Ca2+/calmodulin-dependent protein kinase II: identification of autophosphorylation sites responsible for generation of Ca2+/calmodulin-independence.

Abstract

Ca2+/calmodulin-dependent protein kinase II contains two types of subunit, a (Mr 50,000) and (3 (Mr 60,000/58,000), both of which undergo Ca2+/calmodulindependent autophosphorylation. Autophosphorylation is known to convert the enzyme to a Ca2+/calmodulin-independent form. In the present study, we have characterized the autophosphorylation sites on rat forebrain Ca2+/calmodulindependent protein kinase II that are most likely to be responsible for the generation of Ca2+/calmodulin-independence. Under coniditious (00C, low concentrations of ATP) sufficient to generate close to maximal Ca2+/calmodulin-independence, only a few of the phosphorylatable sites on the enzyme became phosphorylated. These autophosphorylation sites were examined by phospho amino acid analysis, two-dimensional thermolytic phosphopeptide mapping, and high-performance liquid chromatography. The time course of plosphorylation of threonine in both a and fJ subunits was similar to the time course of the generation of Ca2+/calmodulin-independence.Moreover, the time course of phosphorylation of one set of peptides, referred to as peptide 1/1', present in both a and ,3 subunits was similar to the time course of the generation of Ca2+/calmodulin-independence. Threonine was the only amino acid phosphorylated in peptide 1/1'. An additional peptide, referred to as peptide 2, was phosphorylated in the .8 subunit.The time course of phosphorylation of peptide 2, which also contained only phosphothreonine, did not parallel the time course of the generation of Ca2+/calmodulin-independence. It is likely that the phosphorylation of a threonine residue on peptide 1/1' is responsible for the generation of Ca2 /calmodulin-independence of Ca2+/calmodulin-dependent protein kinase IL.

Autophosphorylation of Ca2+/calmodulin-dependent protein kinase. II. Effects on interaction between enzyme and substrate.

“…a) An increase in kinase activity was observed as early as 15 sec after autophosphorylation. This is consistent with the finding that Thr 286 is phosphorylated as early as 15 sec (33,45) and faster than the serine residue. b) The patterns of the increase in kinase activity and generation of Ca 2+/calmodulin-independent activity were almost identical during the time course (Figs.…”

Calcium/Calmodulin-Dependent Protein Kinase II

“…This partially Ca2"-independent form of CaM-kinase II can be converted back to the completely Ca2"-dependent form by treatment with phosphoprotein phosphatase types 1 [116] or 2A [115]. Under suitable limiting autophosphorylation conditions (5°C with 5-10 /M-ATP and 0.5 mM-magnesium acetate) the generation of the partially Ca2"-independent form temporally correlates with the phosphorylation of threonine residues [118,119]. Analysis of CNBr digests of the [32P]Pi-labelled rat brain kinase shows that approx.…”

S-Nitrosylation Induces Both Autonomous Activation and Inhibition of Calcium/Calmodulin-dependent Protein Kinase II δ

“…However, CaMKII␦ is subject to a number of posttranslational modifications that prevent reassociation of the catalytic and regulatory domains of the kinase and, therefore, lead to autonomous activation of CaMKII␦ (7). These modifications, described previously, include autophosphorylation at the Thr-287 site (8), oxidation at the Met-281/282 sites (9), and O-GlcNAc modification at the Ser-280 site (10). Each of these modifications prolongs CaMKII activation in the heart and can induce cardiac pathology, including adverse structural remodeling, arrhythmia, and cell death.…”