Allosteric activation of CaMK4 by Calmodulin enables phosphorylation of WNK2 kinase

The Ca2+-Calmodulin (CaM)-dependent protein kinase (CAMK) family member, CaMK4, has recently attracted interest owing to its implication in neurodevelopment, learning, memory and synaptic plasticity. However, knowledge of the molecular mechanisms underlying CaMK4 regulation is currently incomplete. Initially, we used crosslinking mass spectrometry and biophysical approaches to define the mode of interaction between CaMK4 and a known activator, the Ca2+ sensor protein, Calmodulin. These studies validated Calmodulin-mediated displacement of the autoinhibitory sequence C-terminal to the CaMK4 kinase domain via binding to the overlapping Calmodulin-binding sequence as the underlying mechanism of CaMK4 activation. Using proximity labelling mass spectrometry and biochemical methods, we identified further CaMK4 interactors, which localized CaMK4 to the microtubule cytoskeleton under basal conditions and validated nuclear translocation upon activation by Ca2+ influx into cells. WNK2 kinase was identified as a prominent CaMK4 interactor, which bound via its kinase domain to the CaMK4 kinase domain in structural mass spectrometry experiments, and enabled CaMK4 to phosphorylate Thr410 within the αG helix in the WNK2 kinase domain C-lobe. These data implicate CaMK4 in cytoplasmic signalling and raise the prospect that CaMK4 may facilitate crosstalk between Ca2+ and Cl- regulated pathways.