Prohtein kinase B kinases that mediate phosphatidylinositol 3,4,5- trisphosphate-dependent activation of protein kinase B

Science

Volumn 279, Issue 5351, 1998, Pages 710-714

  Stephens, Len ^a   Anderson, Karen ^a   Stokoe, David ^a   Erdjument Bromage, Hediye ^a   Painter, Gavin F ^a   Holmes, Andrew B ^a   Gaffney, Piers R J ^a   Reese, Colin B ^a   McCormick, Frank ^a   Tempst, Paul ^a   Coadwell, J ^a   Hawkins, Phillip T ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

PHOSPHATIDYLINOSITOL; PHOSPHATIDYLINOSITOL 3 KINASE; PLECKSTRIN; PROTEIN KINASE; AKT1 PROTEIN, DROSOPHILA; COMPLEMENTARY DNA; DROSOPHILA PROTEIN; LIPOSOME; ONCOPROTEIN; PHOSPHATIDYLINOSITOL 3,4 BISPHOSPHATE; PHOSPHATIDYLINOSITOL 3,4,5 TRISPHOSPHATE; PHOSPHATIDYLINOSITOL 3,4,5-TRIPHOSPHATE; PHOSPHATIDYLINOSITOL 3,4-DIPHOSPHATE; PLATELET DERIVED GROWTH FACTOR; POLYPHOSPHOINOSITIDE; PROTEIN KINASE B; PROTEIN KINASE B KINASE; PROTEIN SERINE THREONINE KINASE; RECOMBINANT PROTEIN;

ARTICLE; ENZYME ACTIVATION; ENZYME ACTIVE SITE; ENZYME PHOSPHORYLATION; PRIORITY JOURNAL; PROTEIN LIPID INTERACTION; SIGNAL TRANSDUCTION; ALTERNATIVE RNA SPLICING; AMINO ACID SEQUENCE; ANIMAL; CELL LINE; CELL MEMBRANE; CHEMISTRY; DROSOPHILA; ENZYMOLOGY; GENETICS; HUMAN; ISOLATION AND PURIFICATION; METABOLISM; MOLECULAR CLONING; MOLECULAR GENETICS; OPEN READING FRAME; PHOSPHORYLATION; RAT; SHEEP;

ALTERNATIVE SPLICING; AMINO ACID SEQUENCE; ANIMALS; CELL LINE; CELL MEMBRANE; CLONING, MOLECULAR; DNA, COMPLEMENTARY; DROSOPHILA; DROSOPHILA PROTEINS; ENZYME ACTIVATION; HUMANS; LIPOSOMES; MOLECULAR SEQUENCE DATA; OPEN READING FRAMES; PHOSPHATIDYLINOSITOL PHOSPHATES; PHOSPHORYLATION; PLATELET-DERIVED GROWTH FACTOR; PROTEIN-SERINE-THREONINE KINASES; PROTO-ONCOGENE PROTEINS; PROTO-ONCOGENE PROTEINS C-AKT; RATS; RECOMBINANT PROTEINS; SHEEP; SIGNAL TRANSDUCTION;

EID: 0032578999     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5351.710     Document Type: Article

References (34)

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17. L. Stephens and N. Huskisson, unpublished data.
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22. 32P-labeled 0.3-kb Eco RI-Hind III fragment derived from IMAGE clone 526583. Positive plaques were identified and purified and the cDNAs were excised as pBluescript-based plasmids. DNA sequencing was performed on an Applied Biosystems automatic sequencer at the Babraham Institute Microchemical Facility.
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25. 2-terminal EE-tag or a myc-tag in the pCMV3 transient expression vector (19) by standard polymerase chain reaction-based cloning strategies. Versions were also constructed with a cDNA missing the nucleotides coding for residues 238 to 263 (this was made with IMAGE clones 510982 and 526583, referred to in the text as the catalytically inactive splice variant). All constructs were verified by sequencing.
26. -1) for 45 s; assays were conducted as described in (6). In parallel experiments to examine the effects of transient expression of PKB kinase on another protein kinase cascade, cells were cotransfected with (myc)-ERK-2. Anti-(myc)-directed immunoprecipitates were assayed as described by S. Cook et al. [EMBO J. 12, 3475 (1993)].
27. K. Anderson, unpublished data.
28. B. A. Hemmings Science 277, 534 (1997).
29. 32P]ATP of defined specific radioactivity by an excess of PKB kinase).
30. -1).
31. 32P]ATP (19)} was sonicated into solution (10 μCi in 10 ml) and applied to a filter for 20 min at room temperature and then washed away with fresh solution (five times over 5 min) and finally with PBS containing 1% NP-40. The filter was air dried and autoradiographed.
32. 32P]ATP (1 μM final concentration) and (EE)-PKB (2.5 μM final concentration). If the assays were to estimate association of the kinases with the lipid vesicles, then after 4 min at 30°C the assay mixtures were centrifuged (200,000g for 20 min). Portions of the supernatants were removed for assays or immunoblotting. The pellets were rinsed rapidly with assay buffer, recentrifuged, and dissolved in SDS sample buffer. Phosphorylation of PKB or PKBs was quantitated as described above.
33. Single-letter abbreviations for 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.
34. K.A. is an Australian National Health and Medical Research Council C. J. Martin/R. G. Menzies Fellow. P.T.H. is a BBSRC Senior Research Fellow. Work in the laboratories of C.B.R. and A.B.H. was supported by grants from the Wellcome Trust and the Biotechnology and Biological Sciences Research Council. DNA sequencing and oligonucleotide synthesis were superbly handled by the Microchemical Facility at the Babraham Institute.