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 the single calponin homology (CH) domain, activates Vav's transforming potential, suggesting an important role of the CH domain in influencing Vav function. 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 association of the Vav protein using enhanced green fluorescent protein (EGFP) fusion constructs. Our data show that both EGFP-tagged full length Vav and the CH domain-depleted EGFPvav 143-845 construct localize throughout the cytoplasm but fail to colocalize with F-actin. However, the latter construct of Vav was more strongly retained in the Triton-insoluble cytoskeleton fraction than full length Vav. Whereas removal of the CH domain had no apparent influence on the subcellular localization of Vav, deletion of the SH domains caused nuclear localization, indicating that Vav contains a functional nuclear localization signal. Expression of N-terminally truncated Vav constructs caused depolarization of fibroblasts and triggered the bundling of actin stress fibers into parallel arrays in NIH 3T3 cells. Notably, the parallel actin bundles showed prolonged resistance to the actin polymerization antagonists cytochalasin B and latrunculin B. These data point towards a regulatory role for the CH domain in Vav and suggest an actin cross-linking or bundling protein as a downstream effector molecule of vav-mediated signalling pathways.
Kranewitter
W J
WJ
Institute of Molecular Biology, Department of Cell Biology, Austrian Academy of Sciences, Salzburg.
Gimona
M
M
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001704:Biopolymers; D018797:Cell Cycle Proteins; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051567:Proto-Oncogene Proteins c-vav; D013347:Subcellular Fractions / Q000378:metabolism
D000199:Actins; D001704:Biopolymers; D018797:Cell Cycle Proteins; D017931:DNA Primers; D008164:Luminescent Proteins; D011518:Proto-Oncogene Proteins; D051567:Proto-Oncogene Proteins c-vav; C496619:Vav1 protein, mouse; D049452:Green Fluorescent Proteins