A. Hudmon et al., Light scattering and transmission electron microscopy studies reveal a mechanism for calcium/calmodulin-dependent protein kinase II self-association, J NEUROCHEM, 76(5), 2001, pp. 1364-1375
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.