Protein Kinase C Isoforms Differentially Phosphorylate Human Choline Acetyltransferase Regulating Its Catalytic Activity

Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons; regulation of its activity or response to physiological stimuli is poorly understood. We show that ChAT is differentially phosphorylated by protein kinase C (PKC) isoforms on four serines (Ser-440, Ser-346, Ser-347, and Ser-476) and one threonine (Thr-255). This phosphorylation is hierarchical, with phosphorylation at Ser-476 required for phosphorylation at other serines. Phosphorylation at some, but not all, sites regulates basal catalysis and activation. Ser-476 with Ser-440 and Ser-346/347 maintains basal ChAT activity. Ser-440 is targeted by Arg-442 for phosphorylation by PKC. Arg-442 is mutated spontaneously (R442H) in congenital myasthenic syndrome, rendering ChAT inactive and causing neuromuscular failure. This mutation eliminates phosphorylation of Ser-440, and Arg-442, not phosphorylation of Ser-440, appears primarily responsible for ChAT activity, with Ser-440 phosphorylation modulating catalysis. Finally, basal ChAT phosphorylation in neurons is mediated predominantly by PKC at Ser-476, with PKC activation increasing phosphorylation at Ser-440 and enhancing ChAT activity. Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons; regulation of its activity or response to physiological stimuli is poorly understood. We show that ChAT is differentially phosphorylated by protein kinase C (PKC) isoforms on four serines (Ser-440, Ser-346, Ser-347, and Ser-476) and one threonine (Thr-255). This phosphorylation is hierarchical, with phosphorylation at Ser-476 required for phosphorylation at other serines. Phosphorylation at some, but not all, sites regulates basal catalysis and activation. Ser-476 with Ser-440 and Ser-346/347 maintains basal ChAT activity. Ser-440 is targeted by Arg-442 for phosphorylation by PKC. Arg-442 is mutated spontaneously (R442H) in congenital myasthenic syndrome, rendering ChAT inactive and causing neuromuscular failure. This mutation eliminates phosphorylation of Ser-440, and Arg-442, not phosphorylation of Ser-440, appears primarily responsible for ChAT activity, with Ser-440 phosphorylation modulating catalysis. Finally, basal ChAT phosphorylation in neurons is mediated predominantly by PKC at Ser-476, with PKC activation increasing phosphorylation at Ser-440 and enhancing ChAT activity. Choline acetyltransferase (ChAT, EC 2.3.1.6) 1The abbreviations used are: ChAT, choline acetyltransferase; ACh, acetylcholine; dhA, dehydroalanine; ESI, electrospray ionization; LC, liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; MS/MS, tandem mass spectrometry, mass sequencing; PKC, protein kinase C; cPKC, conventional PKC; nPKC, novel PKC; aPKC, atypical PKC; ANOVA, analysis of variance. synthesizes the neurotransmitter acetylcholine (ACh) and serves as a phenotypic marker for cholinergic neurons. ChAT expression changes in normal aging and in neurological and psychiatric disorders such as Alzheimer disease and schizophrenia (1Blusztajn J.K. Berse B. Metab. Brain Dis. 2000; 15: 45-64Crossref PubMed Google Scholar, 2Boissiere F. Faucheux B. Agid Y. Hirsch E.C. Neurosci. Lett. 1997; 225: 169-172Crossref PubMed Scopus (21) Google Scholar). 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Clatterbuck R.E. Holtzman D.M. Mobley W.C. Price D.L. J. Neurosci. 1990; 10: 3801-3813Crossref PubMed Google Scholar). Little is known, however, about mechanisms involved in short term regulation of ChAT function, with there being only a few reports of physiological perturbations that modulate its catalytic activity (7Pongrac J.L. Rylett R.J. J. Neurochem. 1996; 66: 804-810Crossref PubMed Scopus (29) Google Scholar, 8Tandon A. Bachoo M. Weldon P. Polosa C. Collier B. J. Neurochem. 