Abstract
Byline: Xinxin Gao (b), Peggy Yo (a), Thomas K. Harris (a) Keywords: Phosphoinositide dependent kinase-1; Protein kinase B; Cloning; Expression; Purification; Sf9 insect cells 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.sub.6-PDK1 and His.sub.6-PKB[beta]/Akt2 were optimized, which significantly improved the yields ([greater than or equal to]5-fold) of the affinity purified enzymes over previously reported values. Isoelectric focusing (IEF) and Western analyses indicated that the apparent V.sub.max =192[+ or -]13U/mg and K.sub.m (PDK-Tide)=55[+ or -]10[mu]M of purified His.sub.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.sub.6-PDK1 (pI =6.8) was generated by treatment with lambda protein phosphatase (I'PP), which decreased V.sub.max to 2.4[+ or -]0.4U/mg and increased K.sub.m (PDK-Tide) to 217[+ or -]61[mu]M. Isoelectric focusing and Western analyses indicated that the apparent V.sub.max =0.21[+ or -]0.03U/mg and K.sub.m (Crosstide)=87[+ or -]30[mu]M of purified His.sub.6-PKB[beta]/Akt2 results from a mixture of the enzyme monophosphorylated either at Ser-474 ([approximately equal to]90%) or at Thr-309 ([approximately equal to]10%). A purely unphosphorylated isoform of His.sub.6-PKB[beta]/Akt2 (pI =6.4) was generated by treatment with I'PP, which decreased V.sub.max [approximately equal to]2-fold. The optimization of high-level production and detailed characterization of purified and I'PP-treated His.sub.6-PDK1 and His.sub.6-PKB[beta]/Akt2 will facilitate detailed structural and kinetic studies aimed at understanding the mechanism of second messenger-induced activation. Author Affiliation: (a) Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, P.O. Box 016129 (R-629), Miami, FL 33101-6129, USA (b) Department of Chemistry, University of Miami, P.O. Box 249118, Coral Gables, FL 33124-0431, USA Article History: Received 1 February 2004; Revised 15 March 2005
