Proteolytic activation of calcium/calmodulin-dependent protein kinase II: Putative function in synaptic plasticity

Rat forebrain Ca²⁺/calmodulin-dependent protein kinase II (CaM-kinase II) in isolated postsynaptic densities (PSD) was subjected to limited proteolysis with chymotrypsin or μ-calpain, a Ca²⁺-dependent protease. Incubation of the kinase with either protease resulted in a three- to fivefold enhancement of total kinase activity and solubilization of Ca²⁺/calmodulin (CaM)-independent activity from the PSD. Maximal enhancement of CaM-kinase II activity was observed when autophosphorylated or Ca²⁺/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, μ-calpain degraded only a small fraction of the intact kinase subunits. ¹²⁵I-labeled CaM overlays indicated a major CaM-binding fragment of approximately 23 kDa in μ-calpain digests of purified cytosolic CaM-kinase II. This fragment was also shown to contain the regulatory autophosphorylation site (Thr-286(α)/287(β)) 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 μ-calpain digests. Analysis of the crude digests on a Superose-6 FPLC column also indicated that the Ca²⁺/CaM-independent activity resided in a fragment of approximately 30 kDa. This catalytic fragment did not bind to CaM-Sepharose. Thus, μ-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.