In this study, we have discovered the first catalytic function of CaMKIIβ in the mammalian brain. Remarkably, CaMKIIβ operates at the centrosome in a CaMKIIα-independent manner to drive dendrite retraction and pruning. CaMKIIβ localizes at the centrosome by forming a complex with the centrosomal trafficking protein PCM1. Accordingly, PCM1 is required for CaMKIIβ-regulation of dendrite morphogenesis and the pruning of dendrites in postnatal rat pups in vivo. We have also identified the ubiquitin ligase Cdc20-APC, which is enriched at the centrosome in neurons, as a novel substrate of CaMKIIβ. CaMKIIβ phosphorylates Cdc20 at Ser51 and thereby triggers the dispersion of Cdc20 from the centrosome, culminating in the inhibition of centrosomal Cdc20-APC activity and consequent dendrite retraction. Collectively, our findings define a novel isoform-specific function of CaMKIIβ that regulates ubiquitin-dependent protein degradation at the centrosome and thereby orchestrates dendrite patterning in mammalian brain neurons.
The identification of an unexpected function for CaMKIIβ in dendrite retraction and pruning bears significant ramifications for our understanding of the major protein kinase CaMKII. As the predominant CaMKII isoforms in the brain, CaMKIIα and CaMKIIβ form holoenzyme complexes in neurons16–18
. However, nearly all of the reported functions of CaMKII have focused on CaMKIIα in homomeric or heteromeric complexes19–22
. Accordingly, CaMKIIβ has been previously relegated to a largely redundant role within CaMKIIα heteromeric complexes or as a scaffold recruiting CaMKIIα to specific cellular locales such as dendritic spines34–36
. Our finding that CaMKIIβ operates at the centrosome in a CaMKIIα-independent manner unveils a biological function for CaMKIIβ homomeric complexes. Thus, rather than simply contributing to CaMKIIα complexes, CaMKIIβ homomers harbor a major biological function at the centrosome as drivers of dendrite retraction and pruning and consequent neuronal connectivity in the mammalian brain. During brain development, CaMKIIβ expression peaks at earlier time points relative to CaMKIIα41
. Therefore, it will be interesting to determine in future studies whether centrosomal CaMKIIβ homomeric complexes might also contribute to earlier aspects of neuronal development in which centrosomal signaling is thought to play a critical role, including neuronal polarization and migration42–43
. Beyond the nervous system, it will be important to identify the role of CaMKIIβ homomers in cellular homeostasis and development in other organ systems.
Elucidation of the CaMKIIβ/Cdc20 signaling link at the centrosome provides a mechanism by which CaMKIIβ promotes dendrite retraction and pruning. By inducing the phosphorylation of Cdc20 and inhibiting Cdc20-APC ubiquitin ligase activity at the centrosome, CaMKIIβ triggers a switch from dendrite growth and elaboration to dendrite retraction and pruning. These findings suggest that the centrosome may represent a critical signaling platform that integrates diverse cellular signals to determine the phase of dendrite morphogenesis in neurons. In view of the fundamental role of centrosome signaling in diverse systems from the control of cell polarity and migration to ciliary morphogenesis to vesicular transport43
, it will be interesting to explore whether centrosomal CaMKIIβ phosphorylation of Cdc20, or of other substrates yet to be identified, might contribute to these diverse biological processes.
The identification of the CaMKIIβ/Cdc20 signaling link also advances our understanding of the regulation of the major ubiquitin ligase Cdc20-APC. CaMKIIβ phosphorylates Cdc20 at Ser51, inducing dispersion of Cdc20 from the centrosome and inhibiting centrosomal Cdc20-APC activity. The phosphorylation-dependent control of the subcellular localization of Cdc20 represents an entirely new mode of regulation of the ubiquitin ligase Cdc20-APC. Interestingly, CaMKIIβ had little or no effect on Cdc20 binding to core APC subunits, the in vitro ubiquitin ligase activity of Cdc20-APC, or Cdc20 polyubiquitination (data not shown), highlighting the significance of Cdc20 dispersion as a critical cellular mechanism for regulating centrosomal Cdc20-APC activity. Future research should explore whether Cdc20 has additional roles outside of the centrosome, bearing in mind the exciting possibility that Cdc20 dispersion might be part of a developmental program that coordinates dendrite patterning with other steps in the establishment of neuronal connectivity.
Although CaMKIIβ and CaMKIIα are similar in their catalytic, autoregulatory, and association domains, they diverge in their variable region41
. We have identified a centrosomal targeting sequence (CTS) in the unique variable region of CaMKIIβ, thus providing spatial specificity for CaMKIIβ function. Further, we have found that the CTS mediates the interaction of CaMKIIβ specifically with the centrosomal targeting protein PCM1. Although PCM1 targets structural proteins to the centrosome44
, our findings suggest that PCM1 may also drive the centrosomal localization of signaling proteins. Notably, in addition to interacting with CaMKIIβ, endogenous PCM1 formed a complex with endogenous Cdc20 in neurons and stimulated CaMKIIβ phosphorylation of Cdc20 (data not shown). These observations suggest that beyond recruiting CaMKIIβ to the centrosome, PCM1 may also operate as a scaffold protein that organizes the CaMKIIβ/Cdc20 signaling pathway at the centrosome.
Although signaling and cell-intrinsic mechanisms that promote dendrite growth have been the subject of substantial interest5–8
, the master regulatory mechanisms that govern the developmental transition from dendrite elaboration to dendrite pruning in the mammalian brain have remained to be elucidated. The identification of centrosomal CaMKIIβ signaling as a mechanism that restricts dendrite elaboration and promotes dendrite pruning suggests that pathways that actively drive dendrite retraction have evolved to sculpt dendrite arbors and thus establish accurate neuronal circuits. Since abnormalities in dendrite development represent prominent pathological features in mental retardation and autism spectrum disorders4,45–46
, it will be interesting to determine if deregulation of centrosomal CaMKIIβ signaling contributes to the pathogenesis of neurodevelopmental disorders of cognition.