Pctaire1 Phosphorylates N-Ethylmaleimide-sensitive Fusion Protein

Pctaire1, a member of the cyclin-dependent kinase (Cdk)-related family, has recently been shown to be phosphorylated and regulated by Cdk5/p35. Although Pctaire1 is expressed in both neuronal and non-neuronal cells, its precise functions remain elusive. We performed a yeast two-hybrid screen to identify proteins that interact with Pctaire1. N-Ethylmaleimide-sensitive fusion protein (NSF), a crucial factor in vesicular transport and membrane fusion, was identified as one of the Pctaire1 interacting proteins. We demonstrate that the D2 domain of NSF, which is required for the oligomerization of NSF subunits, binds directly to and is phosphorylated by Pctaire1 on serine 569. Mutation of this phosphorylation site on NSF (S569A) augments its ability to oligomerize. Moreover, inhibition of Pctaire1 activity by transfecting its kinase-dead (KD) mutant into COS-7 cells enhances the self-association of NSF. Interestingly, Pctaire1 associates with NSF and synaptic vesicle-associated proteins in adult rat brain. To investigate whether Pctaire1 phosphorylation of NSF is involved in regulation of Ca2+-dependent exocytosis, we examined the effect of expressing Pctaire1 or NSF phosphorylation mutants on the regulated secretion of growth hormone from PC12 cells. Interestingly, expression of either Pctaire1-KD or NSF-S569A in PC12 cells significantly increases high K+-stimulated growth hormone release. Taken together, our findings provide the first demonstration that Pctaire1 phosphorylation of NSF regulates the ability of NSF to oligomerize, implicating an unexpected role of this kinase in modulating exocytosis. These findings open a new avenue of research in studying the functional roles of Pctaire1 in the nervous system. Pctaire1, a member of the cyclin-dependent kinase (Cdk)-related family, has recently been shown to be phosphorylated and regulated by Cdk5/p35. Although Pctaire1 is expressed in both neuronal and non-neuronal cells, its precise functions remain elusive. We performed a yeast two-hybrid screen to identify proteins that interact with Pctaire1. N-Ethylmaleimide-sensitive fusion protein (NSF), a crucial factor in vesicular transport and membrane fusion, was identified as one of the Pctaire1 interacting proteins. We demonstrate that the D2 domain of NSF, which is required for the oligomerization of NSF subunits, binds directly to and is phosphorylated by Pctaire1 on serine 569. Mutation of this phosphorylation site on NSF (S569A) augments its ability to oligomerize. Moreover, inhibition of Pctaire1 activity by transfecting its kinase-dead (KD) mutant into COS-7 cells enhances the self-association of NSF. Interestingly, Pctaire1 associates with NSF and synaptic vesicle-associated proteins in adult rat brain. To investigate whether Pctaire1 phosphorylation of NSF is involved in regulation of Ca2+-dependent exocytosis, we examined the effect of expressing Pctaire1 or NSF phosphorylation mutants on the regulated secretion of growth hormone from PC12 cells. Interestingly, expression of either Pctaire1-KD or NSF-S569A in PC12 cells significantly increases high K+-stimulated growth hormone release. Taken together, our findings provide the first demonstration that Pctaire1 phosphorylation of NSF regulates the ability of NSF to oligomerize, implicating an unexpected role of this kinase in modulating exocytosis. These findings open a new avenue of research in studying the functional roles of Pctaire1 in the nervous system. Originally identified as a Cdc2-like kinase (1Meyerson M. Enders G.H. Wu C.L. Su L.K. Gorka C. Nelson C. Harlow E. Tsai L.H. EMBO J. 1992; 11: 2909-2917Crossref PubMed Scopus (787) Google Scholar, 2Okuda T. Cleveland J.L. Downing J.R. Oncogene. 1992; 7: 2249-2258PubMed Google Scholar), Pctaire1 is ubiquitously expressed in mammalian tissues and is particularly abundant in terminally differentiated cells such as postmitotic neurons (1Meyerson M. Enders G.H. Wu C.L. Su L.K. Gorka C. Nelson C. Harlow E. Tsai L.H. EMBO J. 1992; 11: 2909-2917Crossref PubMed Scopus (787) Google Scholar, 2Okuda T. Cleveland J.L. Downing J.R. Oncogene. 1992; 7: 2249-2258PubMed Google Scholar, 3Rhee K. Wolgemuth D.J. Dev. Dyn. 1995; 204: 406-420Crossref PubMed Scopus (73) Google Scholar). In contrast to classical Cdks, 2The abbreviations used are: Cdk, cyclin-dependent kinase; NSF, N-ethylmaleimide-sensitive fusion protein; SNAP, soluble NSF attachment protein; SNARE, soluble NSF attachment protein receptor; hGH, human growth hormone; HA, hemagglutinin; GST, glutathione S-transferase; WT, wild type; ATPγS, adenosine 5′-O-(thiotriphosphate); KD, kinase-dead. which depend on cyclins for activation, Pctaire1 is not activated by cyclins. We have reported previously that Pctaire1 interacts with the Cdk5 activator p35, and phosphorylation by Cdk5/p35 enhances the kinase activity of Pctaire1 (4Cheng K. Li Z. Fu W.Y. Wang J.H. Fu A.K. Ip N.Y. J. Biol. Chem. 2002; 277: 31988-31993Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). An alternative regulatory mechanism to inhibit the activity of Pctaire1 through protein kinase A has also been proposed (5Graeser R. Gannon J. Poon R.Y. Dubois T. Aitken A. Hunt T. J. Cell Sci. 2002; 115: 3479-3490Crossref PubMed Google Scholar). Similar to Cdk5, Pctaire1 is not involved in the regulation of cell cycle progression (1Meyerson M. Enders G.H. Wu C.L. Su L.K. Gorka C. Nelson C. Harlow E. Tsai L.H. EMBO J. 1992; 11: 2909-2917Crossref PubMed Scopus (787) Google Scholar, 3Rhee K. Wolgemuth D.J. Dev. Dyn. 1995; 204: 406-420Crossref PubMed Scopus (73) Google Scholar). To date, the precise functions of Pctaire1 remain elusive. Recent studies indicate that Pctaire1 regulates neurite outgrowth in Neuro-2A cells, whereby overexpression of the kinase-dead (KD) mutant of Pctaire1 leads to enhanced neurite outgrowth (5Graeser R. Gannon J. Poon R.Y. Dubois T. Aitken A. Hunt T. J. Cell Sci. 2002; 115: 3479-3490Crossref PubMed Google Scholar). As a first step to explore the functional roles of Pctaire1 in the nervous system, we have performed a yeast two-hybrid screen to identify proteins that interact with Pctaire1 in adult brain. Our study reveals that Pctaire1 interacts with N-ethylmaleimide-sensitive fusion protein (NSF), a cytosolic protein that is essential for vesicular transport and fusion of synaptic vesicles with presynaptic membrane (6Block M.R. Glick B.S. Wilcox C.A. Wieland F.T. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7852-7856Crossref PubMed Scopus (401) Google Scholar, 7Beckers C.J. Block M.R. Glick B.S. Rothman J.E. Balch W.E. Nature. 1989; 339: 397-398Crossref PubMed Scopus (188) Google Scholar). NSF is recruited to membrane through adaptor proteins known as soluble NSF attachment proteins (SNAPs), which bind to membrane-bound SNAP receptors (SNAREs). The hexameric NSF acts as a molecular chaperone to hydrolyze ATP and alters the conformation of SNARE complex, disassembles it, and recycles SNARE monomers for subsequent membrane fusion (8Morgan A. Burgoyne R.D. Curr. Biol. 2004; 14: 968-970Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Each NSF monomer is comprised of three domains: an N-terminal domain (amino acids 1–205) that is responsible for the interaction with α-SNAP and SNAREs; and two homologous ATP-binding domains, D1 (amino acids 206–477), in which the hydrolytic activity is associated with NSF-driven SNARE complex disassembly, and D2 (amino acids 478–744), which is believed to be responsible for hexamer formation (9Yu R.C. Hanson P.I. Jahn R. Brunger A.T. Nat. Struct. Biol. 1998; 5: 803-811Crossref PubMed Scopus (226) Google Scholar, 10Whiteheart S.W. Schraw T. Matveeva E.A. Int. Rev. Cytol. 2001; 207: 71-112Crossref PubMed Scopus (101) Google Scholar). The activity of NSF is tightly regulated by post-translational modifications to ensure its proper function during membrane fusion. In this study, we provide evidence that NSF interacts with Pctaire1 and is phosphorylated by Pctaire1. Phosphorylation of NSF by Pctaire1 on serine 569 attenuates the hexamerization of NSF. The association of Pctaire1 and NSF with components of the molecular machinery required for transmitter release prompted us to explore whether the phosphorylation of NSF by Pctaire1 plays a role in regulating exocytosis. Ca2+-dependent exocytosis of dense core vesicles in PC12 cells has been reported to exhibit properties similar to that of synaptic vesicle release, allowing PC12 cells to be widely adopted for studies on regulated exocytosis (11Khvotchev M.V. Ren M. Takamori S. Jahn R. Sudhof T.C. J. Neurosci. 