Bacterially synthesized vertebrate calmodulin is a specific substrate for ubiquitination.
Calmodulin purified from bacteria which express a cloned chicken calmodulin gene can be selectively conjugated with ubiquitin, using enzymes present in reticulocyte extracts. Analyses of peptide products generated from limited proteolytic digestion of the calmodulin conjugate containing a single ubiquitin indicate that lysine 115 on calmodulin is the site of linkage. This linkage site is identical to that previously reported for calmodulin purified from Dictyostelium discoideum. Substrate-dependent ATP hydrolysis by a partially purified ubiquitin conjugation enzyme system from reticulocyte extracts was used to determine the enzyme affinity to calmodulin. Km values of 7 and 9 microM were determined for dictyostelium and the bacterially expressed calmodulin, respectively. The bacterially expressed calmodulin, unlike the Dictyostelium protein, can also form conjugates containing a 2-5 molar ratio of ubiquitin but at a slower rate than that observed for conjugation at lysine 115. Results from these studies further support our hypothesis that the post-translational methylation of lysine 115 found in most forms of calmodulin serves the important function of protecting calmodulin from ubiquitination and from degradation by the cytoplasmic ubiquitin-dependent proteolytic pathway. The capability of the bacterially expressed calmodulin to form conjugates with a high molar ratio of ubiquitin suggests that the post-translational acetylation of the N terminus of calmodulin may serve a similar function.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002147:Calmodulin / Q000096:biosynthesis* / Q000235:genetics; D002417:Cattle; D002645:Chickens; D003001:Cloning, Molecular; D004023:Dictyostelium; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D014452:Ubiquitins / Q000378:metabolism*
D002147:Calmodulin; D014452:Ubiquitins; D000255:Adenosine Triphosphate