Plant PIP2-dependent phospholipase D activity is regulated by phosphorylation.
Phospholipase D (PLD) forms the major family of phospholipases that was first discovered and cloned in plants. In this report we have shown, for the first time, that C2 phosphatidylinositol-4,5-bisphosphate (PIP2)-dependent PLD(s) from 5 day hypocotyls of Brassica oleracea associated with plasma membrane is covalently modified-phosphorylated. Pre-incubation of the plasma membrane fraction with acid phosphatase resulted in concentration-dependent inhibition of PIP2-dependent PLD activity. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry of tryptic in-gel digests, the BoPLDgamma(1,2) isoform was identified. Comparing the spectra of the proteins obtained from the plasma membrane fractions treated and non-treated with acid phosphatase, three peptides differing in the mass of the phosphate group (80 Da) were revealed: TMQMMYQTIYK, EVADGTVSVYNSPR and KASKSRGLGK which possess five potential Ser/Thr phosphorylation sites. Our findings suggest that a phosphorylation/dephosphorylation mechanism may be involved in the regulation of plant PIP2-dependent PLDgamma activity.
Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Prague 6, Czech Republic. zuzana.novotna@vscht.cz
Zuzana
Z
Novotná
Jan
J
Linek
Radovan
R
Hynek
Jan
J
Martinec
Martin
M
Potocký
Olga
O
Valentová
D000135:Acid Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D001937:Brassica / Q000201:enzymology; D008565:Membrane Proteins / Q000378:metabolism; D010446:Peptide Fragments; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation; D010940:Plant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D008565:Membrane Proteins; D010446:Peptide Fragments; D019269:Phosphatidylinositol 4,5-Diphosphate; D010940:Plant Proteins; D000135:Acid Phosphatase; D010739:Phospholipase D