Js. Moyers et al., RAD AND RAD-RELATED GTPASES INTERACT WITH CALMODULIN AND CALMODULIN-DEPENDENT PROTEIN-KINASE-II, The Journal of biological chemistry, 272(18), 1997, pp. 11832-11839
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.