RAD AND RAD-RELATED GTPASES INTERACT WITH CALMODULIN AND CALMODULIN-DEPENDENT PROTEIN-KINASE-II

Members of the Rad family of GTPases (including Rad, Gem, and Kir) pos sess several unique features of unknown function in comparison to othe r Ras like proteins, with major N terminal and C-terminal extensions, a lack of typical prenylation motifs, and several nonconservative chan ges in the sequence of the GTP binding domain. Here we show that Rad a nd Gem bind to calmodulin (CaM)-Sepharose in vitro in a calcium-depend ent manner and that Rad can be co-immunoprecipitated with CaM in C2C12 cells. The interaction is influenced by the guanine nucleotide bindin g state of Rad with the GDP-bound form exhibiting 5-fold better bindin g to CaM than the GTP-bound protein. In addition, the dominant negativ e mutant of Rad (S105N) which binds GDP, but not GTP, exhibits enhance d binding to CaM in vivo when expressed in C2C12 cells. Peptide compet ition studies and expression of deletion mutants of Rad localize the b inding site for CaM to residues 278-297 at the C terminus of Rad. This domain contains a motif characteristic of a calmodulin-binding region , consisting of numerous basic and hydrophobic residues. In addition, we have identified a second potential regulatory domain in the extende d N terminus of Rad which, when removed, decreases Rad protein express ion but increases the binding of Rad to CaM. The ability of Rad mutant s to bind CaM correlates with their localization in cytoskeletal fract ions of C2C12 cells. Immunoprecipitates of calmodulin-dependent protei n kinase II, the cellular effector of Ca2+-calmodulin, also contain Ra d, and in vitro both Rad and Gem can serve as substrates for this kina se. Thus, the Rad family of GTP-binding proteins possess unique charac teristics of binding CaM and calmodulin-dependent protein kinase II, s uggesting a role for Rad-like GTPases in calcium activation of serine/ threonine kinase cascades.