PMID- 10521418 OWN - NLM STAT- MEDLINE DCOM- 19991123 LR - 20210209 IS - 0021-9258 (Print) IS - 0021-9258 (Linking) VI - 274 IP - 43 DP - 1999 Oct 22 TI - Involvement of NH(2)-terminal sequences in the negative regulation of Vav signaling and transforming activity. PG - 30410-8 AB - Deletion of the NH(2)-terminal 65 amino acids of proto-Vav (to form onco-Vav) activates its transforming activity, suggesting that these sequences serve a negative regulatory role in Vav function. However, the precise role of these NH(2)-terminal sequences and whether additional NH(2)-terminal sequences are also involved in negative regulation have not been determined. Therefore, we generated additional NH(2)-terminal deletion mutants of proto-Vav that lack the NH(2)-terminal 127, 168, or 186 amino acids, and assessed their abilities to cause focus formation in NIH 3T3 cells and to activate different signaling pathways. Since Vav mutants lacking 168 or 186 NH(2)-terminal residues showed a several 100-fold greater focus forming activity than that seen with deletion of 65 residues, residues spanning 66 to 187 also contribute significantly to negative regulation of Vav transforming activity. The increase in Vav transforming activity correlated with the activation of the c-Jun, Elk-1, and NF-kappaB transcription factors, as well as increased transcription from the cyclin D1 promoter. Tyrosine 174 is a key site of phosphorylation by Lck in vitro and Lck-mediated phosphorylation has been shown to be essential for proto-Vav GEF function in vitro. However, we found that an NH(2)-terminal Vav deletion mutant lacking this tyrosine residue (DeltaN-186 Vav) retained the ability to be phosphorylated by Lck in vivo and Lck still caused enhancement of DeltaN-186 Vav signaling and transforming activity. Thus, Lck can stimulate Vav via a mechanism that does not involve Tyr(174) or removal of NH(2)-terminal regulatory activity. Finally, we found that NH(2)-terminal deletion enhanced the degree of Vav association with the membrane-containing particulate fraction and that an isolated NH(2)-terminal fragment (residues 1-186) could impair DeltaN-186 Vav signaling. Taken together, these observations suggest that the NH(2) terminus may serve as a negative regulator of Vav by intramolecular interaction with COOH-terminal sequences to modulate efficient membrane association. FAU - Abe, K AU - Abe K AD - Department of Pharmacology, University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina 27599, USA. FAU - Whitehead, I P AU - Whitehead IP FAU - O'Bryan, J P AU - O'Bryan JP FAU - Der, C J AU - Der CJ LA - eng GR - CA42978/CA/NCI NIH HHS/United States GR - CA55008/CA/NCI NIH HHS/United States GR - CA63071/CA/NCI NIH HHS/United States PT - Journal Article PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, P.H.S. PL - United States TA - J Biol Chem JT - The Journal of biological chemistry JID - 2985121R RN - 0 (Cell Cycle Proteins) RN - 0 (Oncogene Proteins) RN - 0 (Proto-Oncogene Proteins) RN - 0 (Proto-Oncogene Proteins c-vav) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Recombinant Proteins) RN - 0 (VAV1 protein, human) RN - 0 (Vav1 protein, mouse) SB - IM MH - 3T3 Cells MH - Animals MH - *Cell Cycle Proteins MH - Cell Line MH - Cell Transformation, Neoplastic MH - Genes, Reporter MH - Humans MH - Mice MH - Oncogene Proteins/chemistry/genetics/*metabolism MH - Proto-Oncogene Proteins/*chemistry/genetics/*metabolism MH - Proto-Oncogene Proteins c-vav MH - Recombinant Fusion Proteins/biosynthesis MH - Recombinant Proteins/chemistry/metabolism MH - Restriction Mapping MH - Sequence Deletion MH - Signal Transduction/*physiology MH - Transfection EDAT- 1999/10/16 00:00 MHDA- 1999/10/16 00:01 CRDT- 1999/10/16 00:00 PHST- 1999/10/16 00:00 [pubmed] PHST- 1999/10/16 00:01 [medline] PHST- 1999/10/16 00:00 [entrez] AID - 10.1074/jbc.274.43.30410 [doi] AID - S0021-9258(19)51698-2 [pii] PST - ppublish SO - J Biol Chem. 1999 Oct 22;274(43):30410-8. doi: 10.1074/jbc.274.43.30410.