{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T06:25:32Z","timestamp":1779344732399,"version":"3.51.4"},"reference-count":29,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5350","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[1998,1,23]]},"abstract":"<jats:p>Mitogen stimulation of cytoskeletal changes and c-jun amino-terminal kinases is mediated by Rac small guanine nucleotide-binding proteins. Vav, a guanosine diphosphate (GDP)\u2013guanosine triphosphate (GTP) exchange factor for Rac that stimulates the exchange of bound GDP for GTP, bound to and was directly controlled by substrates and products of phosphoinositide (PI) 3-kinase. The PI 3-kinase substrate phosphatidylinositol-4,5-bisphosphate inhibited activation of Vav by the tyrosine kinase Lck, whereas the product phosphatidylinositol-3,4,5-trisphosphate enhanced phosphorylation and activation of Vav by Lck. Control of Vav in response to mitogens by the products of PI 3-kinase suggests a mechanism for Ras-dependent activation of Rac.<\/jats:p>","DOI":"10.1126\/science.279.5350.558","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:42:47Z","timestamp":1027762967000},"page":"558-560","source":"Crossref","is-referenced-by-count":658,"title":["Role of Substrates and Products of PI 3-kinase in Regulating Activation of Rac-Related Guanosine Triphosphatases by Vav"],"prefix":"10.1126","volume":"279","author":[{"given":"Jaewon","family":"Han","sequence":"first","affiliation":[{"name":"J. Han, B. Das, X. Shu, Y. Xia, R. D. Mosteller, D. Broek, Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033\u20130800, USA."},{"name":"K. Luby-Phelps and M. A. White, Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235\u20139039, USA."},{"name":"U. M. Krishna and J. R. Falck, Department of Biochemistry, University of Texas Southwestern Medical Center,"},{"name":"5323 Harry Hines Boulevard, Dallas, TX 75235\u20139039, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Katherine","family":"Luby-Phelps","sequence":"additional","affiliation":[{"name":"J. Han, B. Das, X. Shu, Y. Xia, R. D. Mosteller, D. Broek, Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033\u20130800, USA."},{"name":"K. Luby-Phelps and M. A. White, Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235\u20139039, USA."},{"name":"U. M. Krishna and J. R. Falck, Department of Biochemistry, University of Texas Southwestern Medical Center,"},{"name":"5323 Harry Hines Boulevard, Dallas, TX 75235\u20139039, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Balaka","family":"Das","sequence":"additional","affiliation":[{"name":"J. Han, B. Das, X. Shu, Y. Xia, R. D. Mosteller, D. Broek, Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033\u20130800, USA."},{"name":"K. Luby-Phelps and M. A. White, Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235\u20139039, USA."},{"name":"U. M. Krishna and J. R. Falck, Department of Biochemistry, University of Texas Southwestern Medical Center,"},{"name":"5323 Harry Hines Boulevard, Dallas, TX 75235\u20139039, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaodong","family":"Shu","sequence":"additional","affiliation":[{"name":"J. Han, B. Das, X. Shu, Y. Xia, R. D. Mosteller, D. Broek, Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033\u20130800, USA."},{"name":"K. Luby-Phelps and M. A. White, Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235\u20139039, USA."},{"name":"U. M. Krishna and J. R. 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Falck, Department of Biochemistry, University of Texas Southwestern Medical Center,"},{"name":"5323 Harry Hines Boulevard, Dallas, TX 75235\u20139039, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Raymond D.","family":"Mosteller","sequence":"additional","affiliation":[{"name":"J. Han, B. Das, X. Shu, Y. Xia, R. D. Mosteller, D. Broek, Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033\u20130800, USA."},{"name":"K. Luby-Phelps and M. A. White, Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235\u20139039, USA."},{"name":"U. M. Krishna and J. R. Falck, Department of Biochemistry, University of Texas Southwestern Medical Center,"},{"name":"5323 Harry Hines Boulevard, Dallas, TX 75235\u20139039, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"U. Murali","family":"Krishna","sequence":"additional","affiliation":[{"name":"J. Han, B. Das, X. Shu, Y. Xia, R. D. Mosteller, D. Broek, Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033\u20130800, USA."},{"name":"K. Luby-Phelps and M. A. White, Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235\u20139039, USA."},{"name":"U. M. Krishna and J. R. Falck, Department of Biochemistry, University of Texas Southwestern Medical Center,"},{"name":"5323 Harry Hines Boulevard, Dallas, TX 75235\u20139039, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"John R.","family":"Falck","sequence":"additional","affiliation":[{"name":"J. Han, B. Das, X. Shu, Y. Xia, R. D. Mosteller, D. Broek, Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033\u20130800, USA."},{"name":"K. Luby-Phelps and M. A. White, Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235\u20139039, USA."},{"name":"U. M. Krishna and J. R. Falck, Department of Biochemistry, University of Texas Southwestern Medical Center,"},{"name":"5323 Harry Hines Boulevard, Dallas, TX 75235\u20139039, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael A.","family":"White","sequence":"additional","affiliation":[{"name":"J. Han, B. Das, X. Shu, Y. Xia, R. D. Mosteller, D. Broek, Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033\u20130800, USA."},{"name":"K. Luby-Phelps and M. A. White, Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235\u20139039, USA."},{"name":"U. M. Krishna and J. R. Falck, Department of Biochemistry, University of Texas Southwestern Medical Center,"},{"name":"5323 Harry Hines Boulevard, Dallas, TX 75235\u20139039, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daniel","family":"Broek","sequence":"additional","affiliation":[{"name":"J. Han, B. Das, X. Shu, Y. Xia, R. D. Mosteller, D. Broek, Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033\u20130800, USA."},{"name":"K. Luby-Phelps and M. A. White, Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235\u20139039, USA."},{"name":"U. M. Krishna and J. R. 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Biol. 17, 1346 (1997).","journal-title":"Mol. Cell. Biol."},{"key":"e_1_3_1_5_2","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1038\/385169a0","volume":"385","author":"Crespo P.","year":"1997","unstructured":"Crespo P., et al., Nature 385, 169 (1997).","journal-title":"Nature"},{"key":"e_1_3_1_6_2","first-page":"6848","volume":"14","author":"Khosravi-Far R.","year":"1994","unstructured":"Khosravi-Far R., et al., Mol. Cell. Biol. 14, 6848 (1994).","journal-title":"Mol. Cell. Biol."},{"key":"e_1_3_1_7_2","first-page":"2405","volume":"9","author":"Bustelo X. R.","year":"1994","unstructured":"Bustelo X. R., et al., Oncogene 9, 2405 (1994).","journal-title":"Oncogene"},{"key":"e_1_3_1_8_2","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1016\/S0167-5699(97)01037-2","volume":"18","author":"Collins T. L.","year":"1997","unstructured":"Collins T. L., Deckert M., Altman A., Immunol. Today 18, 221 (1997).","journal-title":"Immunol. Today"},{"key":"e_1_3_1_9_2","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1016\/S0955-0674(96)80066-4","volume":"8","author":"Marshall C. J.","year":"1996","unstructured":"Marshall C. J., Curr. Opin. Cell Biol. 8, 197 (1996).","journal-title":"Curr. Opin. Cell Biol."},{"key":"e_1_3_1_10_2","doi-asserted-by":"crossref","first-page":"168","DOI":"10.1038\/371168a0","volume":"371","author":"Harlan J. E.","year":"1994","unstructured":"Harlan J. E., Hajduk P. J., Yoon H. S., Fesik S. W., Nature 371, 168 (1994).","journal-title":"Nature"},{"key":"e_1_3_1_11_2","doi-asserted-by":"crossref","first-page":"4676","DOI":"10.1002\/j.1460-2075.1995.tb00149.x","volume":"14","author":"Hyvonen M.","year":"1995","unstructured":"Hyvonen M., et al., EMBO J. 14, 4676 (1995).","journal-title":"EMBO J."},{"key":"e_1_3_1_12_2","doi-asserted-by":"crossref","first-page":"1037","DOI":"10.1016\/0092-8674(95)90219-8","volume":"83","author":"Ferguson K. M.","year":"1995","unstructured":"Ferguson K. M., Lemmon M. A., Schlessinger J., Sigler P. B., Cell 83, 1037 (1995).","journal-title":"Cell"},{"key":"e_1_3_1_13_2","doi-asserted-by":"crossref","first-page":"3385","DOI":"10.1021\/jo00116a023","volume":"60","author":"Reddy K. K.","year":"1995","unstructured":"Reddy K. K., Saady M., Whited G., Falck J. R., J. Org. Chem. 60, 3385 (1995).","journal-title":"J. Org. Chem."},{"key":"e_1_3_1_14_2","doi-asserted-by":"crossref","unstructured":"M. P. Derman et al. J. Biol. Chem. 272 6465 (1997).","DOI":"10.1074\/jbc.272.10.6465"},{"key":"e_1_3_1_15_2","unstructured":"J. Han and D. Broek unpublished data."},{"key":"e_1_3_1_16_2","doi-asserted-by":"crossref","first-page":"21999","DOI":"10.1074\/jbc.272.35.22059","volume":"272","author":"Rameh L. E.","year":"1997","unstructured":"Rameh L. E., et al., J. Biol. Chem. 272, 21999 (1997).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_17_2","doi-asserted-by":"publisher","DOI":"10.1038\/42648"},{"key":"e_1_3_1_18_2","unstructured":"J. Han unpublished data."},{"key":"e_1_3_1_19_2","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1016\/S1074-7613(00)80273-3","volume":"5","author":"Deckert M. S.","year":"1996","unstructured":"Deckert M. S., Tartare-Deckert S., Couture C., Mustelin T., Altman A., Immunity 5, 591 (1996).","journal-title":"Immunity"},{"key":"e_1_3_1_20_2","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1016\/S0092-8674(00)80226-3","volume":"89","author":"Rodriguez-Viciana P.","year":"1997","unstructured":"Rodriguez-Viciana P., et al., Cell 89, 457 (1997).","journal-title":"Cell"},{"key":"e_1_3_1_21_2","doi-asserted-by":"crossref","unstructured":"M. A. White et al. ibid 80 533 (1995).","DOI":"10.1016\/0092-8674(95)90507-3"},{"key":"e_1_3_1_22_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.271.5250.810"},{"key":"e_1_3_1_23_2","doi-asserted-by":"crossref","unstructured":"A. S. Nimnual B. A. Yatsula D. Bar-Sagi ibid. 279 XXX (1998).","DOI":"10.1126\/science.279.5350.560"},{"key":"e_1_3_1_24_2","doi-asserted-by":"crossref","unstructured":"D.Stokoe et al. ibid. 277 567 (1997).","DOI":"10.1126\/science.277.5325.567"},{"key":"e_1_3_1_25_2","first-page":"2862","volume":"14","author":"Raab M.","year":"1994","unstructured":"Raab M., Yamamoto M., Rudd C. E., Mol. Cell. Biol. 14, 2862 (1994).","journal-title":"Mol. Cell. Biol."},{"key":"e_1_3_1_26_2","unstructured":"Either C8PtdIns(4 5)P 2 -NH 2 or C8PtdIns(3 4 5)P 3 -NH 2 (250 \u03bcg) in 10 \u03bcl of 0.1 M sodium borate buffer (pH 8.5) was added to the dried 125 I-labeled Bolton-Hunter reagent (NEN Boston MA) and incubated for 15 min at 0\u00b0C. To obviate subsequent conjugation we reacted any unreacted ester with 0.5 ml of 0.2 M of glycine in 0.1 M borate buffer (pH 8.5) for 5 min at 0\u00b0C. Gel filtration (Sephadex G-10; Pharmacia) was used to separate labeled phospholipids from the glycine conjugates and hydrolysis products. Thin-layer chromatography was used to identify fractions containing the iodinated phosphoinositides. His-tagged Vav protein (10 pmol) bound to Ni-agarose was incubated with 0 1 2.5 5 25 or 50 \u03bcM iodinated C8PtdIns(4 5)P 2 in 100 \u03bcl of 20 mM tris-HCl  (pH 7.5) 50 mM NaCl 5 mM MgCl 2 and 0.2% Triton X-100 for 2 hours at room temperature and washed twice with the same buffer. Bound iodinated C8PtdIns(4 5)P 2 was measured with a gamma counter. For the competitive binding assay 125 I-C8PtdIns(3 4 5)P 3 was incubated with His-tagged Vav protein bound to Ni-agarose in the presence or absence of 50 \u03bcM C8PtdIns(4 5)P 2 or 50 \u03bcM C8PtdIns(3 4 5)P 3 in the same buffer mentioned above and the amount of bound iodinated C8PtdIns(3 4 5)P 3 was measured. His-tagged Vav mutant proteins bound to Ni-agarose were used for incubation with 125 I-C8PtdIns(3 4 5)P 3 and bound C8PtdIns(3 4 5)P 3 was counted."},{"key":"e_1_3_1_27_2","unstructured":"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."},{"key":"e_1_3_1_28_2","unstructured":"His-tagged Vav protein (20 pmol) bound to Ni-agarose was washed with 10 mM Hepes (pH 7.0) 10 \u03bcM [\u03b3- 32 P]ATP and 12 mM MgCl 2 and incubated with 5 ng of recombinant Lck tyrosine kinase (Upstate Biotechnology Lake Placid NY) for various incubation times at 37\u00b0C in the presence or absence of 50 \u03bcM C8PtdIns(4 5)P 2 or 50 \u03bcM C8PtdIns(3 4 5)P 3 . Phosphorylated Vav proteins bound to Ni-agarose were then washed with 10 mM Hepes (pH 7.0) and 12 mM MgCl 2 and the resulting products were separated by SDS-PAGE. At completion of the kinase reactions the stoichiometry of phosphate to Vav was determined to be about 0.4 to 1. For Lck phosphorylation of Vav protein used in GEF assays a similar protocol was used except that Lck kinase purified from a baculovirus expression system (24) was used and the phosphorylated Vav protein was washed and resuspended in 50 mM tris-HCl (pH 7.5) 5 mM MgCl 2 20 mM KCl and 1 mM dithiothreitol."},{"key":"e_1_3_1_29_2","unstructured":"Nuclear injection of REF-52 cells was performed as described (21). Ras 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 \u221220\u00b0C. 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)."},{"key":"e_1_3_1_30_2","unstructured":"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)."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.279.5350.558","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T06:02:04Z","timestamp":1705125724000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.279.5350.558"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1998,1,23]]},"references-count":29,"journal-issue":{"issue":"5350","published-print":{"date-parts":[[1998,1,23]]}},"alternative-id":["10.1126\/science.279.5350.558"],"URL":"https:\/\/doi.org\/10.1126\/science.279.5350.558","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[1998,1,23]]}}}