Phosphorylation of 69-kDa Choline Acetyltransferase at Threonine 456 in Response to Amyloid-β Peptide 1–42

Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons. In the brain, these neurons are especially vulnerable to effects of β-amyloid (Aβ) peptides. Choline acetyltransferase is a substrate for several protein kinases. In the present study, we demonstrate that short term exposure of IMR32 neuroblastoma cells expressing human choline acetyltransferase to Aβ-(1–42) changes phosphorylation of the enzyme, resulting in increased activity and alterations in its interaction with other cellular proteins. Using mass spectrometry, we identified threonine 456 as a new phosphorylation site in choline acetyltransferase from Aβ-(1–42)-treated cells and in purified recombinant ChAT phosphorylated in vitro by calcium/calmodulin-dependent protein kinase II (CaM kinase II). Whereas phosphorylation of choline acetyltransferase by protein kinase C alone caused a 2-fold increase in enzyme activity, phosphorylation by CaM kinase II alone did not alter enzyme activity. A 3-fold increase in choline acetyltransferase activity was found with coordinate phosphorylation of threonine 456 by CaM kinase II and phosphorylation of serine 440 by protein kinase C. This phosphorylation combination was observed in choline acetyltransferase from Aβ-(1–42)-treated cells. Treatment of cells with Aβ-(1–42) resulted in two phases of activation of choline acetyltransferase, the first within 30 min and associated with phosphorylation by protein kinase C and the second by 10 h and associated with phosphorylation by both CaM kinase II and protein kinase C. We also show that choline acetyltransferase from Aβ-(1–42)-treated cells co-immunoprecipitates with valosin-containing protein, and mutation of threonine 456 to alanine abolished the Aβ-(1–42)-induced effects. These studies demonstrate that Aβ-(1–42) can acutely regulate the function of choline acetyltransferase, thus potentially altering cholinergic neurotransmission. Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons. In the brain, these neurons are especially vulnerable to effects of β-amyloid (Aβ) peptides. Choline acetyltransferase is a substrate for several protein kinases. In the present study, we demonstrate that short term exposure of IMR32 neuroblastoma cells expressing human choline acetyltransferase to Aβ-(1–42) changes phosphorylation of the enzyme, resulting in increased activity and alterations in its interaction with other cellular proteins. Using mass spectrometry, we identified threonine 456 as a new phosphorylation site in choline acetyltransferase from Aβ-(1–42)-treated cells and in purified recombinant ChAT phosphorylated in vitro by calcium/calmodulin-dependent protein kinase II (CaM kinase II). Whereas phosphorylation of choline acetyltransferase by protein kinase C alone caused a 2-fold increase in enzyme activity, phosphorylation by CaM kinase II alone did not alter enzyme activity. A 3-fold increase in choline acetyltransferase activity was found with coordinate phosphorylation of threonine 456 by CaM kinase II and phosphorylation of serine 440 by protein kinase C. This phosphorylation combination was observed in choline acetyltransferase from Aβ-(1–42)-treated cells. Treatment of cells with Aβ-(1–42) resulted in two phases of activation of choline acetyltransferase, the first within 30 min and associated with phosphorylation by protein kinase C and the second by 10 h and associated with phosphorylation by both CaM kinase II and protein kinase C. We also show that choline acetyltransferase from Aβ-(1–42)-treated cells co-immunoprecipitates with valosin-containing protein, and mutation of threonine 456 to alanine abolished the Aβ-(1–42)-induced effects. These studies demonstrate that Aβ-(1–42) can acutely regulate the function of choline acetyltransferase, thus