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Structure-Function Relationships of Calcium/Calmodulin-dependent Protein Kinase
(CCaMK): Agricultural and Ecological Implications
Biological nitrogen fixation in legumes involves a complex microbiological process in which a
certain type of bacteria (Rhizobium) fixes atmospheric nitrogen and converts it into a form the
plant can use. This symbiotic system would reduce the cost of heavy use of nitrogen fertilizers
in agriculture. Nitrogen fixation in legumes (bacterial symbiosis) has received considerable
attention and it is becoming clear that calcium/calmodulin-mediated signaling plays a key role in
sensing and transducing the bacterial signal (Nod factor). A calcium/calmodulin-dependent
protein kinase (CCaMK), cloned and characterized in our laboratory, is known to play a critical
role in decoding the calcium signal, and it is required for bacterial symbiosis. CCaMK is an
important effector protein of Ca2+/calmodulin-mediated signaling and has been established as a
critical regulator of both bacterial and fungal symbioses in plants. It contains a Ser/Thr kinase
domain in its N-terminal, and two other regulatory domains: a calmodulin-binding/autoinhibitory
domain (CaMBD/AID) and a visinin-like domain. In addition, recent studies have revealed the
presence of two phosphorylation sites, S343 and S344. Recently, we discovered two different
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mutants were drastically compromised in their affinity to calmodulin (CaM), but our unpublished
data indicated that the synthetic-peptides corresponding to the mutated CaM-binding domains
were able to interact with CaM. These results led us to hypothesize that there is an intramolecular
interaction involving S343 and S344, and other region(s), most likely the kinase domain of
CCaMK, which is critical for its activity. In order to test this hypothesis, 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 affinity to calmodulin. We were able to
identify a critical amino acid (T227) that interacts independently with these two phosphorylation
sites. However, our results also suggest that there must be other interactor(s) that may be
involved during phosphorylation of S344 and S343. The second approach involved altering
CCaMK’s calmodulin-binding capacity. It has been reported that CCaMK is regulated by calcium
and calcium/calmodulin separately. This dual regulation is critical for the activation of this kinase
and its interaction with its substrate protein, IPD3. Furthermore, CCaMK contains a regulatory
area such as 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 CaM. This result suggests that the affinity of CCaMK to
calmodulin 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 calmodulinbinding domain showed a considerable increase in calmodulin binding affinity which resulted in
altered nodule development.