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Background: The activation of protein kinase B (PKB, also known as c-Akt) is stimulated by insulin or growth factors and results from its phosphorylation at Thr308 and Ser473. We recently identified a protein kinase, termed PDK1, that phosphorylates PKB at Thr308 only in the presence of lipid vesicles containing phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) or phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ). Results: We have cloned and sequenced human PDK1. The 556-residue monomeric enzyme comprises a catalytic domain that is most similar to the PKA, PKB and PKC subfamily of protein kinases and a carboxy-terminal pleckstrin homology (PH) domain. The PDK1 gene is located on human chromosome 16p 13.3 and is expressed ubiquitously in human tissues. Human PDK1 is homologous to the Drosophila protein kinase DSTPK61, which has been implicated in the regulation of sex differentiation, oogenesis and spermatogenesis. Expressed PDK1 and DSTPK61 phosphorylated Thr308 of PKB α only in the presence of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 . Overexpression of PDK1 in 293 cells activated PKB α and potentiated the IGF1-induced phosphorylation of PKB α at Thr308. Experiments in which the PH domains of either PDK1 or PKB α were deleted indicated that the binding of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 to PKB α is required for phosphorylation and activation by PDK1. IGF1 stimulation of 293 cells did not affect the activity or phosphorylation of PDK1. Conclusions: PDK1 is likely to mediate the activation of PKB by insulin or growth factors. DSTPK61 is a Drosophila homologue of PDK1. The effect of PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 in the activation of PKB α is at least partly substrate directed.