have shown the importance of one of the subtypes of SphK, SphK2, in the regulation of cargo content in exosomes released from human myeloid leukemia K562 cells. First, SphK2 has been shown to localize with N-Rh-PE-positive late endosomes in the cells. Next, siRNA-mediated knockdown of Sphk2 but not SphK1 resulted in a reduction of cargo content in purified exosomes. The involvement of SphK2 in this phenomenon was further investigated by pharmacological approaches. When cells were treated with N,N-dimethylsphingosine (DMS), one of the most frequently used inhibitors for SphK, cargo contents in purified exosomes were enhanced unexpectedly. Finally, it has been shown that DMS has a potency to stimulate SphK2 activity depending on the substrate sphingosine- and the inhibitor-doses as estimated by in vitro assay systems using a purified SphK2. These findings suggest that SphK2/S1P signaling plays an important role in the regulation of cargo content in exosomes in K562 cells.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6192818PMC
May 2018

Extracellular α-synuclein drives sphingosine 1-phosphate receptor subtype 1 out of lipid rafts, leading to impaired inhibitory G-protein signaling.

J Biol Chem 2018 05 9;293(21):8208-8216. Epub 2018 Apr 9.

From the Division of Biochemistry, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki, Kobe 650-0017, Japan

α-Synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies, are thought to be involved in the pathogenesis of Lewy body diseases, such as Parkinson's disease (PD). Although growing evidence suggests that cell-to-cell transmission of α-Syn is associated with the progression of PD and that extracellular α-Syn promotes formation of inclusion bodies, its precise mechanism of action in the extracellular space remains unclear. Here, as indicated by both conventional fractionation techniques and FRET-based protein-protein interaction analysis, we demonstrate that extracellular α-Syn causes expulsion of sphingosine 1-phosphate receptor subtype 1 (S1PR) from the lipid raft fractions. S1PR regulates vesicular trafficking, and its expulsion involved α-Syn binding to membrane-surface gangliosides. Consequently, the S1PR became refractory to S1P stimulation required for activating inhibitory G-protein (G) in the plasma membranes. Moreover, the extracellular α-Syn also induced uncoupling of the S1PR on internal vesicles, resulting in the reduced amount of CD63 molecule (CD63) in the lumen of multivesicular endosomes, together with a decrease in CD63 in the released exosomes from α-Syn-treated cells. Furthermore, cholesterol-depleting agent-induced S1PR expulsion from the rafts also resulted in S1PR uncoupling. Taken together, these results suggest that extracellular α-Syn-induced expulsion of S1PR from lipid rafts promotes the uncoupling of S1PR from G, thereby blocking subsequent G signals, such as inhibition of cargo sorting into exosomal vesicles in multivesicular endosomes. These findings help shed additional light on PD pathogenesis.
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http://dx.doi.org/10.1074/jbc.RA118.001986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5971450PMC
May 2018

DHHC5-mediated palmitoylation of S1P receptor subtype 1 determines G-protein coupling.

Sci Rep 2017 11 29;7(1):16552. Epub 2017 Nov 29.

Division of Biochemistry, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, 650-0017, Japan.

Sphingosine 1-phosphate (S1P) is a pleiotropic lipid mediator involved in the regulation of immune cell trafficking and vascular permeability acting mainly through G-protein-coupled S1P receptors (S1PRs). However, mechanism underlying how S1PRs are coupled with G-proteins remains unknown. Here we have uncovered that palmitoylation of a prototypical subtype S1PR is prerequisite for subsequent inhibitory G-protein (Gi) coupling. We have identified DHHC5 as an enzyme for palmitoylation of S1PR. Under basal conditions, S1PR was functionally associated with DHHC5 in the plasma membranes (PM) and was fully palmitoylated, enabling Gi coupling. Upon stimulation, the receptor underwent internalisation leaving DHHC5 in PM, resulting in depalmitoylation of S1PR. We also revealed that while physiological agonist S1P-induced endocytosed S1PR readily recycled back to PM, pharmacological FTY720-P-induced endocytosed S1PR-positive vesicles became associated with DHHC5 in the later phase, persistently transmitting Gi signals there. This indicates that FTY720-P switches off the S1P signal in PM, while switching on its signal continuously inside the cells. We propose that DHHC5-mediated palmitoylation of S1PR determines Gi coupling and its signalling in a spatio/temporal manner.
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http://dx.doi.org/10.1038/s41598-017-16457-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5707436PMC
November 2017

