Abstract
We have found that deletion of residues 2-8 from the N-terminal leader sequence: Ala1-Asp2-Gln-Leu4-Thr-Glu6-Glu-Gln8, in calmodulin abolishes calmodulin-dependent activation of skeletal muscle myosin light chain kinase activity and reduces calmodulin-dependent activation of smooth muscle myosin light chain kinase activity to approximately 50% of the maximum level measured at a saturating calmodulin concentration. Calmodulin-dependent activation of cerebellar nitric oxide synthase activity is not affected by this deletion. Overlapping tripeptide deletions from the leader sequence indicate that the acidic cluster, Glu6-Glu-Gln8, contains the determinants necessary for activation of myosin light chain kinase activity. Deletion of Asp2-Gln-Leu4 has no effect on activation of enzyme activity. Based on enzyme kinetic analyses, deletions in the leader sequence have little or no effect on the apparent affinities of calmodulin for the synthase or the two kinases. Since the N-terminal leader does not appear to play a significant structural role in the complexes between calmodulin and peptides representing the calmodulin-binding domains in the two kinases, our results indicate that it participates in secondary interactions with these enzymes that are important to activation, but not to recognition or binding of calmodulin.
Highlights
Calmodulin (CaM)1 plays a key role in cellular regulation by virtue of its Ca2ϩ-dependent activation of numerous enzyme activities, as well as its modulation of a variety of non-enzymatic processes in the cell [1,2,3,4,5]
Since CaMNN is a poor activator of skeletal muscle myosin light chain kinase (skMLCK) and smooth muscle myosin light chain kinase (smMLCK) activities, we have not examined the ability of ⌬NCaMNN to activate these enzyme activities [11]
We have demonstrated that the N-terminal leader in CaM is important for CaM-dependent activation of both smMLCK and skMLCK activities; CaM-dependent activation of skMLCK activity is completely abolished by deletion of this region
Summary
Calmodulin (CaM) plays a key role in cellular regulation by virtue of its Ca2ϩ-dependent activation of numerous enzyme activities, as well as its modulation of a variety of non-enzymatic processes in the cell [1,2,3,4,5]. The structures of CaM bound to peptides representing the CaM-binding domains in smooth muscle and skeletal muscle myosin light chain kinase and in CaM-dependent protein kinase II have been determined and show globular structures in which CaM enfolds the bound peptides [7, 8] This requires a substantial conformational change in CaM, which is facilitated by the flexibility of the central helix linker [7,8,9,10]. Structures that have been determined of CaM either alone or in its complexes with peptides representing the CaM-binding domains in smooth or skeletal muscle myosin light chain kinase indicate little or no structural role for the N-terminal leader sequence (6 – 8) For this reason the possible involvement of this region in CaM-dependent enzyme activation has not been explored. In contrast with the kinases, we find that activation of neural nitric oxide synthase activity by CaM does not require the N-terminal leader sequence
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