Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate

Science

Volumn 275, Issue 5300, 1997, Pages 665-668

  Franke, Thomas F ^a,b,c   Kaplan, David R ^a,b   Cantley, Lewis C ^d   Toker, Alex ^d,e  

^a NATIONAL CANCER INSTITUTE   (United States)

^b MCGILL UNIVERSITY   (Canada)

^c BETH ISRAEL DEACONESS MEDICAL CENTER   (United States)

^d Division of Signal Transduction   (United States)

^e BOSTON BIOMEDICAL RESEARCH INSTITUTE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

PHOSPHATIDYLINOSITOL 4,5 BISPHOSPHATE; PLECKSTRIN; PROTEIN SERINE THREONINE KINASE;

APOPTOSIS; ARTICLE; CELL STIMULATION; DIMERIZATION; ENZYME REGULATION; PRIORITY JOURNAL; PROTEIN DOMAIN; PROTO ONCOGENE; SEQUENCE HOMOLOGY;

1-PHOSPHATIDYLINOSITOL 3-KINASE; 3T3 CELLS; ANIMALS; COS CELLS; DIMERIZATION; ENZYME ACTIVATION; MICE; PHOSPHATIDYLINOSITOL PHOSPHATES; PHOSPHORYLATION; PHOSPHOTRANSFERASES (ALCOHOL GROUP ACCEPTOR); PLATELET-DERIVED GROWTH FACTOR; POINT MUTATION; PROTEIN-SERINE-THREONINE KINASES; PROTO-ONCOGENE PROTEINS; PROTO-ONCOGENE PROTEINS C-AKT; RECOMBINANT FUSION PROTEINS;

EID: 0031039024     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5300.665     Document Type: Article

References (25)

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10. T. F. Franke, D. R. Kaplan, L. C. Cantley, A. Toker, unpublished data.
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13. Human platelets were stimulated as described (11) and lysed in 2× NP-40 lysis buffer [1 % NP-40, 10% glyoerol, 137 mM NaCl, 20 mM tris-HCI (pH 7.4)] containing inhibitors. Akt kinase activity was determined by immunoprecipitation (4).
14. 3 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′ -ACCGGATCCTCAGGCTGTGCCACTG-GCTGA-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 KCI 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.
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16. 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.
17. Constructs in pGEX-2T (Pharmacia) were generated from fragments encoding the PH domain of Akt (amino acids 1 through 106) with the primers 5′-TCTG-GATCCAACGACGTAGCCATTGTGAA-3′ and 5′-ACCGAATTCCACAGTCTGAATGGCGGT-3′. The primers 5′-TCTGGATCCAACGACGTAGCCATT-GTGAA-3′ and 5′-CATGAATTCCATGGTCACACG-GTGCTT-3′ were used to generate GST-Akt AH (amino acids 1 through 147).
18. 32P-labeled phosphoinositides. Bound lipids were resolved by TLC.
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24. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lle; 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.
25. 3; T. Copeland (NCl-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. AT. 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.