Involvement of Gβγ subunits of G protein coupled with S1P receptor on multivesicular endosomes in F-actin formation and cargo sorting into exosomes.

J Biol Chem 2018 01 13;293(1):245-253. Epub 2017 Nov 13.

From the Department of Biochemistry and Molecular Biology, Division of Biochemistry, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki, Kobe 650-0017, Japan

Exosomes play a critical role in cell-to-cell communication by delivering cargo molecules to recipient cells. However, the mechanism underlying the generation of the exosomal multivesicular endosome (MVE) is one of the mysteries in the field of endosome research. Although sphingolipid metabolites such as ceramide and sphingosine 1-phosphate (S1P) are known to play important roles in MVE formation and maturation, the detailed molecular mechanisms are still unclear. Here, we show that Rho family GTPases, including Cdc42 and Rac1, are constitutively activated on exosomal MVEs and are regulated by S1P signaling as measured by fluorescence resonance energy transfer (FRET)-based conformational changes. Moreover, we detected S1P signaling-induced filamentous actin (F-actin) formation. A selective inhibitor of Gβγ subunits, M119, strongly inhibited both F-actin formation on MVEs and cargo sorting into exosomal intralumenal vesicles of MVEs, both of which were fully rescued by the simultaneous expression of constitutively active Cdc42 and Rac1. Our results shed light on the mechanism underlying exosomal MVE maturation and inform the understanding of the physiological relevance of continuous activation of the S1P receptor and subsequent downstream G protein signaling to Gβγ subunits/Rho family GTPases-regulated F-actin formation on MVEs for cargo sorting into exosomal intralumenal vesicles.
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http://dx.doi.org/10.1074/jbc.M117.808733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766922PMC
January 2018

Phospholipase D is Dispensable for Epidermal Growth Factor-Induced Chemotaxis.

Kobe J Med Sci 2017 May 9;62(6):E162-E167. Epub 2017 May 9.

Division of Biochemistry, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.

α-Synuclein (α-Syn) is implicated in several neurodegenerative disorders, including Parkinson's disease, known collectively as the synucleinopathies. α-Syn is known to be secreted from the cells and may contribute to the progression of the disease. Although extracellular α-Syn is shown to impair platelet-derived growth factor-induced chemotaxis, molecular mechanism of α-Syn-induced motility failure remains elusive. Here we have aimed at phospholipase D (PLD) as a potential target for α-Syn and examined the involvement of this enzyme in α-Syn action. Indeed, extracellular α-Syn caused inhibition of agonist-induced PLD activation. However, inhibition of hydrolytic activity of PLD by 1-butanol treatment showed little or no effect on agonist-induced chemotaxis. These results suggest that some signaling pathways other than PLD may be involved in α-Syn-induced inhibition of chemotaxis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5436530PMC
May 2017

Extracellular α-synuclein induces sphingosine 1-phosphate receptor subtype 1 uncoupled from inhibitory G-protein leaving β-arrestin signal intact.

Sci Rep 2017 03 16;7:44248. Epub 2017 Mar 16.

Division of Biochemistry, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.

Parkinson's disease (PD) is the second most common neurodegenerative disorder. The presence of α-synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies, is the cytopathological hallmark of PD. Increasing bodies of evidence suggest that cell-to-cell transmission of α-Syn plays a role in the progression of PD. Although extracellular α-Syn is known to cause abnormal cell motility, the precise mechanism remains elusive. Here we show that impairment of platelet-derived growth factor-induced cell motility caused by extracellular α-Syn is mainly attributed to selective inhibition of sphingosine 1-phosphate (S1P) signalling. Treatment of human neuroblastoma cells with recombinant α-Syn caused S1P type 1 (S1P) receptor-selective uncoupling from inhibitory G-protein (Gi) as determined by both functional and fluorescence resonance energy transfer (FRET)-based structural analyses. By contrast, α-Syn caused little or no effect on S1P receptor-mediated signalling. Both wild-type and α-Syn(A53T), a mutant found in familiar PD, caused uncoupling of S1P receptor, although α-Syn(A53T) showed stronger potency in uncoupling. Moreover, S1P receptor-mediated β-arrestin signal was unaltered by α-Syn(A53T). These results suggest that exogenous α-Syn modulates S1P receptor-mediated signalling from both Gi and β-arrestin signals into β-arrestin-biased signal. These findings uncovered a novel function of exogenous α-Syn in the cells.
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http://dx.doi.org/10.1038/srep44248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5353548PMC
March 2017

Impairment of PDGF-induced chemotaxis by extracellular α-synuclein through selective inhibition of Rac1 activation.

Sci Rep 2016 11 25;6:37810. Epub 2016 Nov 25.

Division of Biochemistry, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.

Parkinson's disease (PD) is characterized by α-synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies. Although it is known that extracellular α-Syn is detected in the plasma and cerebrospinal fluid, its physiological significance remains unclear. Here, we show that extracellular α-Syn suppresses platelet-derived growth factor (PDGF)-induced chemotaxis in human neuroblastoma SH-SY5Y cells. The inhibitory effect was stronger in the mutant α-Syn(A53T), found in hereditary PD, and the degree of inhibition was time-dependent, presumably because of the oligomerization of α-Syn. PDGF-induced activation of Akt or Erk was not influenced by α-Syn(A53T). Further studies revealed that α-Syn(A53T) inhibited PDGF-induced Rac1 activation, whereas Cdc42 activation remained unaffected, resulting in unbalanced actin filament remodeling. These results shed light on the understanding of pathological as well as physiological functions of extracellular α-Syn in neuronal cells.
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http://dx.doi.org/10.1038/srep37810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5122898PMC
November 2016

Ongoing activation of sphingosine 1-phosphate receptors mediates maturation of exosomal multivesicular endosomes.

Nat Commun 2013 ;4:2712

Division of Biochemistry, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki, Kobe 650-0017, Japan.

During late endosome maturation, cargo molecules are sorted into intralumenal vesicles (ILVs) of multivesicular endosomes (MVEs), and are either delivered to lysosomes for degradation or fused with the plasma membranes for exosome release. The mechanism underlying formation of exosomal ILVs and cargo sorting into ILVs destined for exosome release is still unclear. Here we show that inhibitory G protein (Gi)-coupled sphingosine 1-phosphate (S1P) receptors regulate exosomal MVE maturation. Gi-coupled S1P receptors on MVEs are constitutively activated through a constant supply of S1P via autocrine activation within organelles. We also found that the continuous activation of Gi-coupled S1P receptors on MVEs is essential for cargo sorting into ILVs destined for exosome release. Our results reveal a mechanism underlying ESCRT-independent maturation of exosomal MVEs.
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http://dx.doi.org/10.1038/ncomms3712DOI Listing
July 2014

Genetically encoded fluorescent thermosensors visualize subcellular thermoregulation in living cells.

Nat Methods 2013 Dec 13;10(12):1232-8. Epub 2013 Oct 13.

1] Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan. [2] Department of Technology and Ecology, Hall of Global Environmental Studies, Kyoto University, Kyoto, Japan. [3] Core Research for Evolution Science and Technology, Japan Science and Technology Agency, Tokyo, Japan.

