Light scattering and transmission electron microscopy studies reveal a mechanism for calcium/calmodulin-dependent protein kinase II self-association

Calmodulin (CaM)-kinase II holoenzymes composed of either alpha or beta sub units were analyzed using light scattering to determine a mechanism for sel f-association. Under identical reaction conditions, only alpha CaM-kinase I I holoenzymes self-associated. Self-association was detected at a remarkabl y low enzyme concentration (0.14 muM or 7 mug/mL). Light scattering reveale d two phases of self-association: a rapid rise that peaked, followed by a s lower decrease that stabilized after 2-3 min. Electron microscopy identifie d that the rapid rise in scattering was due to the formation of loosely pac ked clusters of holoenzymes that undergo further association into large com plexes of several microns in diameter over time. Self-association required activation by Ca2+/CaM and was strongly dependent on pH. Self-association w as not detected at pH 7.5, however, the extent of this process increased as reaction pH decreased below 7.0. A peptide substrate (autocamtide-2) and i nhibitor (AIP) designed from the autoregulatory domain of CaM-kinase II pot ently prevented self-association, whereas the peptide substrate syntide-2 d id not. Thus, CaM-kinase II self-association is isoform specific, regulated by the conditions of activation, and is inhibited by peptides that bind to the catalytic domain likely via their autoregulatory-like sequence. A mode l for CaM-kinase II self-association is presented whereby catalytic domains in one holoenzyme interact with the regulatory domains in neighboring holo enzymes. These intersubunit-interholoenzyme autoinhibitory interactions cou ld contribute to both the translocation and inactivation of CaM-kinase II p reviously reported in models of ischemia.