title: Identification of novel in vitro PKA phosphorylation sites on the low and middle molecular mass neurofilament subunits by mass spectrometry.
abstract: Phosphorylation of the head domains of intermediate filament proteins by second messenger-dependent kinases is important in regulating filament assembly. In the case of neurofilaments, head domain phosphorylation is known to be important in assembly, but few sites have been identified. Using matrix-assisted laser desorption-ionization (MALDI) and nano-electrospray mass spectrometry, we report the identification of several novel in vitro cAMP-dependent protein kinase (PKA) phosphorylation sites in the low (NF-L) and middle (NF-M) molecular mass neurofilament subunits. Neurofilament polypeptides were purified from adult rat brain, and fractions containing a mixture of NF-L and NF-M were nonradioisotopically phosphorylated with PKA prior to proteolytic digestion of the polypeptides in situ in polyacrylamide excised from SDS gels. Sites of phosphorylation were determined by mass spectrometric analysis of mixtures enriched in tryptic phosphopeptides. In NF-L, four novel sites were identified: serines 12, 41, and 49 in the head domain and serine 435 in the carboxyl-terminal tail domain, and data consistent with phosphorylation of serine 2 were obtained. Recombinant rat NF-L protein was also phosphorylated with PKA, and the same serines were identified as phosphorylation sites, with two additional sites, serine 43 and probable phosphorylation of serine 55. In NF-M, one novel site, serine 1 in the amino-terminal head domain, was found to be phosphorylated, and serine 46, also in the amino-terminal head domain, was confirmed as a PKA phosphorylation site.
authors.affiliation: Department of Neuroscience, Institute of Psychiatry, London, England.
authors.forename: K E
authors.initials: KE
authors.lastname: Cleverley
authors.forename: J C
authors.initials: JC
authors.lastname: Betts
authors.forename: W P
authors.initials: WP
authors.lastname: Blackstock
authors.forename: J M
authors.initials: JM
authors.lastname: Gallo
authors.forename: B H
authors.initials: BH
authors.lastname: Anderton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016900:Neurofilament Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010748:Phosphopeptides / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D016900:Neurofilament Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; C044882:neurofilament protein L; C061195:neurofilament protein M; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases