N-terminally truncated Vav induces the formation of depolymerization-resistant actin filaments in NIH 3T3 cells

The Dbl family proto-oncogene vav is a guanine nucleotide exchange factor ( GEF) for Rho family GTPases, Deletion of the N-terminus of Vav, harboring t he single calponin homology (CH) domain, activates Vav's transforming poten tial, suggesting an important role of the CH domain in influencing Vav func tion, Since calponin binds actin, it has been suggested that the CH domain may mediate association with the actin cytoskeleton. In this study we have analyzed the subcellular localization and investigated the putative actin a ssociation of the Vav protein using enhanced green fluorescent protein (EGF P) fusion constructs, Our data show that both EGFP-tagged full length Vav a nd the CH domain-depleted EGFPvav 143-845 construct localize throughout the cytoplasm but fail to colocalize with F-actin. However, the latter constru ct of Vav was more strongly retained in the Triton-insoluble cytoskeleton f raction than full length Vav, Whereas removal of the CH domain had no appar ent influence on the subcellular localization of Vav, deletion of the SH do mains caused nuclear localization, indicating that Vav contains a functiona l nuclear localization signal. Expression of N-terminally truncated Vav con structs caused depolarization of fibroblasts and triggered the bundling of actin stress fibers into parallel arrays in MH 3T3 cells, Notably, the para llel actin bundles showed prolonged resistance to the actin polymerization antagonists cytochalasin B and latrunculin B, These data point towards a re gulatory role for the CH domain in Vav and suggest an actin cross-linking o r bundling protein as a downstream effector molecule of vav-mediated signal ling pathways. (C) 1999 Federation of European Biochemical Societies.