CHARACTERIZATION OF THE PHOSPHORYLATION SITES OF HUMAN HIGH-MOLECULAR-WEIGHT NEUROFILAMENT PROTEIN BY ELECTROSPRAY-IONIZATION TANDEM MASS-SPECTROMETRY AND DATABASE SEARCHING

Hyperphosphorylated high molecular weight neurofilament protein (NF-H) exhibits extensive phosphorylation on lysine serine-proline (KSP) rep eats in the C-terminal domain of the molecule. Specific phosphorylatio n sites in human NF-H were identified by proteolytic digestion and ana lysis of the resulting digests by a combination of microbore liquid ch romatography, electrospray ionization tandem (MS/MS) ion trap mass spe ctrometry, and database searching. The computer programs utilized (PEP SEARCH and SEQUEST) are capable of identifying peptides and phosphoryl ation sites from uninterpreted MS/MS spectra, and by use of these meth ods, 27 phosphopeptides and their phosphorylated residues were identif ied. On the basis of these phosphopeptides, 38 phosphorylation sites i n human NF-H were characterized. These include 33 KSP, lysine-threonin e-proline (KTP) or arginine-serine proline (RSP) sites and four unphos phorylated sites, all of which occur in the KSP repeat domain (residue s 502-823), and one threonine phosphorylation site observed in a KVPTP EK motif. Six KSP sites were not characterized because of the failure to isolate and identify corresponding phosphopeptides, Heterogeneity i n serine and threonine phosphorylation was observed at three sites or deduced to occur at three sites on the basis of enzyme specificity. As a result of the phosphorylated motifs identified (KSPEKEE, KSPVKAE, K SPAEAK, KSPPEAK, KSPEAKT, KSPAEVK, and KVPTPEK), human NF-H tail domai n is postulated to be a substrate of proline-directed kinases, The thr eonine-phosphorylated KVPTPEK motif suggested the existence of a novel proline-directed kinase.