potentially altering cholinergic neurotransmission. Cholinergic neurons in brain are especially vulnerable to effects of both soluble/oligomeric and deposited/fibrillar forms of β-amyloid (Aβ) 1The abbreviations used are: Aβ, β-amyloid; ACh, acetylcholine; APP, amyloid precursor protein; ChAT, choline acetyltransferase; CTab, anti-ChAT carboxyl-terminal peptide antibody; CaM kinase II, calcium/calmodulin-dependent protein kinase II; ESI, electrospray ionization; MS, mass spectrometry; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; PKC, protein kinase C; VCP, valosin-containing protein peptides released from amyloid precursor protein (APP). Shifts in production of soluble APPα by α-secretase to production of Aβ-(1–40) and Aβ-(1–42) with activation of β- and γ-secretase in Alzheimer's disease and following traumatic head injury are associated with decreased function and communication by cholinergic neurons (1Selkoe D.J. J. Am. Med. Assoc. 2000; 283: 1615-1617Google Scholar, 2Emmerling M.R. Morganti-Kossmann M.C. Kossmann T. Stahel P.F. Watson M.D. Evans L.M. Mehta P.D. Spiegel K. Kuo Y.M. Roher A.E. Raby C.A. Ann. N. Y. Acad. Sci. 2000; 903: 118-122Google Scholar, 3Uryu K. Laurer H. McIntosh T. Pratico D. Martinez D. Leight S. Lee V.M.Y. Trojanowski J.Q. J. Neurosci. 2002; 22: 446-454Google Scholar). A complex relationship exists between cholinergic neuron function and APP processing and Aβ peptide production (4Auld D.S. Kar S. Quirion R. Trends Neurosci. 1998; 21: 43-49Google Scholar, 5Rossner S. Ueberham U. Schliebs R. Perez-Polo J.R. Bigl V. Prog. Neurobiol. 1998; 56: 541-569Google Scholar, 6Ehrenstein G. Galdzicki Z. Lange G.D. Ann. N. Y. Acad. Sci. 2000; 899: 283-291Google Scholar). Short term exposure to low (picomolar or nanomolar) concentrations of soluble/oligomeric Aβ peptide leads to presynaptic cholinergic dysfunction with a reduction in the availability of acetylcholine (ACh) precursors choline (7Kar S. Issa A. Seto D. Auld D.S. Collier B. Quirion R. J. Neurochem. 1998; 70: 2179-2187Google Scholar) and acetyl-coenzyme A (8Hoshi M. Takashima A. Noguchi K. Murayama M. Sato M. Kondo S. Saitoh Y. Ishiguro K. Hoshino T. Imahori K. Proc. Nat. Acad. Sci. 1996; 93: 2719-2723Google Scholar, 9Hoshi M. Takashima A. Murayama M. Yasutake K. Yoshida N. Ishiguro K. Hoshino T. Imahori K. J. Biol. Chem. 1997; 272: 2038-2041Google Scholar) coupled to decreased ACh synthesis and release from hippocampal slices or neuronal cultures (9Hoshi M. Takashima A. Murayama M. Yasutake K. Yoshida N. Ishiguro K. Hoshino T. Imahori K. J. Biol. Chem. 1997; 272: 2038-2041Google Scholar, 10Pedersen W.A. Kloczewiak M.A. Blusztajn J.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8068-8071Google Scholar, 11Kar S. Seto D. Gaudreau P. Quirion R. J. Neurosci. 1996; 16: 1034-1040Google Scholar, 12Pedersen W.A. Blusztajn J.K. Neurosci. Lett. 1997; 239: 77-80Google Scholar, 13Satoh Y. Hirakura Y. Shibayama S. Hirashima N. Suzuki T. Kirino Y. Neurosci. Lett. 2001; 302: 97-100Google Scholar). These acute effects of Aβ peptides on neurotransmission and synaptic efficacy probably differ from the neurotoxicity produced by long term exposure and high (micromolar) concentrations of the peptides that cause death of cholinergic neurons. Mechanisms underlying acute and long term effects of Aβ peptides on cholinergic function have not been resolved. Choline acetyltransferase (ChAT; EC 2.3.1.6) produces the neurotransmitter ACh in cholinergic neurons. ChAT undergoes covalent modification post-translationally by protein kinase-mediated phosphorylation (14Bruce G. Hersh L.B. Neurochem. Res. 1989; 14: 613-620Google Scholar, 15Schmidt B.M. Rylett R.J. J. Neurochem. 1993; 61: 1774-1781Google Scholar, 16Habert E. Birman S. Mallet J. J. Neurochem. 