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

Area of Science:

• Neuroscience
• Molecular Biology
• Biochemistry

Background:

• Ca(2+)/calmodulin-dependent protein kinase II (CaM-kinase II) is crucial for synaptic plasticity.
• Its activity is tightly regulated by Ca(2+) and calmodulin (CaM) binding and autophosphorylation.
• Proteolytic modification could represent an alternative regulatory mechanism.

Purpose of the Study:

• To investigate the effect of limited proteolysis on CaM-kinase II activity and structure.
• To identify the specific fragments generated by proteases like mu-calpain.
• To elucidate the functional consequences of proteolytic cleavage on CaM-kinase II.

Main Methods:

• Limited proteolysis of rat forebrain CaM-kinase II using chymotrypsin and mu-calpain.
• Activity assays to measure total and Ca(2+)/CaM-independent kinase activity.
• Western blotting and (125)I-labeled CaM overlays to analyze proteolytic fragments.
• Size exclusion chromatography (Superose-6 FPLC) to characterize active fragments.

Main Results:

• Proteolysis by chymotrypsin or mu-calpain enhanced CaM-kinase II activity significantly (3-5 fold).
• Mu-calpain generated a ~30 kDa catalytic fragment and a ~23 kDa CaM-binding regulatory fragment.
• The catalytic fragment exhibited Ca(2+)/CaM-independent activity and did not bind CaM-Sepharose.

Conclusions:

• Mu-calpain cleaves CaM-kinase II into distinct catalytic and regulatory domains.
• This proteolytic activation generates a constitutively active kinase fragment.
• Such cleavage may provide a mechanism for persistent regulation of synaptic events.