In mammals and birds, thermoregulation to conserve body temperature is vital to life. Multiple mechanisms of thermogeneration have been proposed, localized in different subcellular organelles. However, visualizing thermogenesis directly in intact organelles has been challenging. Here we have developed genetically encoded, GFP-based thermosensors (tsGFPs) that enable visualization of thermogenesis in discrete organelles in living cells. In tsGFPs, a tandem formation of coiled-coil structures of the Salmonella thermosensing protein TlpA transmits conformational changes to GFP to convert temperature changes into visible and quantifiable fluorescence changes. Specific targeting of tsGFPs enables visualization of thermogenesis in the mitochondria of brown adipocytes and the endoplasmic reticulum of myotubes. In HeLa cells, tsGFP targeted to mitochondria reveals heterogeneity in thermogenesis that correlates with the electrochemical gradient. Thus, tsGFPs are powerful tools to noninvasively assess thermogenesis in living cells.
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http://dx.doi.org/10.1038/nmeth.2690DOI Listing
December 2013

Receptor signaling integration by TRP channelsomes.

Adv Exp Med Biol 2011 ;704:373-89

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.

Homologues of transient receptor potential (TRP) genes encode a variety of cation channels, most of which conduct Ca(2+) across the plasma membrane. TRP proteins interact with a variety of proteins and other biologically important factors, such as second messengers, and thereby form "channelsomes", most of which function as Ca(2+) signalsomes. Activation mechanisms and final outputs are exquisitely incorporated in the signaling system of TRP channelsomes. In this study, we discuss the channelsomes of TRPC3, TRPC5, and TRPM2, which show unique molecular interactions and modulations of activation. Comparative studies of these specific TRP channelsomes should aid the determination of general rules that govern the formation and regulation of channelsomes and signalsomes.
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http://dx.doi.org/10.1007/978-94-007-0265-3_21DOI Listing
July 2011

Protein kinase C {delta}-specific activity reporter reveals agonist-evoked nuclear activity controlled by Src family of kinases.

J Biol Chem 2010 Dec 19;285(53):41896-910. Epub 2010 Oct 19.

Department of Pharmacology, University of California at San Diego, La Jolla, California 92093, USA.

Conventional and novel protein kinase C (PKC) isozymes transduce the abundance of signals mediated by phospholipid hydrolysis; however redundancy in regulatory mechanisms confounds dissecting the unique signaling properties of each of the eight isozymes constituting these two subgroups. Previously, we created a genetically encoded reporter (C kinase activity reporter (CKAR)) to visualize the rate, amplitude, and duration of agonist-evoked PKC signaling at specific locations within the cell. Here we designed a reporter, δCKAR, that specifically measures the activation signature of one PKC isozyme, PKC δ, in cells, revealing unique spatial and regulatory properties of this isozyme. Specifically, we show two mechanisms of activation: 1) agonist-stimulated activation at the plasma membrane (the site of most robust PKC δ signaling), Golgi, and mitochondria that is independent of Src and can be triggered by phorbol esters and 2) agonist-stimulated activation in the nucleus that requires Src kinase activation and cannot be triggered by phorbol esters. Translocation studies reveal that the G-protein-coupled receptor agonist UTP induces the translocation of PKC δ into the nucleus by a mechanism that depends on the C2 domain and requires Src kinase activity. However, translocation from the cytosol into the nucleus is not required for the Src-dependent regulation of nuclear activity; a construct of PKC δ prelocalized to the nucleus continues to be activated by UTP by a mechanism dependent on Src kinase activity. These data identify the nucleus as a signaling hub for PKC δ that is driven by receptor-mediated signaling pathways (but not phorbol esters) and differs from signaling at plasma membrane and Golgi in that it is controlled by Src family kinases.
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http://dx.doi.org/10.1074/jbc.M110.184028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3009917PMC
December 2010

The protein scaffold NHERF-1 controls the amplitude and duration of localized protein kinase D activity.