1992; 58: 1447-1453Google Scholar, 17Dobransky T. Davis W.L. Xiao G.H. Rylett R.J. Biochem. J. 2000; 349: 141-151Google Scholar, 18Dobransky T. Davis W.L. Rylett R.J. J. Biol. Chem. 2001; 276: 22244-22250Google Scholar), and we showed previously that it is a substrate for a number of protein kinases (17Dobransky T. Davis W.L. Xiao G.H. Rylett R.J. Biochem. J. 2000; 349: 141-151Google Scholar). Catalytic activity of this enzyme, its subcellular distribution, and potentially its interaction with other cellular proteins can be regulated in a phosphorylation-dependent manner. For example, phosphorylation of ChAT by protein kinase C (PKC) on Serine 440 led to a significant increase in its activity and ionic binding to plasma membrane in cells (18Dobransky T. Davis W.L. Rylett R.J. J. Biol. Chem. 2001; 276: 22244-22250Google Scholar). Phosphorylation of ChAT could be by changes in activity or subcellular of protein kinases by neuronal or as Alzheimer's disease and traumatic brain This could alter ACh and cholinergic neurotransmission and cause dysfunction of cholinergic neurons. Aβ peptides a of cellular and protein kinases J. Neurosci. Scholar, T. M. Neurobiol. 2001; 22: Scholar, M. D. K. J. Neurosci. 2001; 21: Scholar). Whereas a number of for Aβ peptides have been identified T. M. Neurobiol. 2001; 22: Scholar, M. D. K. J. Neurosci. 2001; 21: Scholar, M.C. J. Biol. Chem. 2001; 276: Scholar, Z. J. Neurosci. 2001; 21: Scholar), it is these peptides cellular acutely or in the is that Aβ peptides can alter to increased J. Neurosci. 2001; Scholar, A. M. M. Z. J. Biol. Chem. 2000; Scholar). a this could a number of protein kinases as and protein kinase II (CaM kinase II). ChAT is to be a substrate for both of these protein kinases (17Dobransky T. Davis W.L. Xiao G.H. Rylett R.J. Biochem. J. 2000; 349: 141-151Google Scholar), it is that Aβ peptides could cholinergic neurotransmission of function of this In the present study, we the that short term exposure of IMR32 neuroblastoma cells expressing human ChAT to Aβ peptides to function of the we observed that of cells with not the of phosphorylation of ChAT, a new CaM kinase II phosphorylation phosphorylation this with phosphorylation of by PKC, leads to a activation of ChAT and phosphorylation-dependent of the enzyme with other cellular valosin-containing protein for human ChAT in was by H. for was by of in ChAT the with the and the of the mutation was the by and the protein by was previously (18Dobransky T. Davis W.L. Rylett R.J. J. Biol. Chem. 2001; 276: 22244-22250Google Scholar). neuroblastoma IMR32 cells with human ChAT or or in and for ChAT enzyme activity by and ChAT protein by in and in For of cells h was to and Aβ peptides or or peptides or used as in to concentrations of and to cells for to For protein kinase of CaM kinase II protein kinase 10 or to the h the of Aβ peptides. Aβ peptides in and or and and for A. Proc. Natl. Acad. Sci. U. S. A. For protein phosphorylation studies in was to h the of the with Aβ in the of and with Aβ peptides the of the 10 and was to cells and for 30 min on for 10 and used for of activity or phosphorylation of ChAT or for of proteins that with ChAT activity was a modification of the of Biochem. J. Scholar), as previously R.J. S. A. J. Neurochem. 1993; 61: Scholar). of and the peptides on a in a the a of in and or and of Aβ-(1–40) and Aβ-(1–42) peptide by on a of to peptides to and on or and and by in a with anti-ChAT (17Dobransky T. Davis W.L. Xiao G.H. Rylett R.J. Biochem. J. 2000; 349: 141-151Google Scholar) of or of for h on was from of M. J. Biol. Chem. Scholar). for h and with was to the and by for for proteins by on Scholar). from to in a For of ChAT or VCP, with in and with anti-ChAT or for h with and by for h with and the recombinant human ChAT was as previously (18Dobransky T. Davis W.L. Rylett R.J. J. Biol. Chem. 2001; 276: 22244-22250Google Scholar). phosphorylated by CaM kinase II, of purified ChAT