PDK1 and PKB/Akt have a pleckstrin homology (PH) domain at the C-terminus and N-terminus, respectively, which stabilizes an unphosphorylated, autoinhibited conformation. Binding of the PH domain to a phospholipid second messenger causes relief of autoinhibition, which results in kinase phosphorylation and activation. Baculovirus-mediated expression in Sf9 insect cells of both His(6)-PDK1 and His(6)-PKB beta/Akt2 were optimized, which significantly improved the yields (>= 5-fold) of the affinity purified enzymes over previously reported values. Isoelectric focusing (IEF) and Western analyses indicated that the apparent V-max = 192 +/- 13 U/mg and K-m (PDK Tide) = 55 +/- 10 M of purified His(6)-PDK1 results from a mixture of at least three different phospho-specific isoforms (pI values of 6.8, 6.5, and 6.4). A purely unphosphorylated isoform of His(6)-PDK1 (pI= 6.8) was generated by treatment with lambda protein phosphatase (lambda PP), which decreased V-max to 2.4 +/- 0.4 U/mg and increased K-m (PDK-Tide) to 217 +/- 61 mu M. Isoelectric focusing and Western analyses indicated that the apparent V-max = 0.21 +/- 0.03 U/mg and K-m (Crosstide) = 87 +/- 30 mu M of purified His(6)-PKB beta/Akt2 results from a mixture of the enzyme monophosphorylated either at Ser-474 (similar to 90%) or at Thr-309 (similar to 10%). A purely unphosphorylated isoform of His(6)-PKB beta/Akt2 (pI= 6.4) was generated by treatment with lambda PP, which decreased V-max similar to 2-fold. The optimization of high-level production and detailed characterization of purified and lambda PP-treated His(6)-PDK1 and His(6)-PKB beta/Akt2 will facilitate detailed structural and kinetic studies aimed at understanding the mechanism of second messenger-induced activation. (c) 2005 Elsevier Inc. All rights reserved.