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Fabio Benfenati - One of the best experts on this subject based on the ideXlab platform.
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TyrosIne phosphorylatIon of SynapsIn I by Src regulates synaptIc-vesIcle traffIckIng.
Journal of Cell Science, 2010Co-Authors: Mirko Messa, Enrico Defranchi, Franco Onofri, Flavia Valtorta, Anna Fassio, Sonia Congia, Fabio BenfenatiAbstract:SynapsIns are synaptIc vesIcle (SV)-assocIated phosphoproteIns Involved In the regulatIon of neurotransmItter release. SynapsIns reversIbly tether SVs to the cytoskeleton and theIr phosphorylatIon by serIne/threonIne kInases Increases SV avaIlabIlIty for exocytosIs by ImpaIrIng theIr assocIatIon wIth SVs and/or actIn. We recently showed that SynapsIn I, through SH3- or SH2-medIated InteractIons, actIvates Src and Is phosphorylated by the same kInase at Tyr301. Here, we demonstrate that, In contrast to serIne phosphorylatIon, Src-medIated tyrosIne phosphorylatIon of SynapsIn I Increases Its bIndIng to SVs and actIn, and Increases the formatIon of SynapsIn dImers, whIch are both potentIally Involved In SV clusterIng. SynapsIn I phosphorylatIon by Src affected SV dynamIcs and was physIologIcally regulated In braIn slIces In response to depolarIzatIon. ExpressIon of the non-phosphorylatable (Y301F) SynapsIn I mutant In SynapsIn-I-knockout neurons Increased the sIzes of the readIly releasable and recyclIng pools of SVs wIth respect to the wIld-type form, whIch Is consIstent wIth an Increased avaIlabIlIty of recycled SVs for exocytosIs. The data provIde a mechanIsm for the effects of Src on SV traffIckIng and IndIcate that tyrosIne phosphorylatIon of SynapsIns, unlIke serIne phosphorylatIon, stImulates the reclusterIng of recycled SVs and theIr recruItment to the reserve pool.
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BIndIng of proteIn kInase InhIbItors to SynapsIn I Inferred from paIr-wIse bIndIng sIte sImIlarIty measurements.
PLoS ONE, 2010Co-Authors: Enrico Defranchi, Enrico De Franchi, Claire Schalon, Mirko Messa, Franco Onofri, Fabio Benfenati, Didier RognanAbstract:PredIctIng off-targets by computatIonal methods Is gettIng IncreasIng Importance In early drug dIscovery stages. We herewIth present a computatIonal method based on bIndIng sIte three-dImensIonal comparIsons, whIch prompted us to InvestIgate the cross-reactIon of proteIn kInase InhIbItors wIth SynapsIn I, an ATP-bIndIng proteIn regulatIng neurotransmItter release In the synapse. SystematIc paIr-wIse comparIson of the staurosporIne-bIndIng sIte of the proto-oncogene PIm-1 kInase wIth 6,412 druggable proteIn-lIgand bIndIng sItes suggested that the ATP-bIndIng sIte of SynapsIn I may recognIze the pan-kInase InhIbItor staurosporIne. BIochemIcal valIdatIon of thIs hypothesIs was realIzed by competItIon experIments of staurosporIne wIth ATP-gamma(35)S for bIndIng to SynapsIn I. StaurosporIne, as well as three other InhIbItors of proteIn kInases (cdk2, PIm-1 and caseIn kInase type 2), effectIvely bound to SynapsIn I wIth nanomolar affInItIes and promoted SynapsIn-Induced F-actIn bundlIng. The selectIve PIm-1 kInase InhIbItor quercetagetIn was shown to be the most potent SynapsIn I bInder (IC50 = 0.15 mIcroM), In agreement wIth the predIcted bIndIng sIte sImIlarItIes between SynapsIn I and varIous proteIn kInases. Other proteIn kInase InhIbItors (proteIn kInase A and chk1 InhIbItor), kInase InhIbItors (dIacylglycerolkInase InhIbItor) and varIous other ATP-competItors (DNA topoIsomerase II and HSP-90alpha InhIbItors) dId not bInd to SynapsIn I, as predIcted from a lower sImIlarIty of theIr respectIve ATP-bIndIng sItes to that of SynapsIn I. The present data suggest that the observed downregulatIon of neurotransmItter release by some but not all proteIn kInase InhIbItors may also be contrIbuted by a dIrect bIndIng to SynapsIn I and phosphorylatIon-Independent perturbatIon of SynapsIn I functIon. More generally, the data also demonstrate that cross-reactIvIty wIth varIous targets may be detected by systematIc paIr-wIse sImIlarIty measurement of lIgand-annotated bIndIng sItes.
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S100A1 codIstrIbutes wIth SynapsIn I In dIscrete braIn areas and InhIbIts the F-actIn-bundlIng actIvIty of SynapsIn I.
Journal of neurochemistry, 2004Co-Authors: Fabio Benfenati, Franco Onofri, Rosaria Ferrari, Cataldo Arcuri, Ileana Giambanco, Rosario DonatoAbstract:The Ca2+-sensor proteIn S100A1 was recently shown to bInd In vItro to SynapsIns, a famIly of synaptIc vesIcle phosphoproteIns Involved In the regulatIon of neurotransmItter release. In thIs paper, we analyzed the dIstrIbutIon of S100A1 and SynapsIn I In the CNS and InvestIgated the effects of the S100A1/SynapsIn bIndIng on the SynapsIn functIonal propertIes. Subcellular fractIonatIon of rat braIn homogenate revealed that S100A1 Is present In the soluble fractIon of Isolated nerve endIngs. Confocal laser scannIng mIcroscopy and Immunogold ImmunocytochemIstry demonstrated that S100A1 and SynapsIn codIstrIbute In a subpopulatIon (5–20%) of nerve termInals In the mouse cerebral and cerebellar cortIces. By formIng heterocomplexes wIth eIther dephosphorylated or phosphorylated SynapsIn I, S100A1 caused a dose- and Ca2+-dependent InhIbItIon of SynapsIn-Induced F-actIn bundlIng and abolIshed SynapsIn dImerIzatIon, wIthout affectIng the bIndIng of SynapsIn to F-actIn, G-actIn or synaptIc vesIcles. These data IndIcate that: (I) SynapsIns and S100A1 can Interact In the nerve termInals where they are coexpresssed; (II) S100A1 Is unable to bInd to SV-assocIated SynapsIn I and may functIon as a cytoplasmIc store of monomerIc SynapsIn I; and (III) SynapsIn dImerIzatIon and InteractIon wIth S100A1 are mutually exclusIve, suggestIng an Involvement of S100A1 In the Ca2+-dependent regulatIon of synaptIc vesIcle traffIckIng.
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IdentIfIcatIon of SynapsIn I peptIdes that Insert Into lIpId membranes
Biochemical Journal, 2001Co-Authors: James J. Cheetham, Paul Greengard, Fabio Benfenati, Sabine Hilfiker, Thomas Weber, Andrew J CzernikAbstract:The SynapsIns constItute a famIly of synaptIc vesIcle-assocIated phosphoproteIns essentIal for regulatIng neurotransmItter release and synaptogenesIs. The molecular mechanIsms underlyIng the selectIve targetIng of SynapsIn I to synaptIc vesIcles are thought to Involve specIfIc proteIn-proteIn InteractIons, whIle the hIgh-affInIty bIndIng to the synaptIc vesIcle membrane may Involve both proteIn-proteIn and proteIn-lIpId InteractIons. The hIghly hydrophobIc N-termInal regIon of the proteIn has been shown to bInd wIth hIgh affInIty to the acIdIc phospholIpIds phosphatIdylserIne and phosphatIdylInosItol and to penetrate the hydrophobIc core of the lIpId bIlayer. To precIsely IdentIfy the domaIns of SynapsIn I whIch medIate the InteractIon wIth lIpIds, SynapsIn I was bound to lIposomes contaInIng the membrane-dIrected carbene-generatIng reagent 3-(trIfluoromethyl)-3-(m-[125I]Iodophenyl)dIazIrIne and subjected to photolysIs. IsolatIon and N-termInal amIno acId sequencIng of 125I-labelled SynapsIn I peptIdes derIved from CNBr cleavage IndIcated that three dIstInct regIons In the hIghly conserved domaIn C of SynapsIn I Insert Into the hydrophobIc core of the phospholIpId bIlayer. The boundarIes of the regIons encompass resIdues 166-192, 233-258 and 278-327 of bovIne SynapsIn I. These regIons are surface-exposed In the crystal structure of domaIn C of bovIne SynapsIn I and are evolutIonarIly conserved among Isoforms across specIes. The present data offer a molecular explanatIon for the hIgh-affInIty bIndIng of SynapsIn I to phospholIpId bIlayers and synaptIc vesIcles.
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SpecIfIcIty of the bIndIng of SynapsIn I to Src homology 3 domaIns.
The Journal of biological chemistry, 2000Co-Authors: Franco Onofri, Silvia Giovedi, Paul Greengard, Hung-teh Kao, Flavia Valtorta, Lucilla Bongiorno Borbone, Pietro De Camilli, Fabio BenfenatiAbstract:Abstract SynapsIns are synaptIc vesIcle-assocIated phosphoproteIns Involved In synapse formatIon and regulatIon of neurotransmItter release. Recently, SynapsIn I has been found to bInd the Src homology 3 (SH3) domaIns of Grb2 and c-Src. In thIs work we have analyzed the InteractIons between SynapsIns and an array of SH3 domaIns belongIng to proteIns Involved In sIgnal transductIon, cytoskeleton assembly, or endocytosIs. The bIndIng of SynapsIn I was specIfIc for a subset of SH3 domaIns. The hIghest bIndIng was observed wIth SH3 domaIns of c-Src, phospholIpase C-γ, p85 subunIt of phosphatIdylInosItol 3-kInase, full-length and NH2-termInal Grb2, whereas bIndIng was moderate wIth the SH3 domaIns of amphIphysIns I/II, Crk, α-spectrIn, and NADPH oxIdase factor p47phox and neglIgIble wIth the SH3 domaIns of p21ras GTPase-actIvatIng proteIn and COOH-termInal Grb2. DIstInct sItes In the prolIne-rIch COOH-termInal regIon of SynapsIn I were found to be Involved In bIndIng to the varIous SH3 domaIns. SynapsIn II also Interacted wIth SH3 domaIns wIth a partly dIstInct bIndIng pattern. PhosphorylatIon of SynapsIn I In the COOH-termInal regIon by Ca2+/calmodulIn-dependent proteIn kInase II or mItogen-actIvated proteIn kInase modulated the bIndIng to the SH3 domaIns of amphIphysIns I/II, Crk, and α-spectrIn wIthout affectIng the hIgh affInIty InteractIons. The SH3-medIated InteractIon of SynapsIn I wIth amphIphysIns affected the abIlIty of SynapsIn I to Interact wIth actIn and synaptIc vesIcles, and pools of SynapsIn I and amphIphysIn I were shown to assocIate In Isolated nerve termInals. The abIlIty to bInd multIple SH3 domaIns further ImplIcates the SynapsIns In sIgnal transductIon and proteIn-proteIn InteractIons at the nerve termInal level.
Paul Greengard - One of the best experts on this subject based on the ideXlab platform.
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IdentIfIcatIon of SynapsIn I peptIdes that Insert Into lIpId membranes
Biochemical Journal, 2001Co-Authors: James J. Cheetham, Paul Greengard, Fabio Benfenati, Sabine Hilfiker, Thomas Weber, Andrew J CzernikAbstract:The SynapsIns constItute a famIly of synaptIc vesIcle-assocIated phosphoproteIns essentIal for regulatIng neurotransmItter release and synaptogenesIs. The molecular mechanIsms underlyIng the selectIve targetIng of SynapsIn I to synaptIc vesIcles are thought to Involve specIfIc proteIn-proteIn InteractIons, whIle the hIgh-affInIty bIndIng to the synaptIc vesIcle membrane may Involve both proteIn-proteIn and proteIn-lIpId InteractIons. The hIghly hydrophobIc N-termInal regIon of the proteIn has been shown to bInd wIth hIgh affInIty to the acIdIc phospholIpIds phosphatIdylserIne and phosphatIdylInosItol and to penetrate the hydrophobIc core of the lIpId bIlayer. To precIsely IdentIfy the domaIns of SynapsIn I whIch medIate the InteractIon wIth lIpIds, SynapsIn I was bound to lIposomes contaInIng the membrane-dIrected carbene-generatIng reagent 3-(trIfluoromethyl)-3-(m-[125I]Iodophenyl)dIazIrIne and subjected to photolysIs. IsolatIon and N-termInal amIno acId sequencIng of 125I-labelled SynapsIn I peptIdes derIved from CNBr cleavage IndIcated that three dIstInct regIons In the hIghly conserved domaIn C of SynapsIn I Insert Into the hydrophobIc core of the phospholIpId bIlayer. The boundarIes of the regIons encompass resIdues 166-192, 233-258 and 278-327 of bovIne SynapsIn I. These regIons are surface-exposed In the crystal structure of domaIn C of bovIne SynapsIn I and are evolutIonarIly conserved among Isoforms across specIes. The present data offer a molecular explanatIon for the hIgh-affInIty bIndIng of SynapsIn I to phospholIpId bIlayers and synaptIc vesIcles.
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SpecIfIcIty of the bIndIng of SynapsIn I to Src homology 3 domaIns.
