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 His6-PDK1 and His6-PKBβ/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 Vmax = 192 ± 13 U/mg and Km (PDK-Tide) = 55 ± 10 μM 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 His6-PDK1 (pI = 6.8) was generated by treatment with lambda protein phosphatase (λPP), which decreased V max to 2.4 ± 0.4 U/mg and increased Km (PDK-Tide) to 217 ± 61 μM. Isoelectric focusing and Western analyses indicated that the apparent Vmax = 0.21 ± 0.03 U/mg and Km (Crosstide) = 87 ± 30 μM of purified His6-PKBβ/Akt2 results from a mixture of the enzyme monophosphorylated either at Ser-474 (∼90%) or at Thr-309 (∼10%). A purely unphosphorylated isoform of His6-PKBβ/Akt2 (pI = 6.4) was generated by treatment with λPP, which decreased Vmax ∼2-fold. The optimization of high-level production and detailed characterization of purified and λPP-treated His6-PDK1 and His6-PKBβ/Akt2 will facilitate detailed structural and kinetic studies aimed at understanding the mechanism of second messenger-induced activation.
- Phosphoinositide dependent kinase-1
- Protein kinase B
- Sf9 insect cells
ASJC Scopus subject areas