Fluorescence Polarization and Fluctuation Analysis Reveals CAMKIIα Catalytic Domain Pair Extension Triggered by Ca2+/Calmodulin

The autoinhibited calcium/calmodulin-dependent protein kinases-II (CaMKII) assembles to form a holoenzyme structure with multiple catalytic domain pairs radiating out from a central core composed of oligomerization domains. Activation of CaMKII upon binding calcium2+/calmodulin (CaM) is known to initiate a biochemical cascade resulting in CaMKII translocation to synaptic spines and the induction of long-term potentiation. Little is known about the structural changes in the intact CaMKII holoenzyme responsible for this activation. Fluorescence polarization and fluctuation analysis (FPFA) microscopy, a method that combines time-resolved anisotropy and fluorescence correlation spectroscopy (FCS), was used to monitor Venus-CaMKIIα (where a Venus fluorescent-protein is tagged onto the N-terminus of CaMKIIα) before and after activation with CaM. FPFA simultaneously monitors brightness (a measure of the number of subunits in the holoenzyme), homo-FRET (a catalytic domain proximity detector) and diffusion time (a parameter sensitive to the mass and hydrodynamic volume of the holoenzyme). Together these measurements can potentially detect an array of different covert structural changes in the CaMKII holoenzyme triggered by CaM. In homogenates prepared from HEK cells expressing Venus-CaMKIIα, the addition of CaM triggered T286 autophosphorylation, an indicator of kinase activation. FPFA revealed a large increase in the holoenzyme diffusion time. Because changes in holoenzyme subunit stoichiometry were not observed, the change in diffusion time most likely indicates a large change in hydrodynamic volume, such as catalytic domain pair extension. This change was not observed in Venus-CaMKIIα-TT305/6DD, a mutant that cannot bind calmodulin. Holoenzyme activation by CaM did trigger a small, dose-dependent biphasic change in anisotropy (homo-FRET between Venus-tagged catalytic domains), but its magnitude was too small to be interpreted as catalytic domain un-pairing. Interpretation of these experiments will be discussed.