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

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Abstract
In the presence of Ca2+ and ATP/Mg2+ mammalian calmodulin can be covalently coupled to ubiquitin by ubiquityl calmodulin synthetase (uCaMsynthetase). Three ubiquitin derivatives 125I‐CT‐ubiquitin (prepared by the chloramine‐T method), 125I‐BH‐ubiquitin (prepared by the BoltonHunter 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 polyubiquitin chain is linked. Since it can be shown that the mono‐ubiquitination product of calmodulin still contains ca. 1 mol calmodulin, the polyubiquitin 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.