Abstract
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is an oligomeric enzyme with crucial roles in neuronal signaling and cardiac function. Previously, we showed that activation of CaMKII triggers the exchange of subunits between holoenzymes, potentially increasing the spread of the active state (Stratton et al., 2014; Bhattacharyya et al., 2016). Using mass spectrometry, we show now that unphosphorylated and phosphorylated peptides derived from the CaMKII-α regulatory segment bind to the CaMKII-α hub and break it into smaller oligomers. Molecular dynamics simulations show that the regulatory segments dock spontaneously at the interface between hub subunits, trapping large fluctuations in hub structure. Single-molecule fluorescence intensity analysis of CaMKII-α expressed in mammalian cells shows that activation of CaMKII-α results in the destabilization of the holoenzyme. Our results suggest that release of the regulatory segment by activation and phosphorylation allows it to destabilize the hub, producing smaller assemblies that might reassemble to form new holoenzymes.
Highlights
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is an oligomeric serine/threonine kinase that is important in neuronal signaling and cardiac function (Bhattacharyya et al, 2019; Kennedy, 2013; Lisman et al, 2002)
Each subunit of CaMKII has an N-terminal kinase domain that is followed by a regulatory segment and an unstructured linker that leads into a C-terminal hub domain (Figure 1A, B)
Mass spectrometry shows that peptides derived from the regulatory segment can break the CaMKII-α hub assembly
Summary
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is an oligomeric serine/threonine kinase that is important in neuronal signaling and cardiac function (Bhattacharyya et al, 2019; Kennedy, 2013; Lisman et al, 2002). The activation of CaMKII by Ca2+/calmodulin triggers the co-localization of subunits from different CaMKII holoenzymes, indicating that activation results in subunits being exchanged between holoenzymes (Bhattacharyya et al, 2016; Stratton et al, 2014) This conclusion was based on the results of experiments in which two samples of CaMKII were labeled separately with fluorophores of two different colors, mixed, and activated. Separate CaMKII samples were labeled with FRET donor and acceptor fluorophore pairs, respectively, and mixing these samples after activation led to increased FRET compared to mixing unactivated samples (Bhattacharyya et al, 2016). These data are consistent with subunit exchange. These experiments showed that activated CaMKII holoenzymes could phosphorylate subunits of unactivated ones, thereby spreading the activation signal (Stratton et al, 2014)
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