Modulation of calmodulin function by ubiquitin-calmodulin ligase and identification of the responsible ubiquitylation site in vertebrate calmodulin.
Calmodulin is the universal calcium modulator in eukaryotic cells. Its biological activity is closely regulated by the second messenger Ca2+. Previous studies in cell-free extracts [Laub, M. & Jennissen, H. P. (1997) Biochim. Biophys. Acta 1357, 173-191] have shown that calmodulin is reversibly ubiquitylated by ubiquityl-calmodulin synthetase (ubiquitin-calmodulin ligase, EC 6.3.2.21) in the presence of Ca2+ without being channeled to degradation by the 26S proteasome. As shown here monoubiquitylation strongly decreases the biological activity of calmodulin towards phosphorylase kinase by reducing its affinity approximately threefold and the maximal degree of activation approximately twofold. Thus, a structural clarification of the ubiquitylation site on calmodulin has become crucial for advancing our knowledge in this field on a molecular level. As demonstrated by sequence analysis and mass spectrometry of conjugates, the ubiquitylation site is located in the first Ca2+-binding loop of calmodulin and has the octapeptide structure -L-F-D-K21-D-G-D-G- with Lys21 being the ubiquitylated residue in vertebrate and other calmodulins. This catalytic recognition sequence is, however, not the only structural requirement for calmodulin ubiquitylation by ubiquityl-calmodulin synthetase. Removal of the 41 C-terminal amino acids (fourth Ca2+-binding loop) separated by several nanometers from Lys21 drastically decreases the affinity and reactivity of the synthetase for calmodulin, indicating a more extensive structural requirement for the substrate binding site i.e. binding recognition. This allows the enzyme to discriminate in a site-specific manner between two nearly identical catalytic recognition sites in vertebrate calmodulin of which the second site -V-F-D-K94-D-G-N-G- in the third Ca2+-binding loop is apparently not ubiquitylated by the synthetase.
Laub
M
M
Institut für Physiologische Chemie, AG Biochemische Endokrinologie, Universität-GHS-Essen, Germany.
Steppuhn
J A
JA
Blüggel
M
M
Immler
D
D
Meyer
H E
HE
Jennissen
H P
HP
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D003488:Cyanogen Bromide; D004789:Enzyme Activation; D007700:Kinetics; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010447:Peptide Hydrolases / Q000378:metabolism; D010449:Peptide Mapping; D010453:Peptide Synthases / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D013737:Testis / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000737:chemistry / Q000378:metabolism*; D014714:Vertebrates
D002147:Calmodulin; D010446:Peptide Fragments; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; C059404:ubiquitin-calmodulin ligase; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D044764:Ubiquitin-Activating Enzymes; D010453:Peptide Synthases; D003488:Cyanogen Bromide