J Biol Chem 2009 Sep 6;284(36):24653-61. Epub 2009 Jul 6.

Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0721, USA.

Protein kinase D (PKD) transduces an abundance of signals downstream of diacylglycerol production. The mammalian PKD family consists of three isoforms, PKD1, PKD2, and PKD3; of these PKD1 and PKD2 contain PDZ-binding motifs at their carboxyl termini. Here we show that membrane-localized NHERF scaffold proteins provide a nexus for tightly controlled PKD signaling via a PDZ domain interaction. Using a proteomic array containing 96 purified PDZ domains, we have identified the first PDZ domain of NHERF-1 as an interaction partner for the PDZ-binding motifs of both PKD1 and PKD2. A fluorescence resonance energy transfer-based translocation assay reveals a transient association of PKD1 and PKD2 with NHERF-1 in live cells that is triggered by phorbol ester stimulation and, importantly, differs strikingly from the sustained translocation to plasma membrane. Targeting a fluorescence resonance energy transfer-based kinase activity reporter for PKD to NHERF scaffolds reveals a unique signature of PKD activation at the scaffold that is distinct from that of general cytosolic or plasma membrane activity. Specifically, agonist-evoked activation of PKD at the scaffold is rapid and sustained but blunted in magnitude when compared with cytosolic PKD. Thus, live cell imaging of PKD activity demonstrates ultrasensitive control of kinase signaling at the scaffold compared with bulk activity in the cytosol or at the plasma membrane.
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http://dx.doi.org/10.1074/jbc.M109.024547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2782054PMC
September 2009

Sphingosine kinase/sphingosine 1-phosphate signalling in central nervous system.

Cell Signal 2009 Jan 22;21(1):7-13. Epub 2008 Jul 22.

Division of Biochemistry, Department of Biochemistry/Molecular Biology, Kobe University Graduate School of Medicine, Kobe, 650-0017, Japan.

Sphingolipids were once regarded as inert structural components of cell membranes. Now these metabolites are generally believed to be important bioactive molecules that control a wide repertoire of cellular processes such as proliferation and survival of cells. Along with these ubiquitous cell functions observed in many peripheral tissues sphingolipid metabolites, especially sphingosine 1-phosphate, exert important neuron-specific functions such as regulation of neurotransmitter release. This review summarizes physiological and pathological roles of sphingolipid metabolites emphasizing the role of sphingosine 1-phosphate in the central nervous system.
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http://dx.doi.org/10.1016/j.cellsig.2008.07.011DOI Listing
January 2009

Protein kinase D-mediated phosphorylation and nuclear export of sphingosine kinase 2.

J Biol Chem 2007 Sep 16;282(37):27493-27502. Epub 2007 Jul 16.

Division of Biochemistry, Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Kusunoki-cho 7-5-1, Chuo-ku, Kobe 650-0017, Japan. Electronic address:

Sphingosine kinase (SPHK) is a key enzyme producing important messenger sphingosine 1-phosphate and is implicated in cell proliferation and suppression of apoptosis. Because the extent of agonist-induced activation of SPHK is modest, signaling via SPHK may be regulated through its localization at specific intracellular sites. Although the SPHK1 isoform has been extensively studied and characterized, the regulation of expression and function of the other isoform, SPHK2, remain largely unexplored. Here we describe an important post-translational modification, namely, phosphorylation of SPHK2 catalyzed by protein kinase D (PKD), which regulates its localization. Upon stimulation of HeLa cells by tumor promoter phorbol 12-myristate 13-acetate, a serine residue in a novel and putative nuclear export signal, identified for the first time, in SPHK2 was phosphorylated followed by SPHK2 export from the nucleus. Constitutively active PKD phosphorylated this serine residue in the nuclear export signal both in vivo and in vitro. Moreover, down-regulation of PKDs through RNA interference resulted in the attenuation of both basal and phorbol 12-myristate 13-acetate-induced phosphorylation, which was followed by the accumulation of SPHK2 in the nucleus in a manner rescued by PKD over-expression. These results indicate that PKD is a physiologically relevant enzyme for SPHK2 phosphorylation, which leads to its nuclear export for subsequent cellular signaling.
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http://dx.doi.org/10.1074/jbc.M701641200DOI Listing
September 2007

Involvement of sphingosine-1-phosphate in glutamate secretion in hippocampal neurons.

