REGULATION OF CA2-DEPENDENT PROTEIN-KINASE-II BY INTERSUBUNIT-CATALYZED AND INTRASUBUNIT-CATALYZED AUTOPHOSPHORYLATIONS( CALMODULIN)

Autophosphorylation of CaM kinase II on Thr(286) is known to occur by an intraholoenzyme mechanism, but it is not known whether this reactio n is intra or intersubunit catalyzed in the native heteromeric enzyme containing 10-12 alpha/beta subunits. In this study inactive CaM kinas e II beta subunit, generated by mutation of Lys(43) to Ala, and active kinase a subunit were expressed separately (homomeric kinases) or co expressed (heteromeric kinase) using the baculovirus/Sf9 cell expressi on system and purified on CaM-Sepharose. Ca2+/CaM-de- pendent autophos phorylation of heteromeric alpha/beta kinase, which activated the enzy me, produced rapid autophosphorylation on Thr(286) in both the active alpha and inactive beta subunits; the latter could only occur by inter subunit catalysis. Ca2+/CaM-independent autophosphorylation of nonacti vated heteromeric kinase was slow, resulted in partial loss of total k inase activity, occurred only in the alpha subunit, and existed on Thr (306) but not Thr(286). This result demonstrates intrasubunit catalysi s of Thr(306) autophosphorylation. These observations that regulatory autophosphorylations of Thr(286) and Thr(306) were inter- and intrasub unit-catalyzed, respectively, have important consequences for structur e/function models of CaM kinase II and for involvement of CaM kinase I I autophosphorylation and activation during synaptic plasticity in neu ral systems.