Intramolecular regulation of a calcium-dependent protein kinase with a calmodulin-like regulatory domain

Ca{dollar}sp{lcub}2+{rcub}{dollar}-dependent protein kinases (CDPKs) differ from other protein kinases by containing an intrinsic calmodulin-like domain (CaM-LD), which is tethered to the carboxyl-terminus of the kinase domain by a conserved junction region (31 residues). To understand intramolecular regulation of CDPKs, I have used isoform CPK-1 from Arabidopsis, (accession #L14771) as a model. Four major conclusions are presented. First, CPK-1 is kept inactive by a pseudosubstrate autoinhibitor in the junction. For example, the kinase activity of a truncated CPK-1 lacking the CaM-LD ({dollar}Delta{dollar}NC-wt) was not stimulated by Ca{dollar}sp{lcub}2+{rcub}{dollar}. Second, Ca{dollar}sp{lcub}2+{rcub}{dollar}-activation of CPK-1 requires intramolecular binding between the CaM-LD and the junction. This was supported by the activation of the {dollar}Delta{dollar}NC-wt by an isolated CaM-LD or exogenous calmodulin (with {dollar}Ksb{lcub}act{rcub}{dollar} of 5 {dollar}mu{dollar}M). In addition, Ca{dollar}sp{lcub}2+{rcub}{dollar}-activation of CPK-1 was affected by a mutation in the junction which disrupts intramolecular binding. Third, a tether, defined as residues between the end of the CaM-LD binding domain and the first EF-hand in the CaM-LD, does not function as a simple flexible linker. This was supported by the results that insertions in the tether disrupt Ca{dollar}sp{lcub}2+{rcub}{dollar}-activation without affecting the CaM-LD binding domain. Fourth, the CaM-LD probably binds the junction in the absence and presence of Ca{dollar}sp{lcub}2+{rcub}{dollar} and Ca{dollar}sp{lcub}2+{rcub}{dollar}-binding to the CaM-LD shifts the equilibrium between the two states, consequently, activates the kinase. This model is supported by the observation that an isolated CaM-LD shows a Ca{dollar}sp{lcub}2+{rcub}{dollar}-induced change in the spectrum when analyzed by CD spectroscopy, whereas JC-1 which contains both the CaM-LD and junction does not. Furthermore, JC-4, a mutant protein with intramolecular binding disrupted, shows a Ca{dollar}sp{lcub}2+{rcub}{dollar}-induced change in its CD spectrum. This model is distinct from the activation of Ca{dollar}sp{lcub}2+{rcub}{dollar}/calmodulin-dependent protein kinases by calmodulin in that Ca{dollar}sp{lcub}2+{rcub}{dollar}-binding shifts the equilibrium between two states of intramolecular binding, corresponding to the autoinhibited and activated forms of the kinase, respectively, rather than inducing a binding.