Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin

Science

Volumn 277, Issue 5322, 1997, Pages 99-101

  Brunn, Gregory J ^a   Hudson, Christine C ^b   Sekulić, Aleksandar ^b   Williams, Josie M ^b   Hosoi, Hajime ^c   Houghton, Peter J ^c   Lawrence Jr , John C ^a   Abraham, Robert T ^b  

^a UNIVERSITY OF VIRGINIA   (United States)

^b MAYO CLINIC   (United States)

^c ST JUDE CHILDREN S RESEARCH HOSPITAL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

INSULIN; PROTEIN KINASE; RAPAMYCIN; REPRESSOR PROTEIN; REPRESSOR PROTEIN PHAS I; UNCLASSIFIED DRUG;

ARTICLE; CELL CYCLE G1 PHASE; EMBRYO; GENE CONTROL; HUMAN; HUMAN CELL; KIDNEY CELL; PRIORITY JOURNAL; PROTEIN PHOSPHORYLATION; RNA TRANSLATION;

ADAPTOR PROTEINS, SIGNAL TRANSDUCING; ANDROSTADIENES; ANIMALS; CARRIER PROTEINS; CELL LINE; DNA-BINDING PROTEINS; EUKARYOTIC INITIATION FACTOR-4E; G1 PHASE; HEAT-SHOCK PROTEINS; HUMANS; INSULIN; PEPTIDE INITIATION FACTORS; PHOSPHOPROTEINS; PHOSPHORYLATION; PHOSPHOTRANSFERASES (ALCOHOL GROUP ACCEPTOR); POLYENES; PROTEIN KINASES; RATS; RECOMBINANT PROTEINS; REPRESSOR PROTEINS; SIGNAL TRANSDUCTION; SIROLIMUS; TACROLIMUS BINDING PROTEINS; TRANSFECTION; TUMOR CELLS, CULTURED;

EID: 0030881836     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5322.99     Document Type: Article

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32. 2035→lle substitution that was created with the Transformer kit (Clontech). Both cDNAs were tagged at their 5′-termini with nucleotide sequences encoding the Asp-Thr-Tyr-Arg-Tyr-lle sequence recognized by mAb AU1 (Babco). The PHAS-I expression vector, pCMV4-PHAS-I, is described by J. Lawrence (Advances in Enzyme Regulation, in press).
33. 5 cells per dish and were cultured for 24 hours in Dulbecco's modified Eagles' medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The cells were transfected with 2 μg of pCMV4-PHAS-I and 4 μg of pcDNA3 only or the mTOR-wt or mTOR-rr expression plasmids. Transfections were done with the TransIT reagent (Pan Vera, Madison, WI). After 12 hours, the cells were rested in DMEM containing 0.1% FBS and were stimulated with insulin and treated with drugs as indicated in the figure legends. Cells were washed with phosphate-buffered saline (PBS), then scraped into lysis buffer [50 mM β-glycerophosphate (pH 7.4), 1.5 mM EGTA, supplemented with 1% NP-40, 1 mM dithiothreitol, 20 nM microcystin-LR, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), leupeptin (10 μg/ml), aprotinin (5 μg/ml). and pepstatin (5 μg/ml)]. After centrifugation at 10,000g, extracts were equalized for protein content and subjected to SDS-PAGE through 12.5 or 7.5% gels for PHAS-I or mTOR immunoblots, respectively. PHAS-I immunoblots were performed as described [T. A. Lin and J. C. Lawrence, J. Biol. Chem. 271, 30199 (1996)]. AU1-tagged mTOR proteins were blotted with mAb AU1 followed by rabbit antibodies to mouse immunoglobulin G (Pierce). The blots were developed with horseradish peroxidase coupled to protein A and the Enhanced Chemiluminescence reagent (Amersham).
34. 32P into PHAS-I was quantitated with an Ambis Imaging system.
35. 2338→Ala) was prepared in pcDNA3 as described in (21). The protocols for 293 cell transfections with mTOR-rr-and mTOR-rr-kd-encoding plasmids and sample preparation are given in (22).
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38. We thank E. O'Neill for providing the FLAG-p38 expression plasmid, S. Sutor for technical assistance, and K. Jensen for assistance with the preparation of this manuscript. Supported by NIH grants DK28312, AR41189, and DK50628 (to J.C.L.), CA23099 (to P.J.H.), and CA52995 (to R.T.A.) and by American Cancer Society grants RPG-95-031-03-DHP (to P.J.H.) and RPG-95-040-03 (to R.T.A.). R.T.A. is a Leukemia Society of America Scholar.