Abstract
Calcium-calmodulin (Ca/CaM) dependent protein Kinase-II (CaMKII) is a major mediator of various cellular signaling processes and has been implicated in schizophrenia, heart failure, arthritis, and certain types of cancers. CaMKII subunits assemble to form a large dodecameric holoenzyme with which numerous proteins are known to interact. Among these regulators are CaMKIIN-alpha and -beta, small 8 kDa proteins that are expressed almost exclusively in brain and testis. CaMKIIN proteins are highly specific inhibitors of CaMKII activity, but the physiological role and oligomeric state of these proteins are not known. It is also not known if interactions with the holoenzyme are stable or transient, or if these interactions are stoichiometric with kinase subunits. To address these questions we used Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid method that simultaneously measures Homo-FRET and fluorescence correlation spectroscopy (FCS). FPFA studies reveal that C-terminal Venus-tagged CaMKIIN is a monomeric protein that can bind stably to Ca/CaM-activated CaMKII. Up to six CaMKIIN monomers can bind to the holoenzyme, suggesting that one CaMKIIN inhibitory protein interacts with two CaMKII subunits in the holoenzyme. Furthermore, Homo-FRET analysis of Venus-tagged CaMKII holoenzyme co-incubated with untagged CaMKIIN revealed the separation of catalytic domain pairs, but only after activation with Ca/CaM. This is consistent with our previous observation of catalytic domain unpairing in hippocampal neurons upon calcium influx through NMDA receptors. The C-terminus of CaMKIIN is thought to play a key role in inhibition of CaMKII because a reduction on inhibitory potency was observed when its C-terminal was tagged with Venus. Our results suggest that in addition to inhibiting the kinase, CaMKIIN regulates catalytic domain pairing.
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