1996; 66: 1033-1041Crossref PubMed Scopus (16) Google Scholar). Protein kinase-mediated phosphorylation is a common mechanism that regulates enzymatic activity, subcellular compartmentalization, or interaction of a protein with other cellular proteins. ChAT is a substrate for multiple kinases, with phosphorylation by some kinases regulating its activity (9Dobransky T. Davis W.L. Xiao G.H. Rylett R.J. Biochem. J. 2000; 349: 141-151Crossref PubMed Scopus (37) Google Scholar). Phosphorylation of purified 69-kDa human ChAT by protein kinase C (PKC) increases its activity 2-fold; this increase is attenuated in mutant ChAT in which serine-440 is changed to an alanine residue (10Dobransky T. Davis W.L. Rylett R.J. J. Biol. Chem. 2001; 276: 22244-22250Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Phosphorylation of ChAT at Ser-440 by PKC is also associated with altered membrane binding of the enzyme (10Dobransky T. Davis W.L. Rylett R.J. J. Biol. Chem. 2001; 276: 22244-22250Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). PKC comprises a family of serine/threonine kinases produced as isoenzymes that differ in mode of activation, substrate specificity (11Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2368) Google Scholar, 12Dekker L.V. Parker P.J. Trends Biochem. Sci. 1994; 19: 73-77Abstract Full Text PDF PubMed Scopus (920) Google Scholar), cell/tissue-specific expression (13Ohno S. Tsujino A. 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Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1472) Google Scholar). The biological actions and regulation of PKC isoenzymes in neurons are not fully understood, but it is known that different isoenzymes mediate diverse cellular responses. This appears to be defined by different resting and stimulus-induced subcellular localization and translocation patterns for the isoenzymes and different target substrates based on the recognition of different optimal phosphorylation consensus sequences. Moreover, specific PKC isoenzyme expression and/or activity may be altered by pathology, including neurodegenerative diseases (19Battaini F. Pharmacol. Res. 2001; 44: 353-361Crossref PubMed Scopus (96) Google Scholar). Most isoforms of PKC are found in brain where they are involved in regulation of a wide range of neuronal processes, including ion channel gating, receptor desensitization, neurotransmitter release, synaptic efficacy, and some forms of learning/memory (20Tanaka C. Nishizuka Y. Ann. Rev. Neurosci. 1994; 17: 551-567Crossref PubMed Scopus (509) Google Scholar, 21Le Merrer J. Nogues X. Pharmacol. Rev. 2000; 41: 503-514Google Scholar). Importantly, various PKC isoform levels and activities are increased or decreased differentially during neurodegeneration. These changes have been linked to processes involved in cellular but they to and neuronal (19Battaini F. Pharmacol. Res. 2001; 44: 353-361Crossref PubMed Scopus (96) Google Scholar). the phosphorylation of 69-kDa human ChAT by a of PKC isoforms and in the patterns of We that some that are phosphorylated by PKC are required for regulation of catalytic activity of ChAT, with mutation of in or of enzymatic activity. of activity of PKC isoforms in response to physiological or in or ChAT activity and cholinergic and Protein human 69-kDa ChAT into the expression by H. for This used for expression of the protein in and as a for of mutant forms of the of purified ChAT, the protein in and purified by different ChAT in the and purified by as (9Dobransky T. Davis W.L. Xiao G.H. Rylett R.J. Biochem. J. 2000; 349: 141-151Crossref PubMed Scopus (37) Google Scholar). ChAT by linked to the with at into this purified ChAT in a with a of that be at ChAT in as a protein the that a the and This protein purified on a and with to the The the of ChAT by Sci. S. A. PubMed Scopus Google Scholar), a on the to ChAT protein the of ChAT into and expression the The of and at the level by and at the protein level by and with or mutant 69-kDa human ChAT in in and in at PKC, or growth factor receptor to the following at and of to the and for on for and used for analysis of activity. ChAT specific activity a of the of F. Biochem. J. PubMed Scopus Google Scholar), as R.J. S. A. J. Neurochem. PubMed Scopus Google Scholar). of ChAT at a that of enzyme activity to of to in with or mutant ChAT and human as R.J. S. A. J. Neurochem. PubMed Scopus Google Scholar). Choline kinase required for this purified as the protein The expression choline kinase produced by and by and of Protein Phosphorylation and ChAT with PKC isoenzymes α, βI, βII, δ, ϵ, or ζ in a Phosphorylation by and the at for to of into ChAT by different isoforms of PKC for in the of on and to to at for for to ChAT in of the to ChAT protein and by and on to the of Nature. PubMed Scopus Google Scholar). with and or to for with in with (9Dobransky T. Davis W.L. Xiao G.H. Rylett R.J. Biochem. J. 2000; 349: 141-151Crossref