Abstract
Both long-term potentiation (LTP) and depression (LTD) of excitatory synapse strength require the Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) and its autonomous activity generated by Thr-286 autophosphorylation. Additionally, LTP and LTD are correlated with dendritic spine enlargement and shrinkage that are accompanied by the synaptic accumulation or removal, respectively, of the AMPA-receptor regulatory scaffold protein A-kinase anchoring protein (AKAP) 79/150. We show here that the