Mol Cell Biol 2007 May 26;27(9):3429-40. Epub 2007 Feb 26.

Division of Biochemistry, Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.

Neuronal activity greatly influences the formation and stabilization of synapses. Although receptors for sphingosine-1-phosphate (S1P), a lipid mediator regulating diverse cellular processes, are abundant in the central nervous system, neuron-specific functions of S1P remain largely undefined. Here, we report two novel actions of S1P using primary hippocampal neurons as a model system: (i) as a secretagogue where S1P triggers glutamate secretion and (ii) as an enhancer where S1P potentiates depolarization-evoked glutamate secretion. Sphingosine kinase 1 (SK1), a key enzyme for S1P production, was enriched in functional puncta of hippocampal neurons. Silencing SK1 expression by small interfering RNA as well as SK1 inhibition by dimethylsphingosine resulted in a strong inhibition of depolarization-evoked glutamate secretion. Fluorescence recovery after photobleaching analysis showed translocation of SK1 from cytosol to membranes at the puncta during depolarization, which resulted in subsequent accumulation of S1P within cells. Fluorescent resonance energy transfer analysis demonstrated that the S1P(1) receptor at the puncta was activated during depolarization and that depolarization-induced S1P(1) receptor activation was inhibited in SK1-knock-down cells. Importantly, exogenously added S1P at a nanomolar concentration by itself elicited glutamate secretion from hippocampal cells even when the Na(+)-channel was blocked by tetrodotoxin, suggesting that S1P acts on presynaptic membranes. Furthermore, exogenous S1P at a picomolar level potentiated depolarization-evoked secretion in the neurons. These findings indicate that S1P, through its autocrine action, facilitates glutamate secretion in hippocampal neurons both by secretagogue and enhancer actions and may be involved in mechanisms underlying regulation of synaptic transmission.
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http://dx.doi.org/10.1128/MCB.01465-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1899953PMC
May 2007

Involvement of N-terminal-extended form of sphingosine kinase 2 in serum-dependent regulation of cell proliferation and apoptosis.

J Biol Chem 2005 Oct 15;280(43):36318-25. Epub 2005 Aug 15.

Division of Biochemistry, Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.

Sphingosine kinase (SPHK) 1 is implicated in the regulation of cell proliferation and anti-apoptotic processes by catalyzing the formation of an important bioactive messenger, sphingosine 1-phosphate. Unlike the proliferative action of SPHK1, another isozyme, SPHK2, has been shown to possess anti-proliferative or pro-apoptotic action. Molecular mechanisms of SPHK2 action, however, are largely unknown. The present studies were undertaken to characterize the N-terminal-extended form of SPHK2 (SPHK2-L) by comparing it with the originally reported form, SPHK2-S. Real-time quantitative PCR analysis revealed that SPHK2-L mRNA is the major form in several human cell lines and tissues. From sequence analyses it was concluded that SPHK2-L is a species-specific isoform that is expressed in human but not in mouse. At the protein level it has been demonstrated by immunoprecipitation studies that SPHK2-L is the major isoform in human hepatoma HepG2 cells. SPHK2-L, when expressed in human embryonic kidney (HEK) 293 cells, did not show any inhibition of DNA synthesis in the presence of serum, whereas it showed marked inhibition in the absence of serum. Moreover, serum deprivation resulted in the translocation of SPHK2-L into the nuclei. In addition, serum deprivation induced SPHK2-L expression in HEK293 cells. Furthermore, suppression of SPHK2 by small interfering RNA treatment prevented serum deprivation- or drug-induced apoptosis in HEK293 cells. Taken together, these results indicate that a major form of SPHK2 splice variant, SPHK2-L, in human cells does not inhibit DNA synthesis under normal conditions and that SPHK2-L accumulation in the nucleus induced by serum deprivation may be involved in the cessation of cell proliferation or apoptosis depending on the cell type.
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http://dx.doi.org/10.1074/jbc.M504507200DOI Listing
October 2005

Involvement of gamma protein kinase C in estrogen-induced neuroprotection against focal brain ischemia through G protein-coupled estrogen receptor.

J Neurochem 2005 May;93(4):883-91

Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Nada-ku, Japan.

The neuroprotective effects of estrogen were studied in the ischemic model mice by 90 min transient unilateral middle cerebral artery occlusion (MCAO) followed by 22.5 h reperfusion. The total infarct size in C57BL/6 female mice after MCAO and reperfusion was significantly smaller than that in male mice. Intraperitoneal injection of estrogen after the start of reperfusion significantly reduced the infarct volume in the male mice. However, no significant gender difference was found in total infarct size in gamma protein kinase C (PKC)-knockout mice, suggesting that the neuroprotective effects of estrogen are due to the activation of a specific subtype of PKC, gammaPKC, a neuron-specific PKC subtype, in the brain. We demonstrated that exogenous estrogen-induced neuroprotection was attenuated in gammaPKC-knockout mice. Immunocytochemical study showed that gammaPKC was translocated to nerve fiber-like structures when observed shortly after MCAO and reperfusion. We also visualized the rapid and reversible translocation of gammaPKC-GFP (green fluorescent protein) by estrogen stimulation in living CHO-K1 cells. These results suggest that the activation of gammaPKC through the G-protein-coupled estrogen receptors on the plasma membrane is involved in the estrogen-induced neuroprotection against focal brain ischemia.
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http://dx.doi.org/10.1111/j.1471-4159.2005.03080.xDOI Listing
May 2005

Propagation of gammaPKC translocation along the dendrites of Purkinje cell in gammaPKC-GFP transgenic mice.

Genes Cells 2004 Oct;9(10):945-57

Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.

To elucidate spatial and temporal profiles of the protein kinase C (PKC) activation in relation to neuronal functions including synaptic plasticity, we tried to detect PKC translocation in living brain slices. We first developed brain region-specific and inducible gammaPKC-GFP transgenic mice using a tetracycline (tet)-regulated system. In the transgenic mice, the expression of gammaPKC-GFP was region-specifically regulated by the promoter and abolished by the administration of doxycycline. Cerebellar slices from the mice were utilized for intracellular recording and fluorescence imaging of gammaPKC-GFP in Purkinje cells. GFP fluorescence was uniformly distributed from soma to dendritic arbor. When mGluR agonists were applied, the intensity was transiently increased at the edge of the dendrite and concomitantly decreased in the cytoplasm, indicating that gammaPKC translocated to the plasma membrane. This transient change in the pattern of GFP fluorescence simultaneously occurred throughout the Purkinje cell dendrites by agonist stimulation. Translocation of gammaPKC-GFP was also induced by electrical stimulation of parallel fibres. However, the event was not restricted at the distal dendrites, propagated forwardly along the dendritic tree and reached to the proximal trunk close to the soma. Time course of the propagation was slower than the electrical signal and Ca(2+) waves and faster than conveying molecules through microtubules. The present results indicate that PKC signals activated locally by parallel fibre input could propagate to the soma through dendrites in living Purkinje neurones. The findings may provide us with a new insight for understanding molecular mechanisms of the synaptic plasticity including cerebellar long-term depression.
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http://dx.doi.org/10.1111/j.1365-2443.2004.00779.xDOI Listing
October 2004

Ceramide-induced apoptosis by translocation, phosphorylation, and activation of protein kinase Cdelta in the Golgi complex.

