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.
Ziegenhagen
R
R
Institut für Physiologische Chemie, Universität-GHS-Essen, FRG.
Goldberg
M
M
Rakutt
W D
WD
Jennissen
H P
HP
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D003432:Cross-Linking Reagents / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008297:Male; D008745:Methylation; D010453:Peptide Synthases / Q000378:metabolism*; D012156:Reticulocytes / Q000201:enzymology*; D013379:Substrate Specificity; D013737:Testis / Q000737:chemistry; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000138:chemical synthesis / Q000378:metabolism*
D000596:Amino Acids; D002147:Calmodulin; D003432:Cross-Linking Reagents; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; C059404:ubiquitin-calmodulin ligase; D044764:Ubiquitin-Activating Enzymes; D010453:Peptide Synthases; D008239:Lysine