In vitro phosphorylation of purified glycosylphosphatidylinositol-specific phospholipase D.
Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) was phosphorylated in vitro by cAMP-dependent protein kinase (PKA) and by tyrosine kinase. Phosphorylation by PKA occurred in the 110 kDa native form of GPI-PLD as well as in multiple proteolytic degradation products and caused a significant decrease in enzyme activity. Dephosphorylation by treatment with alkaline phosphatase completely restored GPI-PLD activity. In addition, incubation of GPI-PLD with trypsin, which results in the generation of distinct peptide fragments, resulted in complete dephosphorylation of radiolabeled GPI-PLD. The site of phosphorylation by PKA was assigned to Thr-286. Tyrosine phosphorylation was only observed in a proteolytically processed fragment of GPI-PLD but not in the 110 kDa native form and had no effect on GPI-PLD activity.
Civenni
G
G
Institute of Biochemistry and Molecular Biology, University of Bern, Switzerland.
Bütikofer
P
P
Stadelmann
B
B
Brodbeck
U
U
D000818:Animals; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D010739:Phospholipase D / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism
D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D010739:Phospholipase D; C065017:glycoprotein phospholipase D