J Biol Chem 2004 Mar 10;279(13):12668-76. Epub 2004 Jan 10.

Laboratories of Molecular Pharmacology and Biochemistry, Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan.

Protein kinase C (PKC), a Ca(2+)/phospholipid-dependent protein kinase, is known as a key enzyme in various cellular responses, including apoptosis. However, the functional role of PKC in apoptosis has not been clarified. In this study, we focused on the involvement of PKCdelta in ceramide-induced apoptosis in HeLa cells and examined the importance of spatiotemporal activation of the specific PKC subtype in apoptotic events. Ceramide-induced apoptosis was inhibited by the PKCdelta-specific inhibitor rottlerin and also was blocked by knockdown of endogenous PKCdelta expression using small interfering RNA. Ceramide induced the translocation of PKCdelta to the Golgi complex and the concomitant activation of PKCdelta via phosphorylation of Tyr(311) and Tyr(332) in the hinge region of the enzyme. Unphosphorylatable PKCdelta (mutants Y311F and Y332F) could translocate to the Golgi complex in response to ceramide, suggesting that tyrosine phosphorylation is not necessary for translocation. However, ceramide failed to activate PKCdelta lacking the C1B domain, which did not translocate to the Golgi complex, but could be activated by tyrosine phosphorylation. These findings suggest that ceramide translocates PKCdelta to the Golgi complex and that PKCdelta is activated by tyrosine phosphorylation in the compartment. Furthermore, we utilized species-specific knockdown of PKCdelta by small interfering RNA to study the significance of phosphorylation of Tyr(311) and Tyr(332) in PKCdelta for ceramide-induced apoptosis and found that phosphorylation of Tyr(311) and Tyr(332) is indispensable for ceramide-induced apoptosis. We demonstrate here that the targeting mechanism of PKCdelta, dual regulation of both its activation and translocation to the Golgi complex, is critical for the ceramide-induced apoptotic event.
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http://dx.doi.org/10.1074/jbc.M312350200DOI Listing
March 2004

Subtype- and species-specific knockdown of PKC using short interfering RNA.

Biochem Biophys Res Commun 2002 Nov;298(5):738-43

Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Japan.

RNA interference (RNAi), the targeted mRNA degradation induced by double-stranded RNA (dsRNA), is a powerful tool for analyzing gene function in many organisms. Recently, it has been shown that RNAi is also applicable to cultured mammalian cells by using short interfering RNA (siRNA) [Nature 411 (2001) 494]. To examine whether this siRNA method is useful for analyzing the subtype-specific functions of protein kinase C (PKC), we first prepared siRNAs which target human alphaPKC and human deltaPKC and applied them into mammalian cells to suppress the expression of endogenous alphaPKC and deltaPKC, respectively. Each siRNA for alpha or deltaPKC specifically suppressed the endogenous expression of corresponding PKC subtype in human-derived cell lines such as HEK-293 and HeLa cells, but not in cells derived from rat species. The suppression level of deltaPKC reached maximum 48-72h after the transfection of siRNA. In addition, the siRNA targeting rat deltaPKC suppressed endogenous and exogenous rat deltaPKCs but not human deltaPKC, suggesting that siRNAs targeting PKCs effectively knocked down endogenous/exogenous PKCs in mammalian cells, in subtype- and species-specific manner. Furthermore, we also developed the method to discriminate the siRNA-transfected cells using the antibody recognizing thymine dimer. Our present results strongly suggest that siRNA method enable us to examine the subtype-specific function of PKC, not only by knockdown of the endogenous target PKC subtype, but also by subsequent compensation with the exogenous corresponding wild/mutant PKC derived from other species.
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http://dx.doi.org/10.1016/s0006-291x(02)02531-7DOI Listing
November 2002