In vitro phosphorylation of purified glycosylphosphatidylinositol-specificphospholipase D

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) was phospho rylated in vitro by cAMP-dependent protein kinase (PKA) and by tyrosine kin ase, Phosphorylation by PKA occurred in the 110 kDa native form of GPI-PLD as well as in multiple proteolytic degradation products and caused a signif icant decrease in enzyme activity, Dephosphorylation by treatment with alka line phosphatase completely restored GPI-PLD activity. In addition, incubat ion of GPI-PLD with trypsin, which results in the generation of distinct pe ptide 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 o f GPI-PLD but not in the 110 kDa native form and had no effect on GPI-PLD a ctivity.