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) 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.
Moyers
J S
JS
Research Division, Joslin Diabetes Center, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA.
Bilan
P J
PJ
Zhu
J
J
Kahn
C R
CR
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins; D010446:Peptide Fragments / Q000737:chemistry; D017368:Protein Prenylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D018631:ras Proteins
D002147:Calmodulin; D017874:Immediate-Early Proteins; D010446:Peptide Fragments; C084355:RRAD protein, human; D011993:Recombinant Fusion Proteins; C490030:Rrad protein, mouse; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C490245:GEM protein, human; D020559:Monomeric GTP-Binding Proteins; D018631:ras Proteins; D002118:Calcium