2003; 23: 10531-10539Crossref PubMed Google Scholar, 12Sugita S. Janz R. Sudhof T.C. J. Biol. Chem. 1999; 274: 18893-18901Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Our results with the human growth hormone (hGH) release assay in PC12 cells suggest that the serine phosphorylation of NSF by Pctaire1 plays an important role in regulating the Ca2+-dependent exocytosis. Taken together, the identification of NSF as an interacting protein and substrate for Pctaire1 provides critical insights into how the regulation of NSF phosphorylation by Pctaire1 might impact on its oligomerization and modulate the process of transmitter release. Plasmids and Antibodies—Complementary DNA (cDNA) encoding mouse Pctaire1, Cdk5, and p35 was subcloned into the vector, pAS2-1 (Clontech). Full-length, partial cDNA fragments and point mutations (e.g. serine 569 to alanine (S569A)) of mouse NSF were amplified by PCR and subcloned into different expression vectors including pGEX-6P-2, pETH-32, pcDNA3-His6, and pcDNA3-3HA. pXGH5 containing the hGH gene was a gift from Prof. Masami Takahashi (Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan), and synaptotagmin antibody was generously provided by Prof. Benjamin Peng (The Hong Kong University of Science and Technology). Pctaire1 antibody was raised against the peptide GEAPTRVAPGELRSIR, and NSF antibody was purchased from Calbiochem. Antibodies specific for synapsin I and synaptophysin were purchased from Cell Signaling Technology (Beverly, MA) and Chemicon (Temecula, CA), respectively. Antibodies against SV-2 and α-tubulin were from Sigma, whereas antibodies specific for HA, His, and syntaxin1 (HPC-1) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-Ser/Thr-Pro (MPM-2) antibody was from Upstate Biotechnology (Lake Placid, NY). Yeast Two-hybrid Screen—Yeast two-hybrid screen was performed following the Matchmaker two-hybrid screen protocol (Clontech). The NSF clone isolated from the two-hybrid screen encoded the protein that lacked the five amino acids at the N terminus. Subsequent two-hybrid interaction analyses were carried out by co-transformation of plasmids encoding the GAL4 DNA-binding (pAS2-1) and activation (pACT2) domains into yeast. An o-nitrophenyl-β-d-galactopyranoside-based β-galactosidase activity assay was performed according to the manufacturer's protocol (Clontech). Cell Culture and Transfection—COS-7 and HEK 293T cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum plus antibiotics. PC12 cells were cultured in Dulbecco's modified Eagle's medium supplemented with heat-inactivated horse serum (6%, v/v), heat-inactivated fetal bovine serum (6%, v/v) plus antibiotics and maintained at 37 °C in a humidified atmosphere with 7.5% CO2. Transient transfection was carried out using Lipofectamine PLUS reagents (Invitrogen) according to the manufacturer's instructions. Protein Purification, Protein Extraction, Western Blot, Immunoprecipitation, and in Vitro Phosphorylation Analysis—The recombinant GST-Pctaire1 and His6-tagged NSF and its mutants were purified following the instructions of the manufacturer (Amersham Biosciences) and by nickel-nitrilotriacetic acid-agarose affinity chromatography (Qiagen) (13Nagiec E.E. Bernstein A. Whiteheart S.W. J. Biol. Chem. 1995; 270: 29182-29188Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), respectively. Protein extracts from cells and brain tissues were prepared using different lysis buffers supplemented with various protease inhibitors. COS-7 cells were lysed in lysis buffer A (in mm: Tris, pH 7.6, 20; NaCl, with tissues were in lysis buffer (in mm: Tris, pH NaCl, with HEK 293T cells were lysed in lysis buffer (in mm: Tris, pH NaCl, 20; with of adult rat brain were prepared as previously J. Cell Biol. PubMed Scopus Google Scholar, M. R. Peng J. J. M. Tsai L.H. Full Text Full Text PDF PubMed Scopus Google Scholar). of COS-7 cell were with the antibody at °C and with of protein at °C for The were with buffer A and in and proteins were using Western In studies using brain were performed as previously (4Cheng K. Li Z. Fu W.Y. Wang J.H. Fu A.K. Ip N.Y. J. Biol. Chem. 2002; 277: 31988-31993Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, T. Whiteheart S.W. M. S. Rothman J.E. Nature. PubMed Scopus Google Scholar, S. S. Hanson P.I. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Hong J. PubMed Scopus Google Scholar). Pctaire1 was from adult rat brain with Pctaire1 antibody in assay buffer and five with assay The kinase assay was performed at °C for in kinase buffer as previously A.K. Fu W.Y. J. Ip Wang J.H. Ip N.Y. Nat. Neurosci. 2001; PubMed Scopus Google Scholar). of recombinant proteins were as the in the kinase The were on and by and in Vitro chromatography was performed using a (Amersham Biosciences) at °C with a of The was first with at pH NaCl, and various protease inhibitors. The assay was carried out as previously (4Cheng K. Li Z. Fu W.Y. Wang J.H. Fu A.K. Ip N.Y. J. Biol. Chem. 2002; 277: 31988-31993Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). adult brain membrane was prepared as previously K. Ip N.Y. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), and the was performed in supplemented with bovine serum and in with and HEK 293T cells were with Pctaire1 or Cdk5, and cell were using which was on at °C for The were with of adult rat brain at °C for and the were with buffer containing The were in with Pctaire1 and Cdk5 were by Western hGH in PC12 release in PC12 cells were carried out at 37 °C as previously R. M. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. K. K. S. Takahashi M. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). PC12 cells were with a and maintained in the for To the or high hGH the cells were either in the or in the high for The medium was and the of hGH was using the hGH assay was used for and the results as NSF with clone encoding NSF was identified to interact with Pctaire1 on the yeast two-hybrid screen of an adult brain The cDNA clone NSF that the first five amino acids at the N terminus. The NSF cDNA was by and the interaction of NSF with Pctaire1 in yeast was with the β-galactosidase activity that the interaction of Pctaire1 with NSF was that with p35, which was reported previously to with Pctaire1 K. Li Z. Fu W.Y. Wang J.H. Fu A.K. Ip N.Y. J. Biol. Chem. 2002; 277: 31988-31993Abstract Full Text Full Text PDF PubMed Scopus (51) Google the NSF to interact with Cdk5 and p35 in yeast. We the interaction NSF and Pctaire1 using in GST-Pctaire1 protein was on and with recombinant to GST-Pctaire1 not the To the specific of NSF that with Pctaire1, recombinant proteins encoding various domains of NSF were and ability to interact with Pctaire1 was using Our results that the D2 domain of NSF with Pctaire1 The interaction Pctaire1 and NSF was examined in COS-7 cells that were with Pctaire1 and NSF cDNA We that NSF or Pctaire1 antibody NSF and Pctaire1 from the of COS-7 cells both cDNA Pctaire1 be from brain using the and NSF was by the antibody against Pctaire1 the in interaction Pctaire1 and NSF was in adult rat brain. has been reported that the interaction of NSF with of its proteins is on the of NSF T. Whiteheart S.W. M. S. Rothman J.E. Nature. PubMed Scopus Google Scholar, S. S. Hanson P.I. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Hong J. PubMed Scopus Google Scholar). To whether the interaction NSF and Pctaire1 is on the the was carried out different in the of or The interaction of Pctaire1 and NSF three Phosphorylation of NSF by Pctaire1 Pctaire1 is a kinase (1Meyerson M. Enders G.H. Wu C.L. Su L.K. Gorka C. Nelson C. Harlow E. Tsai L.H. EMBO J. 1992; 11: 2909-2917Crossref PubMed Scopus (787) Google Scholar), we that Pctaire1 might a phosphorylation similar to that of were identified in NSF, serine and serine 569. We that Pctaire1 the D2 not the N-terminal or D1 of NSF in a A and These with the of serine to the N-terminal suggest that serine 569 the D2 domain is a phosphorylation site for Pctaire1. As of serine 569 to alanine (S569A) the phosphorylation of D2 domain by Pctaire1 The of Cdk, Cdk5, to NSF the of Pctaire1 phosphorylation on NSF The ability of Pctaire1 to NSF was by in phosphorylation assay using recombinant His6-tagged NSF protein purified from Although the wild recombinant NSF which was shown previously to as a hexamer R.C. A. Brunger A.T. Hanson P.I. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus (79) Google Scholar), be phosphorylated by Pctaire1, the phosphorylation was not with the NSF-S569A mutant protein To provide evidence that NSF is phosphorylated by Pctaire1 in NSF was with either or its kinase-dead mutant by the involved in the process of phosphorylation S. J. K. S. Cell 1999; Google into HEK 293T cells. NSF protein was and its phosphorylation was examined by Western using Phosphorylation of NSF was in cells that were with not with The D2 domain of NSF that directly with Pctaire1 has been reported to be required for the hexamerization of NSF Whiteheart S.W. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). we whether the interaction of with Pctaire1 phosphorylation by Pctaire1 the oligomerization of NSF. NSF and D2 domain of NSF were into COS-7 cells with or Cell were using antibody and with In the of was an of NSF protein in the complex with in the of a of NSF was in the complex that of Pctaire1 activity is to the oligomerization of NSF. We examined the that phosphorylation on serine 569 of NSF its ability to oligomerize. phosphorylation mutants of NSF, the mutant to and the mutant to were COS-7 cells were with and or NSF. Cell were using antibody and with As the antibody the NSF The oligomerization of NSF-S569A whereas that of with wild NSF These results suggest that phosphorylation of NSF by Pctaire1 on serine 569 its Similar results on the regulation of NSF oligomerization were the mutants were expressed with the wild that the self-association of NSF was in cells with and similar to the results with Interestingly, the oligomerization of NSF was not the cells were with both and that the effect of NSF-S569A mutant is with that of Taken together, findings suggest that the oligomerization of NSF might be regulated by the phosphorylation of NSF on serine 569. To whether the phosphorylation of NSF on serine 569 is critical in regulating the hexamerization of NSF, purified protein and its phosphorylation mutants or were to chromatography by and Western for NSF. The molecular of hexameric NSF is We that in the of both and were in to a molecular Interestingly, the of NSF was in subsequent to a molecular that a at 569 might the of the hexameric NSF. of Pctaire1 to the interaction Pctaire1 and NSF, we examined the of proteins in brain. Pctaire1 as as NSF was shown to be in membrane and synaptic similar to that of the such as or synaptic vesicle-associated proteins including synapsin I and synaptophysin The of Pctaire1 and NSF with proteins to the specific that Pctaire1 might the of vesicles through the phosphorylation of NSF. To explore the of Pctaire1 in synaptic vesicle we performed a and that Pctaire1 with a of synaptic vesicle-associated proteins in components of such as syntaxin1 and be from rat brain membrane by Pctaire1 I and p35 Cdk5 which was previously to interact with Pctaire1 in also be by Pctaire1 (4Cheng K. Li Z. Fu W.Y. Wang J.H. Fu A.K. Ip N.Y. J. Biol. Chem. 2002; 277: 31988-31993Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The were specific synaptic vesicle proteins such as synaptophysin as as not be in the interaction Pctaire1 and syntaxin1 was not in adult brain by in not is that Pctaire1 interacts directly with NSF and with of the SNARE proteins. with our that Pctaire1 activity be regulated by a of the synaptic proteins that interact with Pctaire1 also be by These results with the that Pctaire1 is involved in membrane of NSF Phosphorylation by Pctaire1 on Ca2+-dependent plays a critical role in the process of exocytosis. to the the hexameric NSF from ATP to SNARE membrane fusion, and vesicle-associated membrane protein and allowing the SNARE proteins to be for subsequent of fusion R. Sudhof T.C. Rev. 1999; PubMed Scopus Google Scholar). is that Pctaire1 is a