The pleckstrin homology (PH) domain of the protooncogenic serine/threonine protein kinase PKB/Akt can bind phosphoinositides, A yeast-based two-hybrid system was employed which identified inosine-5' monophosphate dehydrogenase (IMPDH) type II as specifically interacting with PKB/Akts PH domain. IMPDH catalyzes the rate-limiting step of de novo guanosine-triphosphate (GTP) biosynthesis. Using purified fusion proteins, PKB/Akts PH domain and IMPDH associated in vitro and this association moderately activated IMPDH. Purified PKB/Akt also associated with IMPDH in vitro, We could specifically pull-down PKB/Akt or IMPDH from mammalian cell lysates using glutathione-S-transferase (GST)IMPDH or GST-PH domain fusion proteins, respectively, Additionally, PKB/Akt and IMPDH could be co-immunoprecipitated from COS cell lysates and active PKB/Akt could phosphorylate IMPDH in vitro. These results implicate PKB/Akt in the regulation of GTP biosynthesis through its interaction with IMPDH, which is involved in providing the GTP pool used by signal transducing G-proteins. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.