title: Proteolytic activation of calcium/calmodulin-dependent protein kinase II: Putative function in synaptic plasticity.
abstract: Rat forebrain Ca(2+)/calmodulin-dependent protein kinase II (CaM-kinase II) in isolated postsynaptic densities (PSD) was subjected to limited proteolysis with chymotrypsin or mu-calpain, a Ca(2+)-dependent protease. Incubation of the kinase with either protease resulted in a three- to fivefold enhancement of total kinase activity and solubilization of Ca(2+)/calmodulin (CaM)-independent activity from the PSD. Maximal enhancement of CaM-kinase II activity was observed when autophosphorylated or Ca(2+)/CaM-bound forms of the enzyme were proteolyzed. Analysis of the proteolytic products by Western blotting with a polyclonal antibody raised against soluble CaM-kinase II indicated that both proteases generated several immunoreactive fragments between 21 and 32 kDa. However, unlike chymotrypsin, mu-calpain degraded only a small fraction of the intact kinase subunits. (125)I-labeled CaM overlays indicated a major CaM-binding fragment of approximately 23 kDa in mu-calpain digests of purified cytosolic CaM-kinase II. This fragment was also shown to contain the regulatory autophosphorylation site (Thr-286(alpha)/287(beta)) of the kinase. Immunoblotting with antibody to the catalytic domain of the kinase indicated that there was a single active fragment of approximately 30 kDa in the mu-calpain digests. Analysis of the crude digests on a Superose-6 FPLC column also indicated that the Ca(2+)/CaM-independent activity resided in a fragment of approximately 30 kDa. This catalytic fragment did not bind to CaM-Sepharose. Thus, mu-calpain appears to cleave CaM-kinase 11 into a 30-kDa catalytic domain fragment and a 23-kDa regulatory domain fragment. A putative mechanism for persistent regulation of synaptic events by such a proteolytic activation of CaM-kinase 11 is discussed.
authors.affiliation: Department of Molecular Physiology and Biophysics Vanderbilt University School of Medicine, Nashuille, Tennessee 37232 USA.
authors.forename: D P
authors.initials: DP
authors.lastname: Rich
authors.forename: C M
authors.initials: CM
authors.lastname: Schworer
authors.forename: R J
authors.initials: RJ
authors.lastname: Colbran
authors.forename: T R
authors.initials: TR
authors.lastname: Soderling