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.

The ubiquityl-calmodulin synthetase system from rabbit reticulocytes: isolation of the calmodulin-binding second component and enzymatic properties.

Ubiquitin-calmodulin ligase (uCaM synthetase: EC 6.3.2.21), which has been detected in all tissues so far examined, catalyzes the Ca2+-dependent reversible synthesis of ubiquityl-calmodulin which is not directed to degradation by the ATP-dependent 26-S protease [Laub, M. & Jennissen, H. P. (1997) Biochim. Biophys. Acta 1357, 173-191]. As has been shown in the preceding paper in this journal, the uCaM synthetase holosystem can be separated into two essential protein components: uCaM Syn-F1, a ubiquitin-binding protein belonging to the ubiquitin-activating enzyme family (E1) and uCaM Syn-F2 which bestows the reaction specificity leading to the covalent modification of calmodulin with ubiquitin. UCaM Syn-F2, which binds to calmodulin-Sepharose in a Ca2+-dependent manner, has been purified over 3500-fold in seven steps from rabbit reticulocytes and has a native molecular mass of approximately 620 kDa. It binds calmodulin with a Km of 5 microM and to uCaM Syn-F1, i.e. ubiquitin-activating enzyme (E1), with a Km of 3 nM. The maximal specific activity obtained in enriched uCaM Syn-F2 is 6-8 pkat/mg. The pH optimum of uCaM synthetase lies at pH 8.5. In kinetic experiments the Km values for 125I-ubiquitin and ATP/Mg2+ were determined to be 8 microM and 16 nM, respectively, for the uCaM synthetase holosystem. The existence of a third separable protein component of uCaM synthetase, as is the case in E1, E2, E3 systems, is very unlikely since affinity chromatography on calmodulin-Sepharose, two ion-exchange chromatography steps and finally a gel-filtration step failed to indicate any additional protein component essential for synthetase activity. We therefore propose a two-component model for uCaM synthetase. This model is also supported by simple hyperbolic velocity curves in kinetic experiments based on the variation of these two components. The data suggests that uCaM Syn-F2 is neither an E2 nor an E3 but evidently combines the properties of both, making the Ca2+-dependent uCaM synthetase the member of a group of two-component ubiquitin ligase systems.