Multiple ubiquitination of calmodulin results in one polyubiquitin chain linked to calmodulin

In the presence of Ca2+ and ATP/Mg2+ mammalian calmodulin can be covalently coupled to ubiquitin by ubiquityl calmodulin synthetase (uCaM-synthetase). Three ubiquitin derivatives 125I-CT-ubiquitin (prepared by the chloramine-T method), 125I-BH-ubiquitin (prepared by the Bolton-Hunter method) and methylated forms of ubiquitin were tested with native calmodulin. Alternatively native ubiquitin was tested with the Bolton-Hunter derivative of calmodulin (125I-BH-calmodulin). Up to three molecules of ubiquitin can be incorporated into one molecule of calmodulin. Since both native forms of ubiquitin and calmodulin are good substrates of uCaM-synthetase, ubiquitination is not a result of an altered conformation (i.e. denaturation) of either protein. With 125I-BH-calmodulin it is demonstrated that calmodulin is also present in the higher molecular weight ubiquitin conjugates. If methylated ubiquitin is employed as substrate for uCaM-synthetase only one conjugate corresponding to the mono-ubiquitination product of calmodulin is formed. This demonstrates that only a single lysine residue in calmodulin is conjugated to ubiquitin. All other higher molecular weight ubiquitin-calmodulin conjugates must therefore be composed of one molecule of calmodulin to which an oligo- or poly-ubiquitin chain is linked. Since it can be shown that the mono-ubiquitination product of calmodulin still contains ca. 1 mol trimethyllysine/mol calmodulin, the poly-ubiquitin chain is not linked to lysine 115 of calmodulin. In addition a demethylation of trimethyllysine 115 by enzymes in reticulocyte lysate or the DEAE-enriched enzyme fraction with subsequent ubiquitination at this site of calmodulin can also be excluded.