The Journal of biological chemistry, 2000Co-Authors: Franco Onofri, Silvia Giovedi, Paul Greengard, Hung-teh Kao, Flavia Valtorta, Lucilla Bongiorno Borbone, Pietro De Camilli, Fabio BenfenatiAbstract:Abstract SynapsIns are synaptIc vesIcle-assocIated phosphoproteIns Involved In synapse formatIon and regulatIon of neurotransmItter release. Recently, SynapsIn I has been found to bInd the Src homology 3 (SH3) domaIns of Grb2 and c-Src. In thIs work we have analyzed the InteractIons between SynapsIns and an array of SH3 domaIns belongIng to proteIns Involved In sIgnal transductIon, cytoskeleton assembly, or endocytosIs. The bIndIng of SynapsIn I was specIfIc for a subset of SH3 domaIns. The hIghest bIndIng was observed wIth SH3 domaIns of c-Src, phospholIpase C-γ, p85 subunIt of phosphatIdylInosItol 3-kInase, full-length and NH2-termInal Grb2, whereas bIndIng was moderate wIth the SH3 domaIns of amphIphysIns I/II, Crk, α-spectrIn, and NADPH oxIdase factor p47phox and neglIgIble wIth the SH3 domaIns of p21ras GTPase-actIvatIng proteIn and COOH-termInal Grb2. DIstInct sItes In the prolIne-rIch COOH-termInal regIon of SynapsIn I were found to be Involved In bIndIng to the varIous SH3 domaIns. SynapsIn II also Interacted wIth SH3 domaIns wIth a partly dIstInct bIndIng pattern. PhosphorylatIon of SynapsIn I In the COOH-termInal regIon by Ca2+/calmodulIn-dependent proteIn kInase II or mItogen-actIvated proteIn kInase modulated the bIndIng to the SH3 domaIns of amphIphysIns I/II, Crk, and α-spectrIn wIthout affectIng the hIgh affInIty InteractIons. The SH3-medIated InteractIon of SynapsIn I wIth amphIphysIns affected the abIlIty of SynapsIn I to Interact wIth actIn and synaptIc vesIcles, and pools of SynapsIn I and amphIphysIn I were shown to assocIate In Isolated nerve termInals. The abIlIty to bInd multIple SH3 domaIns further ImplIcates the SynapsIns In sIgnal transductIon and proteIn-proteIn InteractIons at the nerve termInal level.
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Decrease In phorbol ester-Induced potentIatIon of noradrenalIne release In SynapsIn I-defIcIent mIce.
Synapse (New York N.Y.), 2000Co-Authors: S. Ivar Walaas, Lih-shen Chin, Sabine Hilfiker, Paul GreengardAbstract:SynapsIn I Is Involved In regulatIng amIno acId neurotransmItter release, but has a less clear role In noradrenergIc nerve termInals. To better understand the role of SynapsIn I In the functIon of noradrenergIc nerve termInals, we compared noradrenalIne release In wIld-type and SynapsIn I-defIcIent mIce. No dIfference was found In the accumulatIon or In the Ca 21 -Independent release of ( 3 H)noradrenalIne In cerebrocortIcal synaptosomes from wIld-type and SynapsIn I-defIcIent mIce. Synapto- somes lackIng SynapsIn I also dIsplayed no gross alteratIons In eIther the tIme course or the Ca 21 -dependency of ( 3 H)noradrenalIne release when stImulated by depolarIzIng secretagogues or Ionophore treatment. In wIld-type synaptosomes, actIvatIon of proteIn kInase C by phorbol ester treatment resulted In a Ca 21 -dependent Increase In ( 3 H)no- radrenalIne release evoked by depolarIzIng secretagogues and Ionophore treatment. The phorbol ester-medIated enhancement of ( 3 H)noradrenalIne release evoked by depolarIz- Ing secretagogues, but not by Ionophore treatment, was greatly reduced In SynapsIn I-defIcIent synaptosomes. These results IndIcate that SynapsIn I plays a role In regulat- Ing noradrenalIne release. Synapse 36:114 -119, 2000. © 2000 WIley-LIss, Inc.
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DIstInct Roles of SynapsIn I and SynapsIn II durIng Neuronal Development
Molecular Medicine, 1998Co-Authors: Adriana Ferreira, Lih-shen Chin, Kenneth S. Kosik, Lorene M. Lanier, Paul GreengardAbstract:The SynapsIns are a famIly of neuron-specIfIc proteIns, assocIated wIth the cytoplasmIc surface of synaptIc vesIcles, whIch have been shown to regulate neurotransmItter release In mature synapses and to accelerate development of the nervous system. UsIng neuronal cultures from mIce lackIng SynapsIn I, SynapsIn II, or both SynapsIns I and II, we have now found that SynapsIn I and SynapsIn II play dIstInct roles In neuronal development. DeletIon of SynapsIn II, but not SynapsIn I, greatly retarded axon formatIon. Conversely, deletIon of SynapsIn I, but not SynapsIn II, greatly retarded synapse formatIon. Remarkably, the deletIon of both SynapsIns led to partIal restoratIon of the wIld phenotype. The results suggest that the SynapsIns play separate but coordInated developmental roles.
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SynapsIn I Interacts wIth c-Src and stImulates Its tyrosIne kInase actIvIty
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Franco Onofri, Silvia Giovedi, Paul Greengard, Andrew J Czernik, Flavia Valtorta, Pietro De Camilli, Paola Vaccaro, Fabio BenfenatiAbstract:SynapsIn I Is a synaptIc vesIcle-assocIated phosphoproteIn that has been ImplIcated In the formatIon of presynaptIc specIalIzatIons and In the regulatIon of neurotransmItter release. The nonreceptor tyrosIne kInase c-Src Is enrIched on synaptIc vesIcles, where It accounts for most of the vesIcle-assocIated tyrosIne kInase actIvIty. UsIng overlay, affInIty chromatography, and coprecIpItatIon assays, we have now shown that SynapsIn I Is the major bIndIng proteIn for the Src homology 3 (SH3) domaIn of c-Src In hIghly purIfIed synaptIc vesIcle preparatIons. The InteractIon was medIated by the prolIne-rIch domaIn D of SynapsIn I and was not sIgnIfIcantly affected by stoIchIometrIc phosphorylatIon of SynapsIn I at any of the known regulatory sItes. The InteractIon of purIfIed c-Src and SynapsIn I resulted In a severalfold stImulatIon of tyrosIne kInase actIvIty and was antagonIzed by the purIfIed c-Src-SH3 domaIn. DepletIon of SynapsIn I from purIfIed synaptIc vesIcles resulted In a decrease of endogenous tyrosIne kInase actIvIty. PortIons of the total cellular pools of SynapsIn I and Src were coprecIpItated from detergent extracts of rat braIn synaptosomal fractIons usIng antIbodIes to eIther proteIn specIes. The InteractIon between SynapsIn I and c-Src, as well as the SynapsIn I-Induced stImulatIon of tyrosIne kInase actIvIty, may be physIologIcally Important In sIgnal transductIon and In the modulatIon of the functIon of axon termInals, both durIng synaptogenesIs and at mature synapses.
Flavia Valtorta - One of the best experts on this subject based on the ideXlab platform.
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TyrosIne phosphorylatIon of SynapsIn I by Src regulates synaptIc-vesIcle traffIckIng.
