Intramolecular activation of a Ca2+-dependent protein kinase is disrupted by insertions in the tether that connects the calmodulin-like domain to thekinase

Ca2+-dependent protein kinases (CDPK) have a calmodulin-like domain (CaM-LD ) tethered to the C-terminal end of the kinase. Activation is proposed to i nvolve intramolecular binding of the CaM-LD to a junction sequence that con nects the CaM-LD to the kinase domain. Consistent with this model, a trunca ted CDPK (Delta NC) in which the CaM-LD has been deleted can be activated i n a bimolecular interaction with an isolated CaM-LD or calmodulin, similar to the activation of a calmodulin-dependent protein kinase (CaMK) by calmod ulin. Here we provide genetic evidence that this bimolecular activation req uires 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, w hereas flanking mutations had only minor effects. Intramolecular activation of a full-length kinase was also disrupted by a VI444/AA mutation, but sur prisingly not by a F436/A mutation (at the N-terminal end of the binding si te). Interestingly, intramolecular but not bimolecular activation was disru pted by insertion mutations placed immediately downstream of I444. To show that mutant enzymes were not misfolded, latent kinase activity was stimulat ed through binding of an antijunction antibody. Results here support a mode l of intramolecular activation in which the tether (A445 to G455) that conn ects the CaM-LD to the kinase provides an important structural constraint a nd is not just a simple flexible connection.