Tyrosine residues at the carboxyl terminus of Vav1 play an important role in regulation of its biological activity.
The guanine nucleotide exchange factor (GEF) Vav1 is an essential signal transducer protein in the hematopoietic system, where it is expressed physiologically. It is also involved in several human malignancies. Tyrosine phosphorylation at the Vav1 amino terminus plays a central role in regulating its activity; however, the role of carboxyl terminal tyrosine residues is unknown. We found that mutation of either Tyr-826 (Y826F) or Tyr-841 (Y841F) to phenylalanine led to loss of Vav1 GEF activity. When these Vav1 mutants were ectopically expressed in pancreatic cancer cells lacking Vav1, they failed to induce growth in agar, indicating loss of transforming potential. Furthermore, although Y841F had no effect on Vav1-stimulated nuclear factor of activated T cells (NFAT) activity, Y826F doubled NFAT activity when compared with Vav1, suggesting that Tyr-826 mediates an autoinhibitory effect on NFAT activity. SH2 profiling revealed that Shc, Csk, Abl, and Sap associate with Tyr-826, whereas SH2-B, Src, Brk, GTPase-activating protein, and phospholipase C-gamma associate with Tyr-841. Although the mutations in the Tyr-826 and Tyr-841 did not affect the binding of the carboxyl SH3 of Vav1 to other proteins, binding to several of the proteins identified by the SH2 profiling was lost. Of interest is Csk, which associates with wild-type Vav1 and Y841F, yet it fails to associate with Y826F, suggesting that loss of binding between Y826F and Csk might relieve an autoinhibitory effect, leading to increased NFAT. Our data indicate that GEF activity is critical for the function of Vav1 as a transforming protein but not for NFAT stimulation. The association of Vav1 with other proteins, detected by SH2 profiling, might affect other Vav1-dependent activities, such as NFAT stimulation.
Lazer
Galit
G
Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada (IMRIC), Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.
Pe'er
Liron
L
Farago
Marganit
M
Machida
Kazuya
K
Mayer
Bruce J
BJ
Katzav
Shulamit
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D002471:Cell Transformation, Neoplastic; D004789:Enzyme Activation; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D050778:NFATC Transcription Factors / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000473:pathology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051567:Proto-Oncogene Proteins c-vav / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013601:T-Lymphocytes / Q000378:metabolism; D014443:Tyrosine
D050778:NFATC Transcription Factors; D051567:Proto-Oncogene Proteins c-vav; D014443:Tyrosine; D051057:Proto-Oncogene Proteins c-akt
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