Regulation of Ca²⁺/calmodulin-dependent protein kinase II by inter- and intrasubunit-catalyzed autophosphorylations

Autophosphorylation of CaM kinase II on Thr²⁸⁶ is known to occur by an intraholoenzyme mechanism, but it is not known whether this reaction is intra- or intersubunit-catalyzed in the native heteromeric enzyme containing 10-12 α/β subunits. In this study inactive CaM kinase II β subunit, generated by mutation of Lys⁴³ to Ala, and active kinase a subunit were expressed separately (homomeric kinases) or co-expressed (heteromeric kinase) using the baculovirus/Sf9 cell expression system and purified on CaM-Sepharose. Ca²⁺/CaM-dependent autophosphorylation of heteromeric α/β kinase, which activated the enzyme, produced rapid autophosphorylation on Thr²⁸⁶ in both the active α and inactive β subunits; the latter could only occur by intersubunit catalysis. Ca²⁺/CaM-independent autophosphorylation of nonactivated heteromeric kinase was slow, resulted in partial loss of total kinase activity, occurred only in the α subunit, and existed on Thr³⁰⁶ but not Thr²⁸⁶. This result demonstrates intrasubunit catalysis of Thr³⁰⁶ autophosphorylation. These observations that regulatory autophosphorylations of Thr²⁸⁶ and Thr³⁰⁶ were inter- and intrasubunit-catalyzed, respectively, have important consequences for structure/function models of CaM kinase II and for involvement of CaM kinase II autophosphorylation and activation during synaptic plasticity in neural systems.