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HA-Akt and the mutant HA-Akt(R25C) have been described (4). The piSH2·MT plasmid is described in [A. Klippel, J. A. Escobedo, Q. Hu, L. T. Williams,Mol. Cell. Biol.13, 5560 (1993)]. p110·MT was generated by inserting bovine p110α into pCMV-6 with the primers 5′-ATGGTCGACATGCCTCCAAGACCATCA-3′ and 5′-CTTCTGCTCTCCCCCGGGGTTCAATGCATGCTGTTTAATTGTGTG-3′ or 5′-TATGGATCCTCAGTTCAGGTCCTCCTCGGAAATCAGCTTCTGCTCTCCCCCGGG-3′.

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Human platelets were stimulated as described (11) and lysed in 2× NP-40 lysis buffer [1% NP-40, 10% glycerol, 137 mM NaCl, 20 mM tris-HCl (pH 7.4)] containing inhibitors. Akt kinase activity was determined by immunoprecipitation (4).

Synthetic DiC₁₆PtdIns-3-P (Matreya, Pleasant Gap, PA), DiC₁₆PtdIns-3,4-P₂ (from C.-S. Chen or from Matreya), DiC₁₆PtdIns-3,4,5-P₃ (5), and PtdIns or PtdIns-4,5-P₂ (Sigma) were dried together with phosphatidylserine (PS) and phosphatidylcholine (PC). Lipids were resuspended in 10 mM Hepes (pH 7.0) containing 1 mM EDTA and sonicated. Mixed vesicles containing PS and PC and PtdIns, PtdIns-4,5-P₂, DiC₁₆PtdIns-3-P, DiC₁₆PtdIns-3,4-P₂, or DiC₁₆PtdIns-3,4,5-P₃ were added to purified Akt at 10 μM. Akt and Akt(R25C) baculovirus expression constructs were generated in pVL1392 (Pharmingen) with the primers 5′-TAACCATGGACGACGTAGCCATTGTGAAGG-3′ and 5′-ACCGGATCCTCAGGCTGTGCCACTGGCTGA-3′. Sf9 cells expressing recombinant Akt were homogenized in 40 mM triethanolamine (TEA; pH 7.6) containing inhibitors. Recombinant protein was purified by fast protein liquid chromatography (FPLC) in three steps on HiLoadQ, Heparin, and MonoQ columns (Pharmacia) equilibrated in 40 mM TEA (pH 7.6). Bound proteins were eluted with linear gradients of 0 to 0.5 M, 0 to 0.7 M, and 0 to 0.3 M KCl in TEA (pH 7.6), respectively, and assayed by immunoblotting (4). The fractions were assayed for Akt activity, and their purity was determined by staining; a greater than 95% purity was achieved.

Derman M. P., et al.,J. Biol. Chem.in press.

Synthetic phosphoinositides were prepared by sonication in the absence of carrier phospholipid and added to serum-starved cells for 10 min. After incubation at 37°C, the cells were harvested and Akt activity was determined.

Constructs in pGEX-2T (Pharmacia) were generated from fragments encoding the PH domain of Akt (amino acids 1 through 106) with the primers 5′-TCTGGATCCAACGACGTAGCCATTGTGAA-3′ and 5′-ACCGAATTCCACAGTCTGAATGGCGGT-3′. The primers 5′-TCTGGATCCAACGACGTAGCCATTGTGAA-3′ and 5′-CATGAATTCCATGGTCACACGGTGCTT-3′ were used to generate GST-Akt AH (amino acids 1 through 147).

PtdIns-4,5-P₂, DiC₁₆PtdIns-3,4-P₂, and DiC₁₆PtdIns3,4,5-P₃ were prepared by sonication in the absence of carrier lipids. Inositol-1,3,4-trisphosphate, inositol-1,4,5-trisphosphate, and inositol-1,3,4,5-tetrakisphosphate were prepared in 10 mM Hepes (pH 7.4). Inositolphosphates and phospholipids were added to bound GST fusion protein on beads together with³²P-labeled phosphoinositides. Bound lipids were resolved by TLC.

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Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

We thank J. Downward (Imperial Cancer Research Fund) for MT-p110(K227E) and for sharing information before publication; A. Couvillon and C. Carpenter (HMS) for purified PI 3-kinase; C.-S. Chen (University of Kentucky) for DiC₁₆PtdIns-3,4-P₂ and DiC₁₆PtdIns-3,4,5-P₃; T. Copeland (NCI-ABL) for synthesizing Akt peptides; T. Chan, R. Friedrich, A. Kazlauskas, Z. Songyang, and P. Tsichlis for critical comments; and G. vande Woude for advice and support. Supported by the National Cancer Institute contract N01-CO-74101 with ABL (D.R.K.), the National Cancer Institute of Canada (D.R.K.), and by USPHS grant GM41890 (L.C.C.). D.R.K. is a recipient of the H. E. Johns and Canadian Cancer Society Research Scientist Award from the National Cancer Institute of Canada. A.T. is supported by the Medical Foundation, Boston, MA. T.F.F. is a recipient of the K. M. Hunter Fellowship in Cancer Research from the National Cancer Institute of Canada supported with funds provided by the Terry Fox Run. T.F.F. and D.R.K. acknowledge the support by the ABL-Basic Research Program.