Regulatory mechanisms of calcium/calmodulin-dependent protein kinase (CCAMK): effects of altered calmodulin binding and intramolecular interaction

Edgard Jauregui

Calcium/calmodulin-dependent protein kinase (CCaMK) is an important effector-protein of Ca2+/calmodulin-mediated signaling and has been established as a critical regulator of bacterial symbioses in legumes. It contains a kinase domain in its N-terminal, and two regulatory domains: a calmodulin-binding/autoinhibitory domain and a visinin-like domain. In addition, recent studies have revealed the presence of two phosphorylation sites, Ser-343 and Ser-344. This study described two new regulatory mechanisms of CCaMK. The first was the discovery of an intramolecular interaction during the phosphorylation of this kinase. Our published data revealed that S343D and S344D mutants were drastically compromised in their capacity to calmodulin, but our recent data indicated that the synthetic peptides corresponding to the mutated calmodulin-binding domains were able to interact with calmodulin. These results suggest that there is an intramolecular interaction involving Ser-343 and Ser-344, and another region(s) of CCaMK. In order to identify this interaction, we created a series of progressive deletions in the CCaMK protein to determine the amino acid in the kinase domain that interacts with these phosphorylation sites. The deletion mutants were subjected to calmodulin-binding assay to observe their recovery of binding to calmodulin. We were able to identify Thr-227as a critical residue for this intramolecular interaction. However, our results also suggest that there must be other residue(s) that is critical for this intramolecular interaction. The second approach involved altering CCaMK's calmodulin-binding capacity. It has been reported that CCaMK is regulated by calcium and calcium/calmodulin and is critical for its activation. Furthermore, CCaMK contains a regulatory area including a calmodulin-binding domain in which the calmodulin binds and promotes substrate phosphorylation. Our data revealed that a site direct mutation in the calmodulin-binding domain of CCaMK increased its binding to calmodulin. This result suggests that the binding of calmodulin to CCaMK could be altered by site-directed mutagenesis, providing an effective approach to study how calmodulin regulates CCaMK in terms of phosphorylation activity; and therefore, the regulation of symbioses in Medicago truncatula. Site direct mutation W342F within the calmodulin-binding domain showed a considerable increase in calmodulin binding capacity which resulted in altered nodule development.

Regulatory mechanisms of calcium/calmodulin-dependent protein kinase (CCAMK): effects of altered calmodulin binding and intramolecular interaction

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