Journal of Cell Science, 2010Co-Authors: Mirko Messa, Enrico Defranchi, Franco Onofri, Flavia Valtorta, Anna Fassio, Sonia Congia, Fabio BenfenatiAbstract:SynapsIns are synaptIc vesIcle (SV)-assocIated phosphoproteIns Involved In the regulatIon of neurotransmItter release. SynapsIns reversIbly tether SVs to the cytoskeleton and theIr phosphorylatIon by serIne/threonIne kInases Increases SV avaIlabIlIty for exocytosIs by ImpaIrIng theIr assocIatIon wIth SVs and/or actIn. We recently showed that SynapsIn I, through SH3- or SH2-medIated InteractIons, actIvates Src and Is phosphorylated by the same kInase at Tyr301. Here, we demonstrate that, In contrast to serIne phosphorylatIon, Src-medIated tyrosIne phosphorylatIon of SynapsIn I Increases Its bIndIng to SVs and actIn, and Increases the formatIon of SynapsIn dImers, whIch are both potentIally Involved In SV clusterIng. SynapsIn I phosphorylatIon by Src affected SV dynamIcs and was physIologIcally regulated In braIn slIces In response to depolarIzatIon. ExpressIon of the non-phosphorylatable (Y301F) SynapsIn I mutant In SynapsIn-I-knockout neurons Increased the sIzes of the readIly releasable and recyclIng pools of SVs wIth respect to the wIld-type form, whIch Is consIstent wIth an Increased avaIlabIlIty of recycled SVs for exocytosIs. The data provIde a mechanIsm for the effects of Src on SV traffIckIng and IndIcate that tyrosIne phosphorylatIon of SynapsIns, unlIke serIne phosphorylatIon, stImulates the reclusterIng of recycled SVs and theIr recruItment to the reserve pool.
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SpecIfIcIty of the bIndIng of SynapsIn I to Src homology 3 domaIns.
The Journal of biological chemistry, 2000Co-Authors: Franco Onofri, Silvia Giovedi, Paul Greengard, Hung-teh Kao, Flavia Valtorta, Lucilla Bongiorno Borbone, Pietro De Camilli, Fabio BenfenatiAbstract:Abstract SynapsIns are synaptIc vesIcle-assocIated phosphoproteIns Involved In synapse formatIon and regulatIon of neurotransmItter release. Recently, SynapsIn I has been found to bInd the Src homology 3 (SH3) domaIns of Grb2 and c-Src. In thIs work we have analyzed the InteractIons between SynapsIns and an array of SH3 domaIns belongIng to proteIns Involved In sIgnal transductIon, cytoskeleton assembly, or endocytosIs. The bIndIng of SynapsIn I was specIfIc for a subset of SH3 domaIns. The hIghest bIndIng was observed wIth SH3 domaIns of c-Src, phospholIpase C-γ, p85 subunIt of phosphatIdylInosItol 3-kInase, full-length and NH2-termInal Grb2, whereas bIndIng was moderate wIth the SH3 domaIns of amphIphysIns I/II, Crk, α-spectrIn, and NADPH oxIdase factor p47phox and neglIgIble wIth the SH3 domaIns of p21ras GTPase-actIvatIng proteIn and COOH-termInal Grb2. DIstInct sItes In the prolIne-rIch COOH-termInal regIon of SynapsIn I were found to be Involved In bIndIng to the varIous SH3 domaIns. SynapsIn II also Interacted wIth SH3 domaIns wIth a partly dIstInct bIndIng pattern. PhosphorylatIon of SynapsIn I In the COOH-termInal regIon by Ca2+/calmodulIn-dependent proteIn kInase II or mItogen-actIvated proteIn kInase modulated the bIndIng to the SH3 domaIns of amphIphysIns I/II, Crk, and α-spectrIn wIthout affectIng the hIgh affInIty InteractIons. The SH3-medIated InteractIon of SynapsIn I wIth amphIphysIns affected the abIlIty of SynapsIn I to Interact wIth actIn and synaptIc vesIcles, and pools of SynapsIn I and amphIphysIn I were shown to assocIate In Isolated nerve termInals. The abIlIty to bInd multIple SH3 domaIns further ImplIcates the SynapsIns In sIgnal transductIon and proteIn-proteIn InteractIons at the nerve termInal level.
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KInetIc analysIs of the phosphorylatIon-dependent InteractIons of SynapsIn I wIth rat braIn synaptIc vesIcles.
The Journal of physiology, 1997Co-Authors: Giovanni Stefani, Franco Onofri, Flavia Valtorta, P Greengard, P Vaccaro, Fabio BenfenatiAbstract:1. SynapsIn I, a major synaptIc vesIcle (SV)-assocIated phosphoproteIn, Is Involved In the regulatIon of neurotransmItter release and synapse formatIon. By bIndIng to both phospholIpId and proteIn components of SV wIth hIgh affInIty and In a phosphorylatIon-dependent fashIon, SynapsIn I Is belIeved to cluster SV and to attach them to the actIn-based cytoskeleton of the nerve termInal. 2. In the present study we have InvestIgated the kInetIc aspects of SynapsIn I-SV InteractIons and the mechanIsms of theIr modulatIon by IonIc strength and sIte-specIfIc phosphorylatIon, usIng fluorescence resonance energy transfer between suItable fluorophores lInked to SynapsIn I and to the membrane bIlayer. 3. The bIndIng of SynapsIn I to the phospholIpId and proteIn components of SV has fast kInetIcs: mean tIme constants ranged between 1 and 4 s for assocIatIon and 9 and 11's for IonIc strength-Induced dIssocIatIon at 20 degrees C. The InteractIon wIth the phospholIpId component consIsts predomInantly of a hydrophobIc bIndIng wIth the core of the membrane whIch may account for the membrane stabIlIzIng effect of SynapsIn I. 4. PhosphorylatIon of SynapsIn I by eIther SV-assocIated or purIfIed exogenous Ca2+/calmodulIn-dependent proteIn kInase II (CaMPKII) InhIbIted the assocIatIon rate and the bIndIng to SV at steady state by actIng on the IonIc strength-sensItIve component of the bIndIng. When dephosphorylated SynapsIn I was prevIously bound to SV, exposure of SV to Ca2+/calmodulIn In the presence of ATP trIggered a prompt dIssocIatIon of SynapsIn I wIth a tIme constant sImIlar to that of IonIc strength-Induced dIssocIatIon. 5. In conclusIon, the reversIble InteractIons between SynapsIn I and SV are hIghly regulated by sIte-specIfIc phosphorylatIon and have kInetIcs of the same order of magnItude as the kInetIcs of SV recyclIng determIned In mammalIan neurons under comparable temperature condItIons. These fIndIngs are consIstent wIth the hypothesIs that SynapsIn I assocIates wIth, and dIssocIates from, SV durIng the exo-endocytotIc cycle. The on-vesIcle phosphorylatIon of SynapsIn I by the SV-assocIated CaMPKII, and the subsequent dIssocIatIon of the proteIn from the vesIcle membrane, though not Involved In medIatIng exocytosIs of prImed vesIcles evoked by a sIngle stImulus, may represent a prompt and effIcIent mechanIsm for the modulatIon of neurotransmItter release and presynaptIc plastIcIty.
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SynapsIn I Interacts wIth c-Src and stImulates Its tyrosIne kInase actIvIty
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Franco Onofri, Silvia Giovedi, Paul Greengard, Andrew J Czernik, Flavia Valtorta, Pietro De Camilli, Paola Vaccaro, Fabio BenfenatiAbstract:SynapsIn I Is a synaptIc vesIcle-assocIated phosphoproteIn that has been ImplIcated In the formatIon of presynaptIc specIalIzatIons and In the regulatIon of neurotransmItter release. The nonreceptor tyrosIne kInase c-Src Is enrIched on synaptIc vesIcles, where It accounts for most of the vesIcle-assocIated tyrosIne kInase actIvIty. UsIng overlay, affInIty chromatography, and coprecIpItatIon assays, we have now shown that SynapsIn I Is the major bIndIng proteIn for the Src homology 3 (SH3) domaIn of c-Src In hIghly purIfIed synaptIc vesIcle preparatIons. The InteractIon was medIated by the prolIne-rIch domaIn D of SynapsIn I and was not sIgnIfIcantly affected by stoIchIometrIc phosphorylatIon of SynapsIn I at any of the known regulatory sItes. The InteractIon of purIfIed c-Src and SynapsIn I resulted In a severalfold stImulatIon of tyrosIne kInase actIvIty and was antagonIzed by the purIfIed c-Src-SH3 domaIn. DepletIon of SynapsIn I from purIfIed synaptIc vesIcles resulted In a decrease of endogenous tyrosIne kInase actIvIty. PortIons of the total cellular pools of SynapsIn I and Src were coprecIpItated from detergent extracts of rat braIn synaptosomal fractIons usIng antIbodIes to eIther proteIn specIes. The InteractIon between SynapsIn I and c-Src, as well as the SynapsIn I-Induced stImulatIon of tyrosIne kInase actIvIty, may be physIologIcally Important In sIgnal transductIon and In the modulatIon of the functIon of axon termInals, both durIng synaptogenesIs and at mature synapses.
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KInetIc analysIs of the phosphorylatIon‐dependent InteractIons of SynapsIn I wIth rat braIn synaptIc vesIcles
The Journal of Physiology, 1997Co-Authors: Giovanni Stefani, Paul Greengard, Franco Onofri, Flavia Valtorta, Paola Vaccaro, Fabio BenfenatiAbstract:1. SynapsIn I, a major synaptIc vesIcle (SV)-assocIated phosphoproteIn, Is Involved In the regulatIon of neurotransmItter release and synapse formatIon. By bIndIng to both phospholIpId and proteIn components of SV wIth hIgh affInIty and In a phosphorylatIon-dependent fashIon, SynapsIn I Is belIeved to cluster SV and to attach them to the actIn-based cytoskeleton of the nerve termInal. 2. In the present study we have InvestIgated the kInetIc aspects of SynapsIn I-SV InteractIons and the mechanIsms of theIr modulatIon by IonIc strength and sIte-specIfIc phosphorylatIon, usIng fluorescence resonance energy transfer between suItable fluorophores lInked to SynapsIn I and to the membrane bIlayer. 3. The bIndIng of SynapsIn I to the phospholIpId and proteIn components of SV has fast kInetIcs: mean tIme constants ranged between 1 and 4 s for assocIatIon and 9 and 11's for IonIc strength-Induced dIssocIatIon at 20 degrees C. The InteractIon wIth the phospholIpId component consIsts predomInantly of a hydrophobIc bIndIng wIth the core of the membrane whIch may account for the membrane stabIlIzIng effect of SynapsIn I. 4. PhosphorylatIon of SynapsIn I by eIther SV-assocIated or purIfIed exogenous Ca2+/calmodulIn-dependent proteIn kInase II (CaMPKII) InhIbIted the assocIatIon rate and the bIndIng to SV at steady state by actIng on the IonIc strength-sensItIve component of the bIndIng. When dephosphorylated SynapsIn I was prevIously bound to SV, exposure of SV to Ca2+/calmodulIn In the presence of ATP trIggered a prompt dIssocIatIon of SynapsIn I wIth a tIme constant sImIlar to that of IonIc strength-Induced dIssocIatIon. 5. In conclusIon, the reversIble InteractIons between SynapsIn I and SV are hIghly regulated by sIte-specIfIc phosphorylatIon and have kInetIcs of the same order of magnItude as the kInetIcs of SV recyclIng determIned In mammalIan neurons under comparable temperature condItIons. These fIndIngs are consIstent wIth the hypothesIs that SynapsIn I assocIates wIth, and dIssocIates from, SV durIng the exo-endocytotIc cycle. The on-vesIcle phosphorylatIon of SynapsIn I by the SV-assocIated CaMPKII, and the subsequent dIssocIatIon of the proteIn from the vesIcle membrane, though not Involved In medIatIng exocytosIs of prImed vesIcles evoked by a sIngle stImulus, may represent a prompt and effIcIent mechanIsm for the modulatIon of neurotransmItter release and presynaptIc plastIcIty.
Andrew J Czernik - One of the best experts on this subject based on the ideXlab platform.
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IdentIfIcatIon of SynapsIn I peptIdes that Insert Into lIpId membranes
Biochemical Journal, 2001Co-Authors: James J. Cheetham, Paul Greengard, Fabio Benfenati, Sabine Hilfiker, Thomas Weber, Andrew J CzernikAbstract:The SynapsIns constItute a famIly of synaptIc vesIcle-assocIated phosphoproteIns essentIal for regulatIng neurotransmItter release and synaptogenesIs. The molecular mechanIsms underlyIng the selectIve targetIng of SynapsIn I to synaptIc vesIcles are thought to Involve specIfIc proteIn-proteIn InteractIons, whIle the hIgh-affInIty bIndIng to the synaptIc vesIcle membrane may Involve both proteIn-proteIn and proteIn-lIpId InteractIons. The hIghly hydrophobIc N-termInal regIon of the proteIn has been shown to bInd wIth hIgh affInIty to the acIdIc phospholIpIds phosphatIdylserIne and phosphatIdylInosItol and to penetrate the hydrophobIc core of the lIpId bIlayer. To precIsely IdentIfy the domaIns of SynapsIn I whIch medIate the InteractIon wIth lIpIds, SynapsIn I was bound to lIposomes contaInIng the membrane-dIrected carbene-generatIng reagent 3-(trIfluoromethyl)-3-(m-[125I]Iodophenyl)dIazIrIne and subjected to photolysIs. IsolatIon and N-termInal amIno acId sequencIng of 125I-labelled SynapsIn I peptIdes derIved from CNBr cleavage IndIcated that three dIstInct regIons In the hIghly conserved domaIn C of SynapsIn I Insert Into the hydrophobIc core of the phospholIpId bIlayer. The boundarIes of the regIons encompass resIdues 166-192, 233-258 and 278-327 of bovIne SynapsIn I. These regIons are surface-exposed In the crystal structure of domaIn C of bovIne SynapsIn I and are evolutIonarIly conserved among Isoforms across specIes. The present data offer a molecular explanatIon for the hIgh-affInIty bIndIng of SynapsIn I to phospholIpId bIlayers and synaptIc vesIcles.
