Calmodulin binds to and inhibits GTP binding of the ras-like GTPase Kir/Gem.
Recently, a new subfamily of Ras-related GTP-binding proteins consisting of Rad (Ras associated with diabetes), Gem (immediate early gene expressed in mitogen-stimulated T-cells), and Kir (tyrosine kinase-inducible Ras-like) was discovered. The C terminus of these proteins contains an extension of approximately 30 amino acids not present in other members of the Ras family and which exhibits all the hallmarks typical for calmodulin (CaM)-binding domains. A peptide corresponding to the putative CaM-binding domain of the Kir/Gem protein was synthesized, and its affinity for CaM was determined by fluorescence spectrometry. Titration of dansyl-CaM with the Kir/Gem peptide gave an affinity constant of 1 nM. Furthermore, a single point mutation of the peptide, W269G, abolished this high affinity interaction. Gel-shift analysis showed that the complex formation between CaM and the Kir/Gem peptide is strictly calcium-dependent. We also demonstrate with a newly developed [32P]CaM overlay technique that full-length Kir/Gem and Rad proteins bind CaM in a Ca2+-dependent fashion. The binding of CaM to glutathione S-transferase-Kir and GST-Gem inhibited the binding of GTP to Kir/Gem significantly. These results suggest the existence of a direct link between Ca2+/CaM and growth factor signal transduction pathways at the level of small Ras-like GTPases.
Fischer
R
R
Department of Veterinary Biochemistry, University Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Wei
Y
Y
Anagli
J
J
Berchtold
M W
MW
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D003619:Dansyl Compounds; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D003619:Dansyl Compounds; D017874:Immediate-Early Proteins; D010446:Peptide Fragments; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C490245:GEM protein, human; D020559:Monomeric GTP-Binding Proteins; D002118:Calcium