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 α 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.
|Original language||English (US)|
|Number of pages||4|
|Journal||Journal of Biological Chemistry|
|State||Published - 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology