title: In vivo phosphorylation site of hexokinase 2 in Saccharomyces cerevisiae.
abstract: Yeast hexokinase 2 is known to be a phosphoprotein in vivo, prominently labeled from 32P-inorganic phosphate after a shift of cells to medium with low glucose concentration [Vojtek, A. B., & Fraenkel D. G. (1990) Eur. J. Biochem, 190, 371-375]. The principal and perhaps sole site of phosphorylation is now identified as residue serine-15, by observation of a single tryptic peptide difference, its sequencing and size determination by mass spectrometry, and by mutation to alanine, which prevents phosphorylation in vivo. Although protein kinase A was unlikely to accomplish the phosphorylation in vivo, serine-15 does belong to a protein kinase A consensus phosphorylation sequence, and in vitro phosphorylation by protein kinase A at serine-15 could be shown by labeling and by peptide determination. The alanine-15 mutant enzyme was not phosphorylated in vitro.
authors.affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: T M
authors.initials: TM
authors.lastname: Kriegel
authors.forename: J
authors.initials: J
authors.lastname: Rush
authors.forename: A B
authors.initials: AB
authors.lastname: Vojtek
authors.forename: D
authors.initials: D
authors.lastname: Clifton
authors.forename: D G
authors.initials: DG
authors.lastname: Fraenkel
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D005800:Genes, Fungal; D006593:Hexokinase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010766:Phosphorylation; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014357:Trypsin
chemical_list: D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D006593:Hexokinase; D014357:Trypsin