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(Received for publication, October 3, 1996, and in revised form, January 31, 1997)
From the Research Division, Joslin Diabetes Center, and Department
of Medicine, Harvard Medical School,
Boston, Massachusetts 02215
Members of the Rad family of GTPases (including
Rad, Gem, and Kir) possess several unique features of unknown function
in comparison to other Ras-like proteins, with major N-terminal and C-terminal extensions, a lack of typical prenylation motifs, and several non-conservative changes in the sequence of the GTP binding domain. Here we show that Rad and Gem bind to calmodulin
(CaM)-Sepharose in vitro in a calcium-dependent
manner and that Rad can be co-immunoprecipitated with CaM in C2C12
cells. The interaction is influenced by the guanine nucleotide binding
state of Rad with the GDP-bound form exhibiting 5-fold better binding
to CaM than the GTP-bound protein. In addition, the dominant negative
mutant of Rad (S105N) which binds GDP, but not GTP, exhibits enhanced
binding to CaM in vivo when expressed in C2C12 cells.
Peptide competition studies and expression of deletion mutants of Rad
localize the binding 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 extended N terminus of Rad which, when removed, decreases
Rad protein expression but increases the binding of Rad to CaM. The ability of Rad mutants to bind CaM correlates with their localization in cytoskeletal fractions of C2C12 cells. Immunoprecipitates of calmodulin-dependent protein kinase II, the cellular
effector of Ca2+-calmodulin, also contain Rad, and in
vitro both Rad and Gem can serve as substrates for this kinase.
Thus, the Rad family of GTP-binding proteins possess unique
characteristics of binding CaM and calmodulin-dependent protein kinase II, suggesting a role for Rad-like GTPases in calcium activation of serine/threonine kinase cascades.
Volume 272, Number 18,
Issue of May 2, 1997
pp. 11832-11839
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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