Peroxovanadate induces tyrosine phosphorylation of phosphoinositide-dependent protein kinase-1 potential involvement of src kinase.
Phosphoinositide-dependent protein kinase-1 (PDK1) is a recently identified kinase that phosphorylates and activates protein kinase B (PKB). Activation of PKB by insulin is linked to its translocation from the cytosol to the plasma membrane. However, no data are available yet concerning the localization of PDK1 in insulin-sensitive tissue. Using isolated adipocytes, we studied the effect of insulin and of an insulin-mimicking agent peroxovanadate on the subcellular localization of PDK1. In unstimulated adipocytes, overexpressed PDK1 was mostly cytosolic with a low amount associated to membranes. Peroxovanadate stimulation induced the redistribution of PDK1 to the membranes while insulin was without effect. This peroxovanadate effect was dependent on phosphatidylinositol 3,4,5 triphosphate [PtdIns(3,4,5)P3] production as inhibition of PtdIns 3-kinase by wortmannin or deletion of the PH domain of PDK1 prevented the peroxovanadate-induced translocation of PDK1. Further, peroxovanadate-treatment induced a tyrosine phosphorylation of PDK1 which was wortmannin insensitive and did not require the PH domain of PDK1. An inhibitor of Src kinase (PP2) decreased the peroxovanadate-induced PDK1 tyrosine phosphorylation and overexpression of v-Src stimulated this phosphorylation. Mutation of tyrosine 373 of PDK1 abolished the v-Src induced PDK1 tyrosine phosphorylation and partially reduced the effect of peroxovanadate. Our findings suggest that PDK1 could be a substrate for tyrosine kinases and identify Src kinase as one of the tyrosine kinases able to phosphorylate PDK1.
Grillo
S
S
INSERM E 99-11 and IFR 50, University of Nice, France; MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, UK.
Grémeaux
T
T
Casamayor
A
A
Alessi
D R
DR
Le Marchand-Brustel
Y
Y
Tanti
J F
JF
D064413:3-Phosphoinositide-Dependent Protein Kinases; D017667:Adipocytes / Q000166:cytology / Q000201:enzymology*; D000818:Animals; D002478:Cells, Cultured; D007700:Kinetics; D008297:Male; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014638:Vanadates / Q000494:pharmacology*; D019061:src-Family Kinases / Q000378:metabolism*
D011994:Recombinant Proteins; C069276:peroxovanadate; D019000:Phosphotyrosine; D014638:Vanadates; D019061:src-Family Kinases; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578579:Pdpk1 protein, rat; D017346:Protein Serine-Threonine Kinases