Regulation of Type VI Adenylyl Cyclase by Snapin, a SNAP25-binding Protein

Abstract

In the present study, we used the N terminus (amino acids 1∼160) of type VI adenylyl cyclase (ACVI) as bait to screen a mouse brain cDNA library and identified Snapin as a novel ACVI-interacting molecule. Snapin is a binding protein of SNAP25, a component of the SNARE complex. Co-immunoprecipitation analyses confirmed the interaction between Snapin and full-length ACVI. Mutational analysis revealed that the interaction domains of ACVI and Snapin were located within amino acids 1∼86 of ACVI and 33–51 of Snapin, respectively. Co-localization of ACVI and Snapin was observed in primary hippocampal neurons. Moreover, expression of Snapin specifically eliminated protein kinase C (PKC)-mediated suppression of ACVI, but not that of cAMP-dependent protein kinase (PKA) or calcium. Mutation of the potential PKC and PKA phosphorylation sites of Snapin did not affect the ability of Snapin to reverse the PKC inhibitory effect on ACVI. Phosphorylation of Snapin by PKC or PKA therefore might not be crucial for Snapin action on ACVI. In contrast, SnapinΔ33–51, which harbors an internal deletion of amino acids 33–51 did not affect PKC-mediated inhibition of ACVI, supporting that amino acids 33–51 of Snapin comprises the ACVI-interacting region. Consistently, Snapin exerted no effect on PKC-mediated inhibition of an ACVI mutant (ACVI-ΔA87), which lacked the Snapin-interacting region (amino acids 1–86). Snapin thus reverses its action via direct interaction with the N terminus of ACVI. Collectively, we demonstrate herein that in addition to its association with the SNARE complex, Snapin also functions as a regulator of an important cAMP synthesis enzyme in the brain. In the present study, we used the N terminus (amino acids 1∼160) of type VI adenylyl cyclase (ACVI) as bait to screen a mouse brain cDNA library and identified Snapin as a novel ACVI-interacting molecule. Snapin is a binding protein of SNAP25, a component of the SNARE complex. Co-immunoprecipitation analyses confirmed the interaction between Snapin and full-length ACVI. Mutational analysis revealed that the interaction domains of ACVI and Snapin were located within amino acids 1∼86 of ACVI and 33–51 of Snapin, respectively. Co-localization of ACVI and Snapin was observed in primary hippocampal neurons. Moreover, expression of Snapin specifically eliminated protein kinase C (PKC)-mediated suppression of ACVI, but not that of cAMP-dependent protein kinase (PKA) or calcium. Mutation of the potential PKC and PKA phosphorylation sites of Snapin did not affect the ability of Snapin to reverse the PKC inhibitory effect on ACVI. Phosphorylation of Snapin by PKC or PKA therefore might not be crucial for Snapin action on ACVI. In contrast, SnapinΔ33–51, which harbors an internal deletion of amino acids 33–51 did not affect PKC-mediated inhibition of ACVI, supporting that amino acids 33–51 of Snapin comprises the ACVI-interacting region. Consistently, Snapin exerted no effect on PKC-mediated inhibition of an ACVI mutant (ACVI-ΔA87), which lacked the Snapin-interacting region (amino acids 1–86). Snapin thus reverses its action via direct interaction with the N terminus of ACVI. Collectively, we demonstrate herein that in addition to its association with the SNARE complex, Snapin also functions as a regulator of an important cAMP synthesis enzyme in the brain. Adenylyl cyclases (ACs) 1The abbreviations used are: AC, adenylyl cyclase; PKC, protein kinase C; PKA, cAMP-dependent protein kinase; SNARE, soluble NSF attachment protein receptors; BSA, bovine serum albumin; MBS, m-maleimidobenzoyl-N-hydroxysuccinimide ester; ACVI, type VI adenylyl cyclase; DIV, days in vitro; MBP, maltose-binding protein.1The abbreviations used are: AC, adenylyl cyclase; PKC, protein kinase C; PKA, cAMP-dependent protein kinase; SNARE, soluble NSF attachment protein receptors; BSA, bovine serum albumin; MBS, m-maleimidobenzoyl-N-hydroxysuccinimide ester; ACVI, type VI adenylyl cyclase; DIV, days in vitro; MBP, maltose-binding protein. are a family of enzymes that produce cyclic AMP (cAMP) from ATP upon extracellular stimulation. To date, at least 9 membrane-bound ACs have been isolated and characterized (1Chern Y. Cell Signal. 2000; 12: 195-204Crossref PubMed Scopus (72) Google Scholar). These enzymes are capable of integrating positive and negative signals that act directly through stimulation of G protein-coupled receptors (GPCRs) or indirectly via intracellular signaling molecules in isozyme-specific patterns. In addition, the regulatory properties and expression patterns of different AC isoforms greatly diverge and may account for the distinctive cell- and tissue-specific responsiveness of ACs. Recently, several different proteins, including RGS2 and the protein associated with Myc (PAM), have been shown to interact and modulate activity of different AC isozymes (2Scholich K. Pierre S. Patel T.B. J. Biol. Chem. 2001; 276: 47583-57489Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 3Salim S. Sinnarajah S. Kehrl J.H. Dessauer C.W. J. Biol. Chem. 2003; 278: 15842-15849Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), adding additional dimensions to the isozyme-specific regulation of the AC superfamily. Except for the newly identified soluble AC, all membrane-bound AC members share a primary structure consisting of 12 transmembrane regions and 3 large cytoplasmic domains (N, C1a/b, and C2). The C1a and C2 domains, which form the catalytic core complex, are highly conserved and are homologous to each other. The N-terminal domains of ACs, in contrast, are variable among ACs, and have been demonstrated to play mostly regulatory roles (4Gu C. Cooper D.M. J. Biol. Chem. 1999; 274: 8012-8021Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 5Lai H.L. Lin T.H. Kao Y.Y. Lin W.J. Hwang M.J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google Scholar). Among the AC isozymes, ACVI is of particular interest, because this enzyme can be suppressed by several intracellular signaling molecules including Ca2+, cAMP-dependent kinase (PKA), and protein kinase C (PKC) (6Mons N. Cooper D.M. Brain. Res. Mol. Brain. Res. 1994; 22: 236-244Crossref PubMed Scopus (94) Google Scholar, 7Chen Y. Harry A. Li J. Smit M.J. Bai X. Magnusson R. Pieroni J.P. Weng G. Iyengar R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14100-14104Crossref PubMed Scopus (123) Google Scholar, 8Lai H.L. Yang T.H. Messing R.O. Ching Y.H. Lin S.C. Chern Y. J. Biol. Chem. 1997; 272: 4970-4977Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Although ACVI has been found in many tissues examined (9Premont R.T. Chen J. Ma H.W. Ponnapalli M. Iyengar R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9809-9813Crossref PubMed Scopus (167) Google Scholar), its expression in the brain is mainly detected in neurons (10Liu F.C. Wu G.C. Hsieh S.T. Lai H.L. Wang H.F. Wang T.W. Chern Y. FEBS Lett. 1998; 436: 92-98Crossref PubMed Scopus (33) Google Scholar). ACVI therefore might play a critical role in integrating multiple signal inputs in neurons. We previously demonstrated that the N-terminal domain of ACVI (designated N1–160) is critical for PKC-mediated suppression of ACVI (5Lai H.L. Lin T.H. Kao Y.Y. Lin W.J. Hwang M.J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google Scholar). Using N1–160 as the bait to screen a mouse brain cDNA library, we report herein that Snapin is a novel ACVI-interacting protein. Snapin has been found on synaptic vesicle membranes and was originally identified as a novel binding protein of SNAP-25, a plasmalemmal component of SNAREs (soluble N-ethylmaleimide-sensitive factor attached protein receptors) (11Hilfiker S. Greengard P. Augustine G.J. Nat. Neurosci. 1999; 2: 104-106Crossref PubMed Scopus (27) Google Scholar, 12Ilardi J.M. Mochida S. Sheng Z.H. Nat. Neurosci. 1999; 2: 119-124Crossref PubMed Scopus (187) Google Scholar). Assembly of the SNAREs complex is the key step for docking and fusion between vesicles and target membranes. Snapin was found to modulate neurotransmitter release by enhancing the interaction between the SNAREs complex and synaptotagmin, a putative calcium sensor, through direct interaction with SNAP-25 (12Ilardi J.M. Mochida S. Sheng Z.H. Nat. Neurosci. 