Intramolecular Activation of a Ca²⁺-Dependent Protein Kinase Is Disrupted by Insertions in the Tether That Connects the Calmodulin-like Domain to the Kinase

Abstract

Ca(2+)-dependent protein kinases (CDPK) have a calmodulin-like domain (CaM-LD) tethered to the C-terminal end of the kinase. Activation is proposed to involve intramolecular binding of the CaM-LD to a junction sequence that connects the CaM-LD to the kinase domain. Consistent with this model, a truncated CDPK (DeltaNC) in which the CaM-LD has been deleted can be activated in a bimolecular interaction with an isolated CaM-LD or calmodulin, similar to the activation of a calmodulin-dependent protein kinase (CaMK) by calmodulin. Here we provide genetic evidence that this bimolecular activation requires a nine-residue binding segment from F436 to I444 (numbers correspond to CPK-1 accession number L14771). Two mutations at either end of this core segment (F436/A and VI444/AA) severely disrupted bimolecular activation, whereas flanking mutations had only minor effects. Intramolecular activation of a full-length kinase was also disrupted by a VI444/AA mutation, but surprisingly not by a F436/A mutation (at the N-terminal end of the binding site). Interestingly, intramolecular but not bimolecular activation was disrupted by insertion mutations placed immediately downstream of I444. To show that mutant enzymes were not misfolded, latent kinase activity was stimulated through binding of an antijunction antibody. Results here support a model of intramolecular activation in which the tether (A445 to G455) that connects the CaM-LD to the kinase provides an important structural constraint and is not just a simple flexible connection.

Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca 2+ affinities

“…C-terminal truncation was shown to render CDPK kinases constitutively active and thus Ca 2+ -insensitive (17,18,30). Interestingly, however, anion currents were only 20% higher with the truncated CPK23 version (Fig.…”

A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca 2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis

“…Two recombinant versions of CDPK isoform CPK1 [KJM23-6H2 (18) and ⌬NC-31 (19)] were used here to provide a constitutively active kinase that was Ca 2ϩ -independent (CDPKci). Kinases were expressed in E. coli as previously described and purified by sequential purification for a C-terminal 6ϫ His motif and an N-terminal GST (18).…”

Autophosphorylation and Subcellular Localization Dynamics of a Salt- and Water Deficit-Induced Calcium-Dependent Protein Kinase from Ice Plant

“…Currently, such conformational changes would be difficult to predict, since the crystal structure of CDPKs is still unknown. However, one of the models proposed by Vitart et al (2000) for the activation of CDPKs suggests that the inactive and active states of the kinase are closely related conformationally. Once Ca 21 binds to the EF hands and/or autophosphorylation takes place, subtle changes in the conformation of the kinase are thought to cause the removal of the pseudosubstrate and the activation of the enzyme.…”

“…The junction domain acts as an autoinhibitor in a pseudosubstrate fashion (Harper et al, 1994;Vitart et al, 2000;Weljie et al, 2000). Binding of Ca 21 to the calmodulin-like domain results in a conformational change leading to the release of the autoinhibitor domain from the active site and kinase activation (Harmon et al, 1994;Harper et al, 1994).…”