of the complex and plays a regulatory role in Ca2+-dependent exocytosis through the phosphorylation of NSF. We examined the effect of Pctaire1 and NSF mutant on vesicle fusion using the hGH release PC12 cells were with pXGH5 encoding hGH and different expression In PC12 cells, hGH is in dense core and high cells Ca2+-dependent exocytosis. The release of dense core vesicles in PC12 cells and was by the of hGH to and high respectively. As shown in A and inhibition of Pctaire1 activity by overexpression of Pctaire1-KD the hGH secretion significantly in both and high K+-stimulated Moreover, expression of NSF-S569A also significantly the hGH release in both and high with that of or the and is to that whereas overexpression of significantly hGH release A and overexpression of effect on hGH secretion and that Pctaire1 also hGH release a in hGH secretion following overexpression of both Pctaire1-KD and NSF-S569A that phosphorylation of NSF at one of the by which Pctaire1 regulates hGH the of membrane fusion, the activity of NSF be regulated by different including the by and of ATP and association with complex, as as oligomerization and phosphorylation E.A. Whiteheart S.W. T.C. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, K. M. C. M.R. J. C.J. 2003; 115: Full Text Full Text PDF PubMed Scopus Google Scholar, Block M.R. Rothman J.E. J. Cell Biol. 1989; PubMed Scopus Google Scholar, S.W. K. M. R. Rothman J.E. J. Cell Biol. PubMed Scopus Google Scholar, S.W. Matveeva E.A. J. Struct. Biol. 2004; PubMed Scopus Google Scholar, S. K. S. E. Wang J. M. E. T. Nat. Cell Biol. 2004; PubMed Scopus Google Scholar). has been reported that phosphorylation of NSF its activity as as its affinity for α-SNAP S. K. S. E. Wang J. M. E. T. Nat. Cell Biol. 2004; PubMed Scopus Google Scholar). phosphorylation of NSF by a protein kinase regulates the function of NSF. Protein kinase NSF on serine in the D1 domain in of serine to attenuates the of NSF to E.A. Whiteheart S.W. T.C. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In this study, we provide evidence that NSF also be phosphorylated and regulated by Pctaire1. phosphorylation of the D2 domain by Pctaire1 on serine 569 the oligomerization of NSF, inhibition of Pctaire1 activity enhances the ability of NSF to on the of NSF, serine 569 is to be on the of NSF monomers in the hexameric complex (9Yu R.C. Hanson P.I. Jahn R. Brunger A.T. Nat. Struct. Biol. 1998; 5: 803-811Crossref PubMed Scopus (226) Google Scholar). is to that phosphorylation of in that the self-association of NSF. Although the precise effect of this phosphorylation on the conformation of NSF to be findings from this study suggest that Pctaire1 the oligomerization of NSF through the phosphorylation of the D2 Pctaire1 activity be regulated by which is known to a role in through the phosphorylation of various synaptic proteins R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. K. T. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. S. M. T. T. M. Li K. S. K. K. J. Cell Biol. 2003; PubMed Scopus Google Scholar, M.R. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). Although Cdk5 not directly interact with or NSF in not Cdk5/p35 associates with Pctaire1 and regulates its Our findings in the study that Pctaire1 not interacts with NSF in adult rat brain also with a of synaptic proteins including components of the SNARE be to whether Pctaire1 and Cdk5 as components of a protein complex and an important role in a of protein to ensure proper transmitter release from synaptic The study reveals a role of Pctaire1 phosphorylation in modulating exocytosis. Recent studies of NSF function in regulated secretion suggest that NSF to synaptic vesicles and the SNARE complex for fusion is required to the of synaptic vesicles J. Neurosci. 1998; PubMed Google Scholar, T. 1998; PubMed Scopus Google Scholar, A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), whereas regulation of NSF phosphorylation by Pctaire1 might modulate the regulated studies on Pctaire1 functions in release might on its roles in neurons during We Masami Takahashi for the pXGH5 We also Nelson and for Prof. Prof. and and of the Ip for