PubMed Scopus (37) Google Scholar) for at with with by and and by with and by with at changes to of three with to ChAT with and with as T. Rylett R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. M. Chem. 1996; PubMed Scopus Google Scholar). and of of ChAT as T. Rylett R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). following of to and in the in at for and in the in by For the with to in a and in and This of used for mass For purified on to the and the with and analysis at also on or on of in of at for at in of and by phosphorylation of ChAT with PKC isoforms by into and to in a of by and to mass analysis in a of or on and mass or and in ion with a of and The in with a in The also used as an for peptide mass and used to the on a or mass a a that a at by for of the the to an that a into the mass used in in for and in liquid for a a of for and for of for the protein or for to of phosphorylated to with to the Phosphorylation of ChAT by PKC human 69-kDa ChAT is a substrate in for of PKC isoforms phosphorylation of ChAT by different isoforms in and of with different by some in phosphorylation of ChAT is with and with and with βII, and into ChAT by of of membrane to ChAT protein by to the that of ChAT protein to sites phosphorylated in ChAT by PKC, phosphorylation patterns on patterns one with α, βI, βII, and and the other with δ, ϵ, and for and are in analysis of that ChAT is phosphorylated on and threonine by cPKC, but only on by and different serine/threonine phosphorylation patterns of ChAT by PKC ChAT with PKC isoforms by ChAT protein to with the by and of ChAT phosphorylated by or with βI, βII, and are to that for and with and are to that for in the on in a for is found in is in and as associated with and are only These are based on of that to to are of at four and three with of the and isoforms of on analysis by mass and four and one threonine in ChAT phosphorylated by various PKC We found that Ser-440 is a PKC phosphorylation (10Dobransky T. Davis W.L. Rylett R.J. J. Biol. Chem. 2001; 276: 22244-22250Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). the four phosphorylated by PKC purified ChAT is phosphorylated differentially by PKC isoforms in with to Ser-346, Ser-347, Ser-440, Ser-476, and and and only Ser-440 and Moreover, based on this appears to of the PKC phosphorylation sites in purified ChAT with PKC in and for ChAT phosphorylated in in in the or of the PKC that be phosphorylated by PKC have to be by of the protein in response to not the the PKC recognition or 1991; PubMed Scopus Google Scholar), there are PKC phosphorylation sites in PKC phosphorylation sites in ChAT in this to other optimal recognition have been for different PKC isoforms in other substrate K. A. Z. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and of PKC phosphorylation peptide phosphorylation associated with and this and also βI, βII, βI, βII, associated with and this and also in a by and of purified ChAT phosphorylated by PKC isoforms are in I. a of four and one threonine as PKC phosphorylation required to phosphorylated to phosphorylated to be in it to Moreover, to be with in a analysis and not be to peptide phosphorylation to the with PKC and by tandem This with one of the by that of which PKC consensus sequences. and of this peptide are in the this peptide phosphorylated at a with a of phosphorylated a and analysis of that to the phosphorylated peptide a ion that as the phosphorylation and of which as for optimal consensus for PKC, found to be to P.J. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). of PKC Phosphorylation and threonine in ChAT as phosphorylation sites for PKC isoforms mutated to or in This to their in regulation of ChAT activity, the and there are phosphorylation sites not by of ChAT phosphorylated by or of associated with specific the of phosphorylated associated with the of associated with of and mutant ChAT phosphorylated by but not by or phosphorylated by but not by or phosphorylated by but not by or in a mutation of Ser-440 of purified ChAT by cPKC, nPKC, and to for and for for ChAT of Ser-346/347 in phosphorylation by cPKC, but not by with this mutation for in The of Ser-476 to phosphorylation by this but not phosphorylation of ChAT by The with mutation of Ser-476 to in Finally, mutation of with mutation of the four produced mutant ChAT that not a substrate for the in the with mutation of to in that are to a phosphorylation one or of the protein during This the phosphorylated residue is to an or that serves as the M. Biochem. 