protein was with of kinase and of purified CaM kinase II from H. for 30 min 30 Phosphorylation by the of effects of phosphorylation of ChAT by and CaM kinase II on ChAT activity, a phosphorylation was for min 30 as previously (17Dobransky T. Davis W.L. Xiao G.H. Rylett R.J. Biochem. J. 2000; 349: 141-151Google Scholar), by phosphorylation by CaM kinase II for ChAT activity was IMR32 cells expressing human ChAT used to CaM kinase II is by with and peptides and the of and J. Biol. Chem. 1989; Scholar). CaM kinase peptide was used as a substrate to CaM kinase II activity in cells. in in the or of Aβ peptides and with with of 10 with CaM kinase substrate peptide was to cells for 10 min 30 A of cells in the substrate peptide to a of a of CaM kinase II, or its was as was by the of 10 of and of cellular was R. Scholar). with and with and and in for of in and as by Biochem. Scholar) to activity of peptide phosphorylation as proteins with and and for h with changes to of with changes of and to ChAT or proteins from and with two changes of slices with 10 for 30 min and by for by with two changes of by and in A. M. M. Chem. 1996; Scholar). a on was of was and for h peptides from the with two changes of of by in for a the with peptides and by to a of with a of of ChAT as previously P. J. Biol. Chem. Scholar). of to by of and in the first in for and in the second in by for from with M. R. J. Biol. Chem. Scholar) and to in a and in and This of peptides was used for mass For peptides purified on to the and from the with and was also on from or on of peptides in of and for or as by P. T. Scholar). on a mass with a was with 1The abbreviations used are: Aβ, β-amyloid; ACh, acetylcholine; APP, amyloid precursor protein; ChAT, choline acetyltransferase; CTab, anti-ChAT carboxyl-terminal peptide antibody; CaM kinase II, calcium/calmodulin-dependent protein kinase II; ESI, electrospray ionization; MS, mass spectrometry; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; PKC, protein kinase C; VCP, valosin-containing and following on a the protein was a A between and was to the in to was the of of and with a of V. that resulted in to and in the of the peptides was also in the of of ChAT from and cells or for of proteins that with ChAT by mass on a mass with a and In of was from the was with 1The abbreviations used are: Aβ, β-amyloid; ACh, acetylcholine; APP, amyloid precursor protein; ChAT, choline acetyltransferase; CTab, anti-ChAT carboxyl-terminal peptide antibody; CaM kinase II, calcium/calmodulin-dependent protein kinase II; ESI, electrospray ionization; MS, mass spectrometry; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; PKC, protein kinase C; VCP, valosin-containing A of in min with was used to peptides from a to the mass in of or precursor for by the mass In identified for was by with in the was on the of the and in the of the peptides was also in the with ChAT identified by mass with with was the in was on a mass was substrate for a In for the was used as with the peptide mass to the by the following on the and mass was to and two with the to human proteins. of Aβ peptide used to cells in the present studies by and to that to be peptide in that the of Aβ-(1–40) and Aβ-(1–42) in that both of by on the of the or was present in these two peptides. on the of Aβ-(1–40) and Aβ-(1–42) used and 30 the peptides and a In of the as in that of both Aβ-(1–40) and Aβ-(1–42) IMR32 cells expressing human ChAT with Aβ-(1–42) for and of and enzyme activity in activity of ChAT was increased within 30 min of the of Aβ-(1–42) to with this 10 h increase in of Aβ-(1–42) on ChAT activity a with the 10 in C demonstrate that cellular ChAT was the Phosphorylation of ChAT was also increased to 3-fold by of cells with Aβ-(1–42) in a that the for in activity of the enzyme, as in the are in cells with peptide ChAT activity in cells