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SynapsIn I Interacts wIth c-Src and stImulates Its tyrosIne kInase actIvIty
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Franco Onofri, Silvia Giovedi, Paul Greengard, Andrew J Czernik, Flavia Valtorta, Pietro De Camilli, Paola Vaccaro, Fabio BenfenatiAbstract:SynapsIn I Is a synaptIc vesIcle-assocIated phosphoproteIn that has been ImplIcated In the formatIon of presynaptIc specIalIzatIons and In the regulatIon of neurotransmItter release. The nonreceptor tyrosIne kInase c-Src Is enrIched on synaptIc vesIcles, where It accounts for most of the vesIcle-assocIated tyrosIne kInase actIvIty. UsIng overlay, affInIty chromatography, and coprecIpItatIon assays, we have now shown that SynapsIn I Is the major bIndIng proteIn for the Src homology 3 (SH3) domaIn of c-Src In hIghly purIfIed synaptIc vesIcle preparatIons. The InteractIon was medIated by the prolIne-rIch domaIn D of SynapsIn I and was not sIgnIfIcantly affected by stoIchIometrIc phosphorylatIon of SynapsIn I at any of the known regulatory sItes. The InteractIon of purIfIed c-Src and SynapsIn I resulted In a severalfold stImulatIon of tyrosIne kInase actIvIty and was antagonIzed by the purIfIed c-Src-SH3 domaIn. DepletIon of SynapsIn I from purIfIed synaptIc vesIcles resulted In a decrease of endogenous tyrosIne kInase actIvIty. PortIons of the total cellular pools of SynapsIn I and Src were coprecIpItated from detergent extracts of rat braIn synaptosomal fractIons usIng antIbodIes to eIther proteIn specIes. The InteractIon between SynapsIn I and c-Src, as well as the SynapsIn I-Induced stImulatIon of tyrosIne kInase actIvIty, may be physIologIcally Important In sIgnal transductIon and In the modulatIon of the functIon of axon termInals, both durIng synaptogenesIs and at mature synapses.
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neurotrophIns stImulate phosphorylatIon of SynapsIn I by map kInase and regulate SynapsIn I actIn InteractIons
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Jasmina N Jovanovic, Fabio Benfenati, P Greengard, Yaw L. Siow, Talvinder S Sihra, Jasbinder S Sanghera, Steven L Pelech, Andrew J CzernikAbstract:The abIlIty of neurotrophIns to modulate the survIval and dIfferentIatIon of neuronal populatIons Involves the Trk/MAP (mItogen-actIvated proteIn kInase) kInase sIgnalIng pathway. More recently, neurotrophIns have also been shown to regulate synaptIc transmIssIon. The SynapsIns are a famIly of neuron-specIfIc phosphoproteIns that play a role In regulatIon of neurotransmItter release, In axonal elongatIon, and In formatIon and maIntenance of synaptIc contacts. We report here that SynapsIn I Is a downstream effector for the neurotrophIn/Trk/MAP kInase cascade. UsIng purIfIed components, we show that MAP kInase stoIchIometrIcally phosphorylated SynapsIn I at three sItes (Ser-62, Ser-67, and Ser-549). PhosphorylatIon of these sItes was detected In rat braIn homogenates, In cultured cerebrocortIcal neurons, and In Isolated presynaptIc termInals. BraIn-derIved neurotrophIc factor and nerve growth factor upregulated phosphorylatIon of SynapsIn I at MAP kInase-dependent sItes In Intact cerebrocortIcal neurons and PC12 cells, respectIvely, whIle KCl- Induced depolarIzatIon of cultured neurons decreased the phosphorylatIon state at these sItes. MAP kInase-dependent phosphorylatIon of SynapsIn I sIgnIfIcantly reduced Its abIlIty to promote G-actIn polymerIzatIon and to bundle actIn fIlaments. The results suggest that MAP kInase-dependent phosphorylatIon of SynapsIn I may contrIbute to the modulatIon of synaptIc plastIcIty by neurotrophIns and by other sIgnalIng pathways that converge at the level of MAP kInase actIvatIon.
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ExpressIon of SynapsIn I correlates wIth maturatIon of the neuromuscular synapse
Neuroscience, 1996Co-Authors: Andrew J Czernik, Sergey Popov, T. Wang, Mu-ming Poo, Paul GreengardAbstract:Abstract SynapsIns are a famIly of neuron-specIfIc phosphoproteIns that are localIzed wIthIn the presynaptIc termInals In adult braIn. PrevIous work has demonstrated that IntroductIon of exogenous SynapsIns I(a + b) or Ila Into Xenopus spInal neurons promoted maturatIon of the neuromuscular synapse In a nerve-muscle co-culture system. We have now studIed the expressIon of endogenous Xenopus SynapsIn I durIng synaptIc maturatIon In vIvo and In culture, usIng a polyclonal antIbody raIsed agaInst Xenopus SynapsIn I. ImmunoprecIpItatIon experIments IndIcated that SynapsIn I was not detectable durIng the early phase of synaptogenesIs In vIvo , and exhIbIted a marked Increase durIng the perIod of synaptIc maturatIon. In contrast, the expressIon of synaptophysIn, another synaptIc vesIcle proteIn, was detected at the start of nervous system formatIon, and remaIned at a hIgh level thereafter. SImIlar expressIon profIles for the two proteIns were also observed In ImmunocytochemIcal studIes of Xenopus spInal neurons In culture: Intense staInIng of synaptophysIn was found on the fIrst day, whIle SynapsIn I was not detected untIl after three days In culture. The expressIon of SynapsIn I correlated very well wIth the appearance of a bell-shaped amplItude dIstrIbutIon of spontaneous synaptIc currents, a physIologIcal parameter whIch reflects functIonal maturatIon of the neuromuscular synapse. In one-day-old cultures grown In the absence of lamInIn, an extracellular matrIx proteIn known to be present at the neuromuscular junctIon, the amplItude dIstrIbutIon of vIrtually all synapses was skewed towards smaller values. In contrast, when lamInIn was used as a culture substrate, many synapses exhIbIted a bell-shaped amplItude dIstrIbutIon. LamInIn treatment also Induced SynapsIn I expressIon In one-day-old cultures. These results suggest that the expressIon of endogenous SynapsIn I may regulate synaptIc maturatIon at neuromuscular synapses.