1999; 2: 119-124Crossref PubMed Scopus (187) Google Scholar). In addition, Snapin is a physiological PKA substrate and can modulate neurotransmitter secretion by the cAMP-dependent pathway (13Chheda M.G. Ashery U. Thakur P. Rettig J. Sheng Z.H. Nat. Cell Biol. 2001; 3: 331-338Crossref PubMed Scopus (137) Google Scholar, 14Shih Y.P. Kung W.M. Chen J.C. Yeh C.H. Wang A.H. Wang Sci. PubMed Scopus Google Scholar). to the expression of Snapin in the we previously demonstrated that ACVI is in many brain regions (10Liu F.C. Wu G.C. Hsieh S.T. Lai H.L. Wang H.F. Wang T.W. Chern Y. FEBS Lett. 1998; 436: 92-98Crossref PubMed Scopus (33) Google Scholar). of ACVI and Snapin shown in the present the of ACVI and Snapin in the brain and in primary hippocampal supporting the physiological of this PKC its ability to ACVI activity in the of These for the a for the cAMP by the in the brain. and amino acids were from PKA, and PKC were from was from and were from and cDNA of ACVI (9Premont R.T. Chen J. Ma H.W. Ponnapalli M. Iyengar R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9809-9813Crossref PubMed Scopus (167) Google was by R. Iyengar of of The the N-terminal domain of ACVI (amino acids N1–160) was by the the and as the bait to screen a mouse brain cDNA library the the The identified cDNA were by and of which the region of Snapin was the of a in of N1–160 or Snapin were by the and the and sites of and the and sites of or the a expression The and were by a as previously 5Lai H.L. Lin T.H. Kao Y.Y. Lin W.J. Hwang M.J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google with as the The were the In and Snapin were in the of the the in binding was by the at for by addition of of the (10Liu F.C. Wu G.C. Hsieh S.T. Lai H.L. Wang H.F. Wang T.W. Chern Y. FEBS Lett. 1998; 436: 92-98Crossref PubMed Scopus (33) Google complex and at for with The were and on PubMed Scopus Google Scholar). of was by the in for were and and were in and were the the interaction between and Snapin or was to and with the N1–160 in of binding and for at The on the were with binding and The of that were to and the of and were by the analysis and respectively. The of the which were and as that an of was used as a negative to the of the binding between and Cell and were from at as G.J. J. Neurosci. Res. PubMed Scopus Google and in with days in were with and and with or for at the of were in for and were for or The M. Res. PubMed Scopus (94) Google was a from J. and was in with bovine serum in and were with the the Y.H. 1994; PubMed Scopus Google or the were for were in with bovine serum and in an with and at were with the at 3 in as and of the to amino acids of mouse was from and to bovine serum m-maleimidobenzoyl-N-hydroxysuccinimide as by Scholar). The was by with the Scholar). The was To the was with in at To the of the was to an as and used for The addition of an of the to an protein the of Snapin in the brain and the of an of the of Snapin, in the of the the was the was by a protein and with at a of the brain the an expression of Snapin as that by the in brain not and of and mutant ACVI were in a the as H.L. Yang T.H. Messing R.O. Ching Y.H. Lin S.C. Chern Y. J. Biol. Chem. 1997; 272: 4970-4977Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). ACVI were by with an as previously H.L. Yang T.H. Messing R.O. Ching Y.H. Lin S.C. Chern Y. J. Biol. Chem. 1997; 272: 4970-4977Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). from with the ACVI were with of and at for and with of the complex or the complex at for with ACVI were with in of binding and at for to complex were on The of that were to membranes and to the expression of ACVI The of the which were and as of ACVI and Snapin was as Lai Lin Wang Lin Chern Y. J. Neurosci. 