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Autophosphorylation of Calmodulin‐Kinase II in Synaptic Junctions Modulates Endogenous Kinase Activity
Author(s) -
Shields Steven M.,
Ver Paula J.,
Kelly Paul T.
Publication year - 1984
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1984.tb06084.x
Subject(s) - autophosphorylation , synapsin i , calmodulin , protein kinase a , biochemistry , mitogen activated protein kinase kinase , biology , kinase , synaptic vesicle , cyclin dependent kinase 9 , phosphorylation , microbiology and biotechnology , chemistry , enzyme , vesicle , membrane
Previous studies have purified from bfain a Ca 2+ /calmodulin‐dependent protein kinase II (designated CaM‐kinase II) that phosphorylates synapsin I, a synaptic vesicle‐associated phosphoprotein. CaM‐kinase II is composed of a major M r 50K polypeptide and a minor M r 60K polypeptide; both bind calmodulin and are phosphorylated in a Ca 2+ /calmodulin‐dependent manner. Recent studies have demonstrated that the 50K component of CaM‐kinase II and the major postsynaptic density protein (mPSDp) in brain synaptic junctions (SJs) are virtually identical and that the CaM‐kinase II and SJ 60K polypeptides are highly related. In the present study the photoaffinity analog [α‐ 32 P]8‐azido‐ATP was used to demonstrate that the 60K and 50K polypeptides of SJ‐associated CaM‐kinase II each bind ATP in the presence of Ca 2+ plus calmodulin. This result is consistent with the observation that these proteins are phosphorylated in a Ca 2+ /calmodulin‐dependent manner. Experiments using 32 P‐labeled peptides obtained by limited proteolysis of 60K and 50K polypeptides from SJs demonstrated that within each kinase polypeptide the same peptide regions contain both autophosphorylation and 125 I‐calmoduhn binding sites. These results suggested that the autophosphorylation of CaM‐kinase II could regulate its capacity to bind calmodulin and, thus, its capacity to phosphorylate substrate proteins. By using 125 I‐calmodulin overlay techniques and sodium dodecyl Solfate‐polyacrylamide gel electrophoresis we found that phosphorylated 50K and 60K CaM‐kinase II polypeptides bound more calmodulin (50–70%) than did unphosphorylated kinase polypeptides. Levels of in vitro CaM‐kinase II activity in SJs were measured by phosphorylation of exogenous synapsin I. SJs containing highly phosphorylated CaMkinase II displayed greater activity in phosphorylating synapsin I (300% at 15 n M calmodulin) relative to control SJs that contained unphosphorylated CaM‐kinase II. The CaM‐kinase II activity in phosphorylated SJs was indistinguishable from control SJs at saturating calmodulin concentrations (300–1,000 n M ). These findings show that the degree of autophosphorylation of CaM‐kinase II in brain SJs modulates its in vitro activity at low and possibly physiological calmodulin concentrations; such a process may represent a mechanism of regulating this kinase's activity at CNS synapses in situ.