Lysine 3 acetylation regulates the phosphorylation of yeast 6-phosphofructo-2-kinase under hypo-osmotic stress.
N-terminal acetylation in the yeast Saccharomyces cerevisiae is catalysed by any of three N-terminal acetyltransferases (NAT), NatA, NatB, and NatC, which contain the catalytic subunits Ard1p, Nat3p and Mak3p, respectively. Yeast 6-phosphofructo-2-kinase (PFK2) was found to be acetylated at the amino acid lysine 3. The Lys3-Arg mutant was not acetylated and the mutation causes a slight decrease in enzyme activity. PFK2 from yeast cells exposed to hypo-osmotic stress is known to be phosphorylated at Ser8 and Ser652 (Dihazi et al., 2001a). We have taken a mass spectrometric approach to investigate the influence of PFK2 acetylation on its phosphorylation. Wild-type PFK2 and the Lys3-Arg mutant were purified from hypo-osmotically stressed cells and analysed with MALDI-TOF MS for phosphorylation. Wild-type PFK2 without any tag sequence was found to be acetylated and two times phosphorylated at the N-terminal peptide T(1-40) carrying the acetylation. The same results were observed with C-terminally His-tagged PFK2. When the His-tag was added to the N-terminus of the protein PFK2, acetylation was found to be incomplete and only one phosphate was incorporated in the peptide T(1-41). The Lys3-Arg mutant of PFK2 was not at all post-translationally modified at the N-terminal peptide. Our data indicate that Lys3 acetylation affects the N-terminal phosphorylation of PFK2 under hypo-osmotic stress.
Dihazi
Hassan
H
Department of Nephrology and Rheumatology, University Hospital Göttingen, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany. dihazi@med.uni-goettingen.de
Kessler
Renate
R
Müller
Gerhard A
GA
Eschrich
Klaus
K
D000107:Acetylation; D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009997:Osmotic Pressure; D025481:Phosphofructokinase-2 / Q000378:metabolism*; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D029701:Saccharomyces cerevisiae Proteins; D025481:Phosphofructokinase-2; D008239:Lysine