The Rho family GTPases Rac, Rho and Cdc42 are critical in regulating the actin-based cytoskeleton, cell migration, growth, survival and gene expression. These GTPases are activated by guanine nucleotide-exchange factors (GEFs). A biochemical search for Cdc42 activators led to the cloning of zizimin1, a new protein whose overexpression induces Cdc42 activation. Sequence comparison combined with mutational analysis identified a new domain, which we named CZH2, that mediates direct interaction with Cdc42. CZH2-containing proteins constitute a new superfamily that includes the so-called 'CDM' proteins that bind to and activate Rac. Together, the results suggest that CZH2 is a new GEF domain for the Rho family of proteins.
Rho family small GTPases are critical regulators of multiple cellular processes and activities. Dbl homology domain-containing proteins are the classical guanine nucleotide exchange factors (GEFs) responsible for activation of Rho proteins. Recently another group of mammalian Rho-GEFs was discovered that includes CDM (Ced-5, DOCK180, Myoblast city) proteins that activate Rac and zizimin1 that activates Cdc42 via a nonconventional GEF module that we named the CZH2 domain. We report here that zizimin1 dimerizes via the CZH2 domain and that dimers are the only form detected. Dimerization was mapped to a ϳ200-amino acid region that overlaps but is distinct from the Cdc42-binding sequences. Rotary shadowing electron microscopy revealed zizimin1 to be a symmetric, V-shaped molecule. Experiments with DOCK180 and homology analysis suggest that dimerization may be a general feature of CZH proteins. Deletion and mutation analysis indicated existence of individual Cdc42-binding sites in the zizimin1 monomers. Kinetic measurements demonstrated increased binding affinity of Cdc42 to zizimin1 at higher Cdc42 concentration, suggesting positive cooperativity. These features are likely to be critical for Cdc42 activation.
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