with the of did not differ from cells with Aβ peptide ChAT activity was not in cells for to h with Aβ-(1–40) or its peptide not a 2-fold increase in ChAT was found in IMR32 cells with Aβ-(1–40) between and h not IMR32 cells expressing ChAT with Aβ peptides for 10 and ChAT was by and to In cells and cells with Aβ-(1–40) and the peptides and a was as in A. In of cells with Aβ-(1–42) resulted in the of a second phosphorylated ChAT of these following showed that phosphorylated serine phosphorylated ChAT from cells with Aβ-(1–42) as as the threonine phosphorylated in ChAT with Aβ-(1–42) ChAT was from IMR32 cells 10 h of with Aβ peptides. by to and for mass by a present in ChAT from cells with Aβ-(1–42) not in cells with Aβ-(1–40) or or in ChAT from cells not other in of with of these to ChAT peptides with phosphorylation of the for this from ChAT from Aβ-(1–42)-treated cells was low with purified ChAT phosphorylated by with protein kinases in vitro This that a low of the enzyme is phosphorylated in was by for of the the as as the This of this peptide as of human ChAT with the and a mass of also the of the for the peptide of human ChAT and This a for CaM kinase II phosphorylation of the for CaM kinase II is J. Biol. Chem. Scholar, J. Biol. Chem. Scholar, J.R. P. J. Biol. Chem. Scholar), with the ChAT of this was to alanine by to a for as the of the mutation was by of the and of the peptide in and of the peptide the in C and of peptides of from IMR32 cells expressing the enzyme and with Aβ-(1–42) by and a with serine the This is in to the with cells expressing ChAT in and B. A was also found in This to the phosphorylated of the peptide of human ChAT This peptide a serine that was found previously to be phosphorylated by (18Dobransky T. Davis W.L. Rylett R.J. J. Biol. Chem. 2001; 276: 22244-22250Google Scholar). ChAT was with CaM kinase II and by and with for by to phosphorylated and Using a was found to a of that is of of a R. C. for for Scholar). This was to in to This peptide to as can be in the of a phosphorylated threonine This to of ChAT that a for CaM kinase II; this is the threonine found to be phosphorylated in IMR32 cells with We CaM kinase II was in IMR32 cells with Aβ peptides. expressing ChAT with or peptides or and and with substrate peptide a CaM kinase II phosphorylation in with Aβ-(1–42) increased phosphorylation of the CaM kinase II substrate peptide by activation of this protein Phosphorylation of the substrate peptide in cells with the other Aβ peptides did not differ from cells. This to the in activity and phosphorylation of ChAT are that phosphorylation of CaM kinase II substrate peptide was not from 30 min or increased with by and to and h the of Aβ-(1–42) not that phosphorylation of the substrate peptide in Aβ-(1–42)-treated cells was to activation of CaM kinase II, we the effects of the CaM kinase II and its of not to cells with Aβ-(1–42) phosphorylation of the CaM kinase II substrate peptide We the relationship between phosphorylation of ChAT by and CaM kinase II and activation of the enzyme both purified recombinant ChAT and IMR32 cells with in purified ChAT activity was increased 3-fold by CaM kinase phosphorylation the enzyme was also phosphorylated by Phosphorylation of ChAT by alone led to a 2-fold increase in enzyme activity, phosphorylation by CaM kinase II alone did not alter ChAT activity. of by activation of ChAT observed the enzyme was with and CaM kinase II of purified ChAT with and CaM kinase II in the of resulted in a 2-fold increase in ChAT activity to that observed for phosphorylation of ChAT by in also activation of ChAT in IMR32 cells with Aβ-(1–42) for 30 min or 10 the other the CaM kinase II activation of ChAT by 10 h of Aβ and on ChAT activation 30 min of Whereas is in a that could be by CaM kinase II, it is also from a the that this threonine could be phosphorylated