hSiah2 Is a New Vav Binding Protein Which Inhibits Vav-Mediated Signaling Pathways

Abstract

The hematopoietic proto-oncogene vav has been characterized as a Rac1-GDP/GTP exchanger protein which regulates cytoskeletal reorganization as well as signaling pathways leading to the activation of stress-activated protein kinases (SAPK/JNKs). Furthermore, vav overexpression enhances basal and T-cell receptor (TCR)-mediated stimulation of the nuclear factor of activated T cells (NFAT). We report here the interaction between Vav and hSiah2, a mammalian homolog of Drosophila Seven in absentia (Sina) that has been implicated in R7 photoreceptor cell formation during Drosophila eye development via the proteasome degradation pathway. Vav and hSiah2 interact in vitro and in vivo and colocalize in the cytoplasm of hematopoietic cells. The Src homology domain of Vav and the C-terminal region of hSiah2 are required for this interaction. We provide evidence for a negative regulation by hSiah2 of Vav-induced basal and TCR-mediated NFAT-dependent transcription. Overexpression of hSiah2 also inhibits the onco-Vav-induced JNK activation. Although the Vav-interacting domain is located in the C-terminal portion of hSiah2, the N-terminal region of hSiah2 is necessary for the inhibitory role that seems to be independent of the proteasome degradation.

ACKNOWLEDGMENTS

We thank G. Baier for T-Ag Jurkat cells, M. Barbacid and X. Bustelo for plasmid pKLS1, A. Altman for pEF-Myc-tagged Vav and pKES-Vav plasmids, O. Acuto for the NFAT reporter construct, P. Crespo for HA-JNK and pGEX-cJun plasmids, R. B. Amson for plasmid pBK-hSiah1, G. Bismuth for anti-CD3 antibody, D. Olive for anti-CD28 antibody, I. Bouchaert for technical assistance in confocal microscopy, and D. Littman and I. Dusanter for critical reading of the manuscript.

This work was supported by the Ligue Nationale Contre le Cancer—Axe oncogénèse and Fondation pour le Recherche Medicale. A.G. was the recipient of a Poste Vert from INSERM (France).