Stabilization of calmodulin-dependent protein kinase II through the autoinhibitory domain.
The active 30-kDa chymotryptic fragment of calmodulin-dependent protein kinase II (CaM kinase II), devoid of the autoinhibitory domain, and the enzyme, autothiophosphorylated at Thr286/Thr287, were much more labile than was the original native enzyme. They were markedly stabilized by synthetic peptides, designed after the sequence around the autophosphorylation site in the autoinhibitory domain, such as autocamtide-2 and CaMK-(281-309), but such marked stabilizations were not observed with the ordinary exogenous substrates, such as syntide-2. These results suggest that the autoinhibitory domain of CaM kinase II plays a crucial role in stabilizing the enzyme. A nonphosphorylatable analog of autocamtide-2, AIP, strongly inhibited the activity of the 30-kDa fragment. Kinetic analysis revealed that the inhibition by AIP was competitive with respect to autocamtide-2 and CaMK-(281-289) and noncompetitive with respect to syntide-2 and ATP/Mg2+, suggesting that CaM kinase II possesses at least two distinct substrate-binding sites; one for ordinary exogenous substrates such as syntide-2 and the other for an endogenous substrate, the autophosphorylation site (Thr286/Thr287) in the autoinhibitory domain. Fluorescence analysis of the binding of 7-nitrobenz-2-oxa-1,3-diazole-4-yl labeled AIP to the 30-kDa fragment also supported this contention. Thus, the autoinhibitory domain appears to play a crucial role in keeping the enzyme stable by binding to the substrate-binding site for the autophosphorylation site.
Department of Biochemistry, Asahikawa Medical College, Japan.
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Ishida
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Fujisawa
D000595:Amino Acid Sequence; D001665:Binding Sites; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D006358:Hot Temperature; D066298:In Vitro Techniques; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D010769:Phosphothreonine / Q000737:chemistry; D011489:Protein Denaturation
D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D010455:Peptides; C085903:autocamtide-2; C063391:syntide-2; D010769:Phosphothreonine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases