Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav

Science

Volumn 279, Issue 5350, 1998, Pages 558-560

  Han, Jaewon ^a   Luby Phelps, Katherine ^b   Das, Balaka ^a   Shu, Xiaodong ^a   Xia, Yi ^a   Mosteller, Raymond D ^a   Krishna, U Murali ^b   Falck, John R ^b   White, Michael A ^b   Broek, Daniel ^a  

^a UNIVERSITY OF SOUTHERN CALIFORNIA   (United States)

^b UNIVERSITY OF TEXAS SOUTHWESTERN MEDICAL CENTER   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

GUANOSINE TRIPHOSPHATASE; MITOGENIC AGENT; PHOSPHOTRANSFERASE;

ARTICLE; CATALYSIS; ENZYME ACTIVATION; ENZYME ACTIVITY; ENZYME PHOSPHORYLATION; PRIORITY JOURNAL; PROTEIN BINDING; SIGNAL TRANSDUCTION;

1-PHOSPHATIDYLINOSITOL 3-KINASE; AMINO ACID SEQUENCE; ANIMALS; CELL LINE; ENZYME ACTIVATION; GTP PHOSPHOHYDROLASES; GTP-BINDING PROTEINS; GUANINE NUCLEOTIDE EXCHANGE FACTORS; GUANOSINE DIPHOSPHATE; GUANOSINE TRIPHOSPHATE; INOSITOL 1,4,5-TRISPHOSPHATE; LYMPHOCYTE SPECIFIC PROTEIN TYROSINE KINASE P56(LCK); MUTAGENESIS, SITE-DIRECTED; ONCOGENE PROTEINS; PHOSPHATIDYLINOSITOL 4,5-DIPHOSPHATE; PHOSPHATIDYLINOSITOL PHOSPHATES; PHOSPHATIDYLINOSITOLS; PHOSPHORYLATION; PROTEINS; PROTO-ONCOGENE PROTEINS C-VAV; RAC GTP-BINDING PROTEINS; RAS GUANINE NUCLEOTIDE EXCHANGE FACTORS; RATS;

EID: 15144353776     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5350.558     Document Type: Article

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25. 3 was counted.
26. Vav expression constructs with point mutations in the PH domain were created by site-directed mutagenesis with polymerase chain reaction (PCR). Overlapping fragments of the Vav PH coding sequence were amplified by PCR from pMB24 (3), a plasmid encoding full-length mouse Vav, with primers containing the point mutations. The fragments were joined by a second round of PCR. The resulting PCR product was digested with Fse I and Nde I and subcloned into pMB24. We created the pRSET-Vav PH domain mutants by replacing the Bgl II fragment of pRSET-Vav(L) (3) with Bgl II fragments from pMB24 PH domain mutants. DNA sequence analysis of the final plasmids confirmed that the mutations indicated in the text were present.
27. 2, 20 mM KCl, and 1 mM dithiothreitol.
28. V12C40 was expressed from the vector pSRalpha (20). Myc-tagged Vav was expressed from the vector pCDNA-3 (Invitrogen) into which the entire wild-type Vav coding region was inserted. For immunofluorescence, injected cells were fixed with 3.7% formaldehyde in phosphate-buffered saline for at least 1 hour at room temperature. Cells were permeabilized with acetone for 3 min at -20°C. Cover slips were incubated for 1 hour at room temperature with a mixture of fluorescein-labeled phalloidin (Molecular Probes) and mouse antibody to Ras (Transduction Labs, Lexington, KY) or mouse monoclonal antibody to Myc (9E10, Santa Cruz Biotechnology, Santa Cruz, CA) followed by rhodamine-conjugated goat antibody to mouse immunoglobulin G (Cappel, Cochranville, PA). The cells were photographed with a Zeiss fluorescence microscope with a chilled Argus charge-coupled-device camera (Hamamatsu).
29. We thank G. Bokoch, T. Roberts, and C. Rudd for providing reagents. Supported by NIH grants CA50261 (D.B.), CA71443 (M.A.W), and GM31278 (J.R.F).