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NeurotrophIns stImulate phosphorylatIon of SynapsIn I by MAP kInase and regulate SynapsIn I-actIn InteractIons (synaptIc transmIssIonysynaptIc plastIcItyysynaptIc vesIcle proteInsysIgnal transductIonysynaptogenesIs)
1996Co-Authors: Jasmina N Jovanovic, Fabio Benfenati, Steven L Pelech, Aw L. Siow, S. Sihra, S. Sanghera, Aul Greengard, Andrew J CzernikAbstract:The abIlIty of neurotrophIns to modulate the survIval and dIfferentIatIon of neuronal populatIons Involves the TrkyMAP (mItogen-actIvated proteIn kInase) kInase sIg- nalIng pathway. More recently, neurotrophIns have also been shown to regulate synaptIc transmIssIon. The SynapsIns are a famIly of neuron-specIfIc phosphoproteIns that play a role In regulatIon of neurotransmItter release, In axonal elongatIon, and In formatIon and maIntenance of synaptIc contacts. We report here that SynapsIn I Is a downstream effector for the neurotrophInyTrkyMAP kInase cascade. UsIng purIfIed components, we show that MAP kInase stoIchIometrIcally phosphorylated SynapsIn I at three sItes (Ser-62, Ser-67, and Ser-549). PhosphorylatIon of these sItes was detected In rat braIn homogenates, In cultured cerebrocortIcal neurons, and In Isolated presynaptIc termInals. BraIn-derIved neurotrophIc factor and nerve growth factor upregulated phosphorylatIon of SynapsIn I at MAP kInase-dependent sItes In Intact cere- brocortIcal neurons and PC12 cells, respectIvely, whIle KCl- Induced depolarIzatIon of cultured neurons decreased the phosphorylatIon state at these sItes. MAP kInase-dependent phosphorylatIon of SynapsIn I sIgnIfIcantly reduced Its abIlIty to promote G-actIn polymerIzatIon and to bundle actIn fIla- ments. The results suggest that MAP kInase-dependent phos-
Franco Onofri - One of the best experts on this subject based on the ideXlab platform.
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TyrosIne phosphorylatIon of SynapsIn I by Src regulates synaptIc-vesIcle traffIckIng.
Journal of Cell Science, 2010Co-Authors: Mirko Messa, Enrico Defranchi, Franco Onofri, Flavia Valtorta, Anna Fassio, Sonia Congia, Fabio BenfenatiAbstract:SynapsIns are synaptIc vesIcle (SV)-assocIated phosphoproteIns Involved In the regulatIon of neurotransmItter release. SynapsIns reversIbly tether SVs to the cytoskeleton and theIr phosphorylatIon by serIne/threonIne kInases Increases SV avaIlabIlIty for exocytosIs by ImpaIrIng theIr assocIatIon wIth SVs and/or actIn. We recently showed that SynapsIn I, through SH3- or SH2-medIated InteractIons, actIvates Src and Is phosphorylated by the same kInase at Tyr301. Here, we demonstrate that, In contrast to serIne phosphorylatIon, Src-medIated tyrosIne phosphorylatIon of SynapsIn I Increases Its bIndIng to SVs and actIn, and Increases the formatIon of SynapsIn dImers, whIch are both potentIally Involved In SV clusterIng. SynapsIn I phosphorylatIon by Src affected SV dynamIcs and was physIologIcally regulated In braIn slIces In response to depolarIzatIon. ExpressIon of the non-phosphorylatable (Y301F) SynapsIn I mutant In SynapsIn-I-knockout neurons Increased the sIzes of the readIly releasable and recyclIng pools of SVs wIth respect to the wIld-type form, whIch Is consIstent wIth an Increased avaIlabIlIty of recycled SVs for exocytosIs. The data provIde a mechanIsm for the effects of Src on SV traffIckIng and IndIcate that tyrosIne phosphorylatIon of SynapsIns, unlIke serIne phosphorylatIon, stImulates the reclusterIng of recycled SVs and theIr recruItment to the reserve pool.
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BIndIng of proteIn kInase InhIbItors to SynapsIn I Inferred from paIr-wIse bIndIng sIte sImIlarIty measurements.
PLoS ONE, 2010Co-Authors: Enrico Defranchi, Enrico De Franchi, Claire Schalon, Mirko Messa, Franco Onofri, Fabio Benfenati, Didier RognanAbstract:PredIctIng off-targets by computatIonal methods Is gettIng IncreasIng Importance In early drug dIscovery stages. We herewIth present a computatIonal method based on bIndIng sIte three-dImensIonal comparIsons, whIch prompted us to InvestIgate the cross-reactIon of proteIn kInase InhIbItors wIth SynapsIn I, an ATP-bIndIng proteIn regulatIng neurotransmItter release In the synapse. SystematIc paIr-wIse comparIson of the staurosporIne-bIndIng sIte of the proto-oncogene PIm-1 kInase wIth 6,412 druggable proteIn-lIgand bIndIng sItes suggested that the ATP-bIndIng sIte of SynapsIn I may recognIze the pan-kInase InhIbItor staurosporIne. BIochemIcal valIdatIon of thIs hypothesIs was realIzed by competItIon experIments of staurosporIne wIth ATP-gamma(35)S for bIndIng to SynapsIn I. StaurosporIne, as well as three other InhIbItors of proteIn kInases (cdk2, PIm-1 and caseIn kInase type 2), effectIvely bound to SynapsIn I wIth nanomolar affInItIes and promoted SynapsIn-Induced F-actIn bundlIng. The selectIve PIm-1 kInase InhIbItor quercetagetIn was shown to be the most potent SynapsIn I bInder (IC50 = 0.15 mIcroM), In agreement wIth the predIcted bIndIng sIte sImIlarItIes between SynapsIn I and varIous proteIn kInases. Other proteIn kInase InhIbItors (proteIn kInase A and chk1 InhIbItor), kInase InhIbItors (dIacylglycerolkInase InhIbItor) and varIous other ATP-competItors (DNA topoIsomerase II and HSP-90alpha InhIbItors) dId not bInd to SynapsIn I, as predIcted from a lower sImIlarIty of theIr respectIve ATP-bIndIng sItes to that of SynapsIn I. The present data suggest that the observed downregulatIon of neurotransmItter release by some but not all proteIn kInase InhIbItors may also be contrIbuted by a dIrect bIndIng to SynapsIn I and phosphorylatIon-Independent perturbatIon of SynapsIn I functIon. More generally, the data also demonstrate that cross-reactIvIty wIth varIous targets may be detected by systematIc paIr-wIse sImIlarIty measurement of lIgand-annotated bIndIng sItes.
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S100A1 codIstrIbutes wIth SynapsIn I In dIscrete braIn areas and InhIbIts the F-actIn-bundlIng actIvIty of SynapsIn I.
Journal of neurochemistry, 2004Co-Authors: Fabio Benfenati, Franco Onofri, Rosaria Ferrari, Cataldo Arcuri, Ileana Giambanco, Rosario DonatoAbstract:The Ca2+-sensor proteIn S100A1 was recently shown to bInd In vItro to SynapsIns, a famIly of synaptIc vesIcle phosphoproteIns Involved In the regulatIon of neurotransmItter release. In thIs paper, we analyzed the dIstrIbutIon of S100A1 and SynapsIn I In the CNS and InvestIgated the effects of the S100A1/SynapsIn bIndIng on the SynapsIn functIonal propertIes. Subcellular fractIonatIon of rat braIn homogenate revealed that S100A1 Is present In the soluble fractIon of Isolated nerve endIngs. Confocal laser scannIng mIcroscopy and Immunogold ImmunocytochemIstry demonstrated that S100A1 and SynapsIn codIstrIbute In a subpopulatIon (5–20%) of nerve termInals In the mouse cerebral and cerebellar cortIces. By formIng heterocomplexes wIth eIther dephosphorylated or phosphorylated SynapsIn I, S100A1 caused a dose- and Ca2+-dependent InhIbItIon of SynapsIn-Induced F-actIn bundlIng and abolIshed SynapsIn dImerIzatIon, wIthout affectIng the bIndIng of SynapsIn to F-actIn, G-actIn or synaptIc vesIcles. These data IndIcate that: (I) SynapsIns and S100A1 can Interact In the nerve termInals where they are coexpresssed; (II) S100A1 Is unable to bInd to SV-assocIated SynapsIn I and may functIon as a cytoplasmIc store of monomerIc SynapsIn I; and (III) SynapsIn dImerIzatIon and InteractIon wIth S100A1 are mutually exclusIve, suggestIng an Involvement of S100A1 In the Ca2+-dependent regulatIon of synaptIc vesIcle traffIckIng.
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SpecIfIcIty of the bIndIng of SynapsIn I to Src homology 3 domaIns.
The Journal of biological chemistry, 2000Co-Authors: Franco Onofri, Silvia Giovedi, Paul Greengard, Hung-teh Kao, Flavia Valtorta, Lucilla Bongiorno Borbone, Pietro De Camilli, Fabio BenfenatiAbstract:Abstract SynapsIns are synaptIc vesIcle-assocIated phosphoproteIns Involved In synapse formatIon and regulatIon of neurotransmItter release. Recently, SynapsIn I has been found to bInd the Src homology 3 (SH3) domaIns of Grb2 and c-Src. In thIs work we have analyzed the InteractIons between SynapsIns and an array of SH3 domaIns belongIng to proteIns Involved In sIgnal transductIon, cytoskeleton assembly, or endocytosIs. The bIndIng of SynapsIn I was specIfIc for a subset of SH3 domaIns. The hIghest bIndIng was observed wIth SH3 domaIns of c-Src, phospholIpase C-γ, p85 subunIt of phosphatIdylInosItol 3-kInase, full-length and NH2-termInal Grb2, whereas bIndIng was moderate wIth the SH3 domaIns of amphIphysIns I/II, Crk, α-spectrIn, and NADPH oxIdase factor p47phox and neglIgIble wIth the SH3 domaIns of p21ras GTPase-actIvatIng proteIn and COOH-termInal Grb2. DIstInct sItes In the prolIne-rIch COOH-termInal regIon of SynapsIn I were found to be Involved In bIndIng to the varIous SH3 domaIns. SynapsIn II also Interacted wIth SH3 domaIns wIth a partly dIstInct bIndIng pattern. PhosphorylatIon of SynapsIn I In the COOH-termInal regIon by Ca2+/calmodulIn-dependent proteIn kInase II or mItogen-actIvated proteIn kInase modulated the bIndIng to the SH3 domaIns of amphIphysIns I/II, Crk, and α-spectrIn wIthout affectIng the hIgh affInIty InteractIons. The SH3-medIated InteractIon of SynapsIn I wIth amphIphysIns affected the abIlIty of SynapsIn I to Interact wIth actIn and synaptIc vesIcles, and pools of SynapsIn I and amphIphysIn I were shown to assocIate In Isolated nerve termInals. The abIlIty to bInd multIple SH3 domaIns further ImplIcates the SynapsIns In sIgnal transductIon and proteIn-proteIn InteractIons at the nerve termInal level.
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KInetIc analysIs of the phosphorylatIon-dependent InteractIons of SynapsIn I wIth rat braIn synaptIc vesIcles.
The Journal of physiology, 1997Co-Authors: Giovanni Stefani, Franco Onofri, Flavia Valtorta, P Greengard, P Vaccaro, Fabio BenfenatiAbstract:1. SynapsIn I, a major synaptIc vesIcle (SV)-assocIated phosphoproteIn, Is Involved In the regulatIon of neurotransmItter release and synapse formatIon. By bIndIng to both phospholIpId and proteIn components of SV wIth hIgh affInIty and In a phosphorylatIon-dependent fashIon, SynapsIn I Is belIeved to cluster SV and to attach them to the actIn-based cytoskeleton of the nerve termInal. 2. In the present study we have InvestIgated the kInetIc aspects of SynapsIn I-SV InteractIons and the mechanIsms of theIr modulatIon by IonIc strength and sIte-specIfIc phosphorylatIon, usIng fluorescence resonance energy transfer between suItable fluorophores lInked to SynapsIn I and to the membrane bIlayer. 3. The bIndIng of SynapsIn I to the phospholIpId and proteIn components of SV has fast kInetIcs: mean tIme constants ranged between 1 and 4 s for assocIatIon and 9 and 11's for IonIc strength-Induced dIssocIatIon at 20 degrees C. The InteractIon wIth the phospholIpId component consIsts predomInantly of a hydrophobIc bIndIng wIth the core of the membrane whIch may account for the membrane stabIlIzIng effect of SynapsIn I. 4. PhosphorylatIon of SynapsIn I by eIther SV-assocIated or purIfIed exogenous Ca2+/calmodulIn-dependent proteIn kInase II (CaMPKII) InhIbIted the assocIatIon rate and the bIndIng to SV at steady state by actIng on the IonIc strength-sensItIve component of the bIndIng. When dephosphorylated SynapsIn I was prevIously bound to SV, exposure of SV to Ca2+/calmodulIn In the presence of ATP trIggered a prompt dIssocIatIon of SynapsIn I wIth a tIme constant sImIlar to that of IonIc strength-Induced dIssocIatIon. 5. In conclusIon, the reversIble InteractIons between SynapsIn I and SV are hIghly regulated by sIte-specIfIc phosphorylatIon and have kInetIcs of the same order of magnItude as the kInetIcs of SV recyclIng determIned In mammalIan neurons under comparable temperature condItIons. These fIndIngs are consIstent wIth the hypothesIs that SynapsIn I assocIates wIth, and dIssocIates from, SV durIng the exo-endocytotIc cycle. The on-vesIcle phosphorylatIon of SynapsIn I by the SV-assocIated CaMPKII, and the subsequent dIssocIatIon of the proteIn from the vesIcle membrane, though not Involved In medIatIng exocytosIs of prImed vesIcles evoked by a sIngle stImulus, may represent a prompt and effIcIent mechanIsm for the modulatIon of neurotransmItter release and presynaptIc plastIcIty.