by other protein kinases as protein we used of and protein kinase to in phosphorylation of in Aβ-(1–42)-treated IMR32 cells. of or to IMR32 cells Aβ also on activation of ChAT this we the relationship of serine and threonine phosphorylation of ChAT in IMR32 cells with Aβ-(1–42) and the of kinase on phosphorylation of ChAT in cells. in serine phosphorylation to both 30 min and 10 threonine phosphorylation in cells with Aβ-(1–42) for 10 or on phosphorylation or We also the of Aβ-(1–42) on activity of and in with that of in activity not differ in the with that of enzyme in the of Aβ-(1–42) For cells expressing ChAT, enzyme activity was increased by 30 min and by 2-fold 10 h the of Aβ-(1–42) of IMR32 cells expressing forms of ChAT did not in a in activity of the of ChAT did increase activity of the of ChAT by both 30 min and 10 ChAT was from IMR32 cells with and proteins and by is from the of ChAT in A that a number of other proteins with both ChAT and from cells with These not observed in to of cells with Aβ-(1–40) or or from cells expressing with A mass and was to to proteins with For mass from and with on peptide mass of the protein, we identified protein with mass of to be human of was and peptides to in two of peptides by also to the of by with in from cells expressing ChAT that with Aβ-(1–42) not other Aβ this was in this was from of IMR32 cells expressing ChAT with Aβ peptides to in a ChAT was observed in the from cells with concentrations of Aβ peptides are released the brain by of APP in as Alzheimer's disease and following traumatic head In the present study, we the that exposure of IMR32 neuroblastoma cells expressing ChAT to Aβ peptides alter function of the We demonstrate for the first that phosphorylation and activity of ChAT are by exposure of cells to exposure of cells to Aβ-(1–42) leads to activation of CaM kinase II and phosphorylation on a threonine in ChAT, in ChAT is phosphorylated by CaM kinase II in vitro and in cells following with the increase in ChAT activity observed with is with phosphorylation of by and this is by CaM kinase phosphorylation of 10 of cells with Aβ-(1–42) leads to phosphorylation-dependent between ChAT and other cellular with of these identified as by Aβ peptides acute or long term on neurons are not it is that with several ACh M. D. K. J. Neurosci. 2001; 21: Scholar, H. Proc. Natl. Acad. Sci. U. S. A. 2001; Scholar), M. S. J. Biol. Chem. 2002; Scholar), Y. A. S. J. Neurosci. 2001; 21: Scholar), and J. M. H. Roher A. T. M. J. A. P. D. 1996; Scholar). and cellular by binding of Aβ peptides to these have not been are by M.C. J. Biol. Chem. 2001; 276: Scholar). of Aβ peptides with cellular with of or release from J. Neurosci. 2001; Scholar, M. Y. Res. 2000; Scholar). Aβ peptides can in plasma to plasma membrane M. Y. Res. 2000; Scholar, H. R. R. J. 2001; Scholar). In Aβ-(1–42) not other Aβ by interaction with the ACh D. C. E. J. Neurochem. Scholar), led to neuronal a death G. R.J. J. Neurochem. 1998; 70: Scholar). In to the present it is that IMR32 cells ACh D. C. E. J. Neurochem. Scholar) and low of G. R.J. J. Neurochem. 1998; 70: it is not other binding for Aβ peptides are by these cells. caused by Aβ peptide could protein kinases as and CaM kinase ChAT is a substrate for both of these kinases (17Dobransky T. Davis W.L. Xiao G.H. Rylett R.J. Biochem. J. 2000; 349: 141-151Google Scholar). present these by a between activation of CaM kinase II and and of ChAT activity and phosphorylation of ChAT and its interaction with other proteins. We also showed that Aβ-(1–42) increased CaM kinase II activity in IMR32 we did not activation in we found increased activity in not of by Aβ peptides was previously J. H. Res. 2000; Scholar, M. S. K. N. T. A. T. H. K. E. C. Neurochem. 2001; Scholar, Y. K. T. B. Res. 1997; Scholar). A in the present is that Aβ-(1–42) caused changes in ChAT activity, and protein Mechanisms underlying this are other studies have by Aβ-(1–42) and not other Aβ peptides. that this did not to in between Aβ-(1–42) and we of peptides by and and found both to be of and to have is that the in cellular to Aβ-(1–42) and Aβ-(1–40) observed in the present to in of the peptides to that the cellular Using and mass spectrometry, we identified as a phosphorylation site in ChAT following short term of IMR32 cells with in ChAT is phosphorylated in Aβ-(1–42)-treated cells a with increased activity of CaM kinase other protein kinases of the protein kinase could be by M. D. K. J. Neurosci. 2001; 21: Scholar), we used to that these are probably not phosphorylation of in this kinase and of in ChAT used to a relationship between phosphorylation of by and phosphorylation of by CaM kinase II in activity of Phosphorylation of ChAT on by alone increased ChAT activity by with this increased to 3-fold is also phosphorylated by CaM kinase II phosphorylation by CaM kinase II alone did not alter ChAT activity. This that phosphorylation of ChAT on leads to the activation of ChAT by 30 This increased ChAT activity is not the a second of activation by h the of Aβ-(1–42) increased CaM kinase II activity, phosphorylation of and increased serine Mechanisms underlying this increase in ChAT activity and activation of CaM kinase II are is that cellular with acute of Aβ-(1–42) in of cellular effects several These effects activation of CaM kinase II, activity of ChAT, and interaction of ChAT with and other cellular proteins. have been proteins that ChAT with in the it was that neurons of ChAT that be proteins C. J. Biol. Chem. Scholar, C. Res. 276: Scholar). that regulate ChAT to be or as C. Neurobiol. Scholar) and N. Biochem. 2002; Scholar). In the present study, a number of proteins with ChAT following of cells expressing ChAT with Aβ-(1–42) and associated with phosphorylation of We identified of these by mass spectrometry, with by to be VCP, a of the of is a protein in and cells T. 1997; Scholar, S. M. Trends Biol. 1998; Scholar, Nat. Biol. 2001; Scholar) with in cellular protein and cellular proteins with is to H. K. Biol. 2000; Scholar) and D. A. S. C. J. S. S. Biol. 2001; 21: Scholar). it that of with ChAT is both and are not with ChAT in Aβ-(1–42)-treated cells expressing of interaction of ChAT with is A cellular function of is its in the between and of proteins D. A. S. C. J. S. S. Biol. 2001; 21: Scholar, Nat. Biol. 2001; Scholar). function for acetyltransferase is to to proteins to from and for D. A. S. C. J. S. S. Biol. 2001; 21: Scholar, 2001; Scholar). a function for the interaction between ChAT and to be two could be In the to VCP, as a to the enzyme to the In the ChAT as to other proteins to alter function or ChAT the of to of choline and potentially other been that ChAT can the that be found with of proteins on in the present a new of Aβ-(1–42) on cholinergic neuron of ChAT activity as a of the effects of Aβ peptides on cholinergic neurons been acute effects of Aβ peptides on function of the enzyme ChAT have not been of cultures of neurons to not concentrations used in the present for h ACh synthesis did not alter ChAT activity (9Hoshi M. Takashima A. Murayama M. Yasutake K. Yoshida N. Ishiguro K. Hoshino T. Imahori K. J. Biol. Chem. 1997; 272: 2038-2041Google Scholar). exposure of cells to Aβ-(1–42) for h ChAT activity W.A. Kloczewiak M.A. Blusztajn J.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8068-8071Google Scholar). in the present study, activation of and of the in to exposure to Aβ-(1–42) leads to short term changes in the phosphorylation of ChAT that could in acute changes in cholinergic neurotransmission. We for the H. for the of CaM kinase II, P. of for of amyloid and and of for of amyloid peptides.

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