2003; PubMed Scopus Google with In were with a by with a to the for and the for and with the by with with with ACVI, Snapin, and in primary hippocampal were with bovine serum for with a at and in a to for of ACVI, and Snapin was as the with ACVI, Snapin, and with ACVI and Snapin was as were with in for at by with to of or were with the of a for the of were by of the primary from the AC and cAMP activity was as previously Y. Lai H.L. J.C. Y. Mol. Pharmacol. Google Scholar). The enzyme activity was for to with to of protein. The ACVI activity was as the between cyclase in the from with the ACVI expression and from with the ACVI, cyclase of the The of ACVI activity observed in different might have to the of and of the of AC regulation was among cAMP was as Y. Lai H.L. J.C. Y. Mol. Pharmacol. Google Scholar, G. Res. Google with were with the and to and in the at in each The effect of calcium on ACVI was as D.M. M. Y. M. R. J. 1994; PubMed Scopus Google Scholar). In were with and with for 3 by for with for at in the or of cAMP was by adding of to each with for on The cAMP was the cAMP from of ACVI was as the between cAMP in ACVI an Snapin with the N-terminal of Although the N terminus is variable among different AC is highly conserved between The N terminus therefore might conserved regulatory of ACVI. with members in the AC the N terminus of ACVI with amino acids is large and might its functions through with To for that interact with the N-terminal domain of ACVI (designated N1–160) in the we used N1–160 as the bait to screen a mouse brain cDNA library the library was because of its and because the N of and mouse ACVI are highly homologous in amino of a full-length and a (amino acids of Snapin were that the amino of mouse and Snapin are an interaction was by the identified or the with the N1–160 bait shown in of the Snapin and N1–160 to expression of the the interaction between Snapin and N1–160 in In binding of the N1–160 and Snapin was demonstrated by an Snapin, but not an was with N1–160 interaction between N1–160 and Snapin was because in the of no Snapin was detected in the complex Moreover, full-length ACVI from also Snapin Collectively, demonstrate that ACVI with Snapin through direct interaction at its N-terminal To the Snapin-interacting of we in binding analyses of the Snapin and of the N-terminal domain of ACVI were the in in the of was identified by demonstrated in the amino acids was with the Snapin as were the full-length N terminus and The Snapin-interacting region of N1–160 therefore might in the region amino acids We to on Snapin N1–160 was the in binding as different of mouse Snapin (amino acids and and were in shown in and N1–160 all of the Snapin for the ability to interact with amino acids of Snapin to be crucial for the interaction with addition of a amino acids of Snapin (designated the interaction between Snapin and N1–160 and that the domain of Snapin with N1–160 in the region amino acids of The amino acids of Snapin (designated did not a effect on the binding of Snapin to N1–160 and thus as a in this and Collectively, the region of Snapin is located within amino acids a region different from that which was used to interact with (12Ilardi J.M. Mochida S. Sheng Z.H. Nat. Neurosci. 1999; 2: 119-124Crossref PubMed Scopus (187) Google or P. A. J. 2003; PubMed Scopus Google Scholar). of ACVI and previously demonstrated that in the ACVI is in examined and is present in with a (10Liu F.C. Wu G.C. Hsieh S.T. Lai H.L. Wang H.F. Wang T.W. Chern Y. FEBS Lett. 1998; 436: 92-98Crossref PubMed Scopus (33) Google Scholar). the expression of Snapin in the has not been we to the expression of Snapin in the brain. analysis revealed that to the expression of ACVI in the Snapin was in many brain with different demonstrated that ACVI was with Snapin in the brain and in primary hippocampal neurons of hippocampal neurons by did not the of ACVI and Snapin which be observed in and Consistently, in binding of ACVI and Snapin by be observed in the and of calcium not between ACVI and Snapin therefore to be of the The ACVI and Snapin were also with a In addition to the of of Snapin and ACVI on the of also be observed an interaction between molecules therefore might play an important role in Although adenylyl cyclases in are to in we found that ACVI also be detected in intracellular F.C. Wu G.C. Hsieh S.T. Lai H.L. Wang H.F. Wang T.W. Chern Y. FEBS Lett. 1998; 436: 92-98Crossref PubMed Scopus (33) Google and this in hippocampal of and were found in in addition to membranes G.C. J. J. PubMed Scopus Google Scholar, G.C. S.T. J. Neurosci. 