Multiple ubiquitination of vertebrate calmodulin by reticulocyte lysate and inhibition of calmodulin conjugation by phosphorylase kinase.
Mammalian calmodulin containing trimethyllysine 115 can be covalently coupled to ubiquitin in a Ca2+-dependent manner in the presence of ATP/Mg2+ by reticulocyte lysate. This conjugation reaction can be quantitated in a novel test employing fluphenazine-Sepharose. It is shown that at least 3 ubiquitin molecules can be coupled to calmodulin indicating that more than one lysine residue is involved in the ubiquitination reaction. In addition only the free form of calmodulin can be ubiquitinated. Neither calmodulin bound to phosphorylase kinase as an integral subunit (delta-subunit) nor that bound as a peripheral subunit (delta'-subunit) is ubiquitinated. A total binding of equimolar calmodulin to phosphorylase kinase occurs since the affinity of binding of calmodulin to phosphorylase kinase as integral (KCaMm unknown) or peripheral subunit (KCaMm ca. 30-50nM) is several order of magnitude higher than the corresponding affinity of calmodulin for the ubiquitin-conjugating enzyme (KCaMm ca. 8 microM). We conclude that the "protective" effect of phosphorylase kinase towards calmodulin conjugation is due to a changed conformation of bound calmodulin and/or inacessibility of the ubiquitination sites (e.g. at subunit-subunit interface). Thus Ca2+-dependent ubiquitination only of free calmodulin may provide an efficient scavanging mechanism (with subsequent breakdown) for all free calmodulin in excess of that amount which can be bound by the calmodulin-binding proteins in the cell.
Ziegenhagen
R
R
Institut für Physiologie, Ludwig-Maximilians-Universität München.
Jennissen
H P
HP
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000097:blood*; D002474:Cell-Free System; D007700:Kinetics; D046911:Macromolecular Substances; D010764:Phosphorylase Kinase / Q000378:metabolism*; D012156:Reticulocytes / Q000378:metabolism*; D013552:Swine; D014452:Ubiquitins / Q000097:blood*
D002147:Calmodulin; D046911:Macromolecular Substances; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D010764:Phosphorylase Kinase