Activation of a membrane-associated phosphatidylinositol kinase through tyrosine-protein phosphorylation by naphthoquinones and orthovanadate.
We have previously reported that several naphthoquinones stimulated tyrosine-specific protein phosphorylation in isolated rat liver membranes. Our more recent study demonstrated a similar effect by orthovanadate, which concomitantly stimulated phosphorylation of protein-tyrosine and phosphatidylinositol (Ptd-Ins). Results presented here show a simultaneous increase in PtdIns phosphorylation along with stimulation of tyrosine-protein phosphorylation by naphthoquinones. This PtdIns kinase resembles the type I PtdIns kinase in that it was insensitive to adenosine inhibition. The product, nevertheless, comigrated with a PtdIns-4-phosphate standard in TLC using three different solvent systems. Stimulation of PtdIns phosphorylation by vanadate or naphthoquinones could be achieved in the following preparations: intact rat liver membranes, Triton X-100-solubilized membranes, solubilized membranes partially purified by Sephacryl chromatography, solubilized membranes purified by wheat germ agglutinin chromatography. The naphthoquinone or vanadate-activated PtdIns kinase activity could be isolated by antiphosphotyrosine antibody-agarose affinity chromatography. The relative potencies of a series of ring-substituted naphthoquinones in the stimulation of tyrosine-protein phosphorylation, PtdIns kinase activity, dithiothreitol-dependent oxygen consumption, and cytochrome c reduction were highly correlated. We conclude that oxidant(s) produced by redox cycling of naphthoquinones stimulated an adenosine-insensitive PtdIns kinase through tyrosine phosphorylation of the enzyme.
Chen
Y X
YX
Institute for Toxicology (Pharmacy), University of Southern California, Los Angeles 90033.
Yang
D C
DC
Brown
A B
AB
Jeng
Y
Y
Tatoyan
A
A
Chan
T M
TM
D019870:1-Phosphatidylinositol 4-Kinase; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D009285:Naphthoquinones / Q000494:pharmacology*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis; D014638:Vanadates / Q000494:pharmacology*
D009285:Naphthoquinones; D019000:Phosphotyrosine; D014638:Vanadates; D014443:Tyrosine; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase; D011505:Protein-Tyrosine Kinases; C014758:plumbagin