1989; PubMed Scopus Google Scholar). of phosphorylation by PKC on ChAT function, the phosphorylation at or of and enzymatic activity. the the and with activity of are in and of ChAT with to and a increase in catalytic activity. of the and have a in to in of only basal activity with Moreover, activation in and but attenuated in and in of the and with or and have basal activity of or with not catalytic activity to the proteins. These decreased by with for mutant with Ser-476 and to about for mutant with sites some for mutant may basal ChAT activity in a by causing a in the protein that catalysis. Finally, mutation of Ser-476 phosphorylation in by not by a in enzyme activity. of to ChAT that phosphorylation of is on this is by with and in which the four mutated in the or of the mutant catalytic activity not different ChAT, phosphorylation by and activity of mutant not by Importantly, phosphorylation of ChAT by PKC mutation of Ser-476 phosphorylation by on Ser-346, Ser-347, and with multiple phosphorylation of ChAT by a with analysis this as of in of this threonine not used to the of phosphorylation of a residue in a biological is to the by an acidic such as or in of the phosphorylated residue S.J. J.M. 1996; PubMed Scopus Google Scholar). We the of phosphorylation of Ser-476 as a for phosphorylation of other by PKC by Ser-476 in of of purified and phosphorylated by that phosphorylation of in is is by and that to phosphorylated Ser-440 and to phosphorylated in ChAT phosphorylated by in but not in phosphorylation of by not in a not recovery of phosphorylation with about of that with there recovery of phosphorylation with recovery of phosphorylation of Ser-440 in not to altered specific activity of this mutant ChAT activity in of or with ChAT This that is not an substrate for PKC isoenzymes to of the phosphorylation of Moreover, recovery of phosphorylation of ChAT in to basal catalytic activity. Several have been in ChAT with some causing and of cholinergic K. Tsujino A. Brengman J.M. Harper C.M. Bajzer Z. Udd B. Beyring R. Robb S. Kirkham F.J. Engel A.G. Sci. S. A. 2001; PubMed Scopus Google Scholar, Chen C. PubMed Scopus Google Scholar, A.G. K. Rev. Neurosci. 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Phosphorylation and of ChAT in by and mutant ChAT in and for analysis to of phosphorylation of the enzyme in activity of ChAT with PKC phosphorylation sites or in in to ChAT is to in with activity of purified in expression of ChAT in is by phosphorylation of the in are not of some and on activity of ChAT in human for purified found for C and with specific activity of being with ChAT in but not in We also the to which basal phosphorylation of ChAT be to PKC. of into and mutant ChAT ChAT with C and in which Ser-476 or PKC phosphorylation sites Moreover, basal phosphorylation of ChAT is mediated predominantly by PKC as with PKC of ChAT by multiple that ChAT phosphorylation in in one with term phosphorylated mutant ChAT in of Ser-476 attenuated basal phosphorylation that this the predominantly phosphorylated residue in of with the of the that to to this is with the of phosphorylation associated with mutant ChAT as PKC phosphorylation in of this of ChAT with but not phosphorylation of the one or with the on as that for C and following of PKC. analysis that this is with threonine and in ChAT in not phosphorylated not Moreover, of PKC in ChAT by different and growth factor receptor ChAT activity of increased into ChAT, predominantly in to of Ser-440 increased with PKC activation to an to that found for Ser-476 basal not the of of some PKC phosphorylation sites on the of the enzyme to in or mutant or and ChAT into that the human choline This it of and mutant forms of the enzyme with which the substrate that increased of to in ChAT, but not (10Dobransky T. 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PubMed Scopus Google the one or protein kinases that ChAT in not The a for regulation of ChAT by and the of PKC in cholinergic PKC to basal phosphorylation of ChAT in this phosphorylation of ChAT by PKC, or protein kinases such as protein kinase that are by PKC; phosphorylation of ChAT by PKC is with phosphorylation of Ser-476 required for phosphorylation by PKC at other to PKC isoforms ChAT differentially with Ser-440, Ser-476, and and only Ser-440 and Ser-440 and/or Ser-346/347 in of basal catalytic activity and in of ChAT activity, Ser-476 and not to be required for this Ser-346/347 are involved in modulating the of phosphorylation of ChAT on other to Ser-346/347 comprises only a of on but of by and the of phosphorylation at specific sites in in and for basal in Ser-440 is phosphorylated predominantly purified ChAT is with PKC, Ser-476 is phosphorylated primarily in and increased phosphorylation of ChAT in is on phosphorylation by kinases to regulate their physiological For example, is a substrate for multiple kinases with in phosphorylation by protein kinase and by protein is not a substrate for protein kinase phosphorylation by protein kinase or phosphorylation by other kinases, including PKC, protein kinase protein kinase and G.A. 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The Ser-476 as a phosphorylation for PKC in phosphorylation of other by PKC in a this Ser-476 not Ser-476 in with Ser-440 and Ser-346/347 is involved in basal ChAT this is in some to regulation of of ChAT with the four PKC sites mutated activity, the activity as however, in the of activity, to of phosphorylation of other by PKC. phosphorylation of Ser-476 phosphorylation of other Ser-440, to increased catalytic activity. specific activity of in is by the of this in catalysis. recovery of phosphorylation at Ser-440 by in purified but this not by of catalytic activity this mutant ChAT in of the acidic residue not phosphorylated Phosphorylation of Ser-440 is and regulate subcellular of the protein by binding of ChAT to and regulating catalytic activity (10Dobransky T. Davis W.L. Rylett R.J. J. Biol. Chem. 2001; 276: 22244-22250Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). of specific phosphorylation sites phosphorylation at other or to consensus regulation of the enzyme and Ser-440 and multiple in of Ser-440 is targeted by Arg-442 for phosphorylation in the PKC consensus Arg-442 in ChAT (R442H) is mutated spontaneously in congenital myasthenic R.J. S. A. J. Neurochem. PubMed Scopus Google Scholar), with this mutation rendering ChAT in of neuromuscular to and K. Tsujino A. Brengman J.M. Harper C.M. Bajzer Z. Udd B. Beyring R. Robb S. Kirkham F.J. Engel A.G. Sci. S. A. 2001; PubMed Scopus Google Scholar). We that the mutation eliminates phosphorylation on Ser-440, as be the kinase not the of an PKC consensus The for phosphorylation of Ser-440 in of basal and ChAT activity. altered enzyme with in for its and of at choline K. Tsujino A. Brengman J.M. Harper C.M. Bajzer Z. Udd B. Beyring R. Robb S. Kirkham F.J. Engel A.G. Sci. S. A. 2001; PubMed Scopus Google Scholar). This may to Arg-442 with the of and being to that in of of ChAT for choline J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The of phosphorylation of Ser-440 in modulating of ChAT not been The of ChAT Y. Engel A.G. K. J. PubMed Scopus Google Scholar) and an to the phosphorylation sites in the of this structural phosphorylation of Ser-440 by PKC (10Dobransky T. Davis W.L. Rylett R.J. J. Biol. Chem. 2001; 276: 22244-22250Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) and by kinase T. Rylett R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Ser-440 is to the binding in the catalytic of the is that phosphorylation of this residue the binding for and increase its of release, an for the catalytic activity with phosphorylation of Ser-440 (10Dobransky T. Davis W.L. Rylett R.J. J. Biol. Chem. 2001; 276: 22244-22250Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). on the ChAT Y. Engel A.G. K. J. PubMed Scopus Google Scholar), human not to be and of the enzyme to be the Ser-476 is in an that is to This is phosphorylation of this is required to phosphorylation to at other PKC other phosphorylation sites Ser-347, and are also phosphorylation of the of a protein phosphorylation a range of for functional including of catalytic activity, interaction with other and cellular and subcellular and compartmentalization. This is in the of neurons to of for of cholinergic neurons is in that is not into the nerve following and be Phosphorylation of ChAT by PKC appears to be an mechanism that a level of required for of cholinergic

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