2003; PubMed Google Scholar). of ACs therefore might be an important regulatory role for the AC is of particular the ACVI-interacting Snapin is to with the SNARE complex, which an important role in intracellular is therefore that Snapin might the of ACVI through with the SNARE complex. including that cAMP have been shown to modulate synaptic We previously that ACVI protein is in the brain and may in regulation of the neurotransmitter (10Liu F.C. Wu G.C. Hsieh S.T. Lai H.L. Wang H.F. Wang T.W. Chern Y. FEBS Lett. 1998; 436: 92-98Crossref PubMed Scopus (33) Google Scholar). between ACVI and Snapin as herein the potential of ACVI in synaptic the cAMP-dependent pathway has been in neurotransmitter release G.J. 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Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Res. 2003; PubMed Scopus Google Scholar), the of functions of Snapin has also been a of intracellular not to neurotransmitter release as (12Ilardi J.M. Mochida S. Sheng Z.H. Nat. Neurosci. 1999; 2: 119-124Crossref PubMed Scopus (187) Google Scholar, M.G. Ashery U. Thakur P. Rettig J. Sheng Z.H. Nat. Cell Biol. 2001; 3: 331-338Crossref PubMed Scopus (137) Google Scholar). ACVI, Snapin, is also in many tissues (9Premont R.T. Chen J. Ma H.W. Ponnapalli M. Iyengar R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9809-9813Crossref PubMed Scopus (167) Google Scholar), be of in the to the association of ACVI and Snapin might also in tissues the and functions intracellular might be by a interaction between the cAMP and SNAREs Snapin the of ACVI by AC isozymes, ACVI is because catalytic activity J.P. Harry A. Chen J. Magnusson Iyengar R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and regulatory of this enzyme to are ACVI can be by PKA, PKC, and protein (1Chern Y. Cell Signal. 2000; 12: 195-204Crossref PubMed Scopus (72) Google Scholar, 7Chen Y. Harry A. Li J. Smit M.J. Bai X. Magnusson R. Pieroni J.P. Weng G. Iyengar R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14100-14104Crossref PubMed Scopus (123) Google Scholar, 8Lai H.L. Yang T.H. Messing R.O. Ching Y.H. Lin S.C. Chern Y. J. Biol. Chem. 1997; 272: 4970-4977Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, D.M. M. Y. M. R. J. 1994; PubMed Scopus Google Scholar, P. A. J. 2003; PubMed Scopus Google Scholar, R. W.J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). We Snapin exerted a effect on the properties of ACVI. shown in expression of Snapin did not affect the or of ACVI in We examined interaction with Snapin the regulation of ACVI by PKA Y. Harry A. Li J. Smit M.J. Bai X. Magnusson R. Pieroni J.P. Weng G. Iyengar R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14100-14104Crossref PubMed Scopus (123) Google Scholar). shown in PKA suppressed the activity of ACVI by in of Snapin did not modulate inhibition of ACVI. To Snapin exerted an important role in inhibition of ACVI, we the of Cooper D.M. M. Y. M. R. J. 1994; PubMed Scopus Google Scholar). In expression of Snapin and ACVI were The intracellular calcium were and shown in calcium through by addition of extracellular calcium a in ACVI activity by of Snapin did not affect the inhibition of ACVI by calcium did not affect the of ACVI were with an expression of ACVI an or an expression of Snapin as were from and AC activity by protein or was the of from in a We previously that PKC the catalytic activity of ACVI and that the N-terminal domain of ACVI to this To interaction with Snapin its to PKC-mediated expression of ACVI and the Snapin were from with the expression were with which at least different PKC isoforms including the and the isoforms that ACVI H.L. Yang T.H. Messing R.O. Ching Y.H. Lin S.C. Chern Y. J. Biol. Chem. 1997; 272: 4970-4977Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), and were for the of Snapin the PKC-mediated inhibition of ACVI activity expression of Snapin did not affect PKC Snapin did not its action through Moreover, of the potential PKC phosphorylation on Snapin its expression did not the mutant ability to reverse the inhibitory effect of PKC on ACVI. Snapin is a substrate of PKA (13Chheda M.G. Ashery U. Thakur P. Rettig J. Sheng Z.H. Nat. Cell Biol. 2001; 3: 331-338Crossref PubMed Scopus (137) Google Scholar), we the PKA phosphorylation of Snapin the Snapin mutant was as as the Snapin in PKC-mediated inhibition of ACVI. Phosphorylation of Snapin by PKC or PKA therefore is not crucial for action on ACVI. which harbors an internal deletion of amino acids 33–51 did not affect PKC-mediated inhibition of ACVI, supporting that amino acids 33–51 of Snapin comprises the ACVI-interacting region as in Moreover, expression of Snapin did not affect PKC-mediated inhibition of an ACVI which lacked the Snapin-interacting region (amino acids and 5Lai H.L. Lin T.H. Kao Y.Y. Lin W.J. Hwang M.J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google Scholar). interaction between Snapin and ACVI therefore is crucial for Snapin To the effect of Snapin on PKC-mediated inhibition of ACVI, Snapin to maltose-binding protein (designated was to its ability to reverse the inhibition of ACVI. shown in but not N1–160 in the in binding that the for interaction as that of that a protein in the protein to be a of because was by but not by which was the C terminus amino acids of the interaction between and N1–160 be the of this was to with the action of Snapin the but not the inhibition of ACVI by PKC that Snapin the inhibition of ACVI by We previously that phosphorylation of ACVI by PKC suppression of its activity and to cAMP signaling stimulation of the in H.L. Yang T.H. Messing R.O. Ching Y.H. Lin S.C. Chern Y. J. Biol. Chem. 1997; 272: 4970-4977Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, Y. Lai H.L. J.C. Y. Mol. Pharmacol. Google Scholar, Y. Lai H.L. Mol. Pharmacol. Google Scholar). analyses that the regulatory domain and the catalytic core complex of ACVI are by PKC, and thus to PKC-mediated inhibition (5Lai H.L. Lin T.H. Kao Y.Y. Lin W.J. Hwang M.J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google Scholar, T.H. Lai H.L. Kao Y.Y. Hwang M.J. Chern Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These that the 3 domains of ACVI might form a regulatory complex T.H. Lai H.L. Kao Y.Y. Hwang M.J. Chern Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and that the N terminus might to its of ACVI by PKC to be to its in at extracellular and ACVI to be to PKC-mediated inhibition G.C. Lai H.L. Lin Chern Y. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). we report that the interaction with Snapin ACVI to to PKC-mediated suppression and is that Snapin, by with the N terminus of ACVI, a of ACVI, which is to PKC-mediated Phosphorylation of Snapin by PKC or PKA did not to be important for the ability of Snapin to the effect of PKC on ACVI We were to Snapin in the action of PKC by with PKC-mediated phosphorylation of ACVI, because the interaction between membrane-bound ACVI and Snapin in was not to a step for of ACVI and Snapin from not of protein and protein is a of protein J. J. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, J.C. 1999; PubMed Scopus Google Scholar). demonstrate that the action of Snapin on PKC-mediated regulation of ACVI is is also of to that but not of the ACVI detected in the brain and primary hippocampal neurons was with Snapin and with Snapin therefore ACVI with a highly regulatory for PKC-mediated herein demonstrate that Snapin is a protein of ACVI, and functions as a of ACVI on PKC-mediated inhibition via with its N-terminal association of the cAMP pathway and SNAREs might be in or the of synaptic proteins, which We for and of primary hippocampal and for this We are also to for the and for and for and the at for the and of the with