Activation of phospholipase D is not mediated by direct phosphorylation on tyrosine residues.
The activation of phospholipase D (PLD) in PC12/PC2 pheochromocytoma cells involves a tyrosine kinase. However, it is not clear whether this is due to direct phosphorylation of the enzyme or some other intermediary protein. In this manuscript, we examined this issue by two methods: (1) immunoprecipitation of phosphotyrosine containing proteins and assay of phospholipase D; (2) overexpression of HA-phospholipase D2 and susbsequent immunoprecipitation. The only agent that caused phosphorylation of phospholipase D on tyrosine residues was the phosphatase inhibitor, peroxyvanadate. Other agents that activate phospholipase D, including bradykinin, ionomycin, and phorbol dibutyrate did not cause phosphorylation of the enzyme. In addition, there was a lack of correlation between the peroxyvanadate-mediated phosphorylation and activation of phospholipase D, both in terms of time course and concentration dependence. These data demonstrate that phospholipase D is directly phosphorylated on tyrosine residues. However, phosphorylation of tyrosine residues does not correlate with activation of the enzyme.
Mehta
Sanjoy
S
Department of Pharmacology and Physiology, MCP Hahnemann School of Medicine, Mail Stop 488, 245 N. 15th Street, Philadelphia, PA 19102-1192, USA.
Maglio
Jeff
J
Kobayashi
Mike S
MS
Sipple
Andrea M
AM
Horwitz
Joel
J
D000818:Animals; D004789:Enzyme Activation; D004791:Enzyme Inhibitors; D007527:Isoenzymes / Q000378:metabolism; D016716:PC12 Cells; D010739:Phospholipase D / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000032:analysis; D011233:Precipitin Tests; D051381:Rats; D014443:Tyrosine / Q000737:chemistry*; D014638:Vanadates
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D019000:Phosphotyrosine; D014638:Vanadates; D014443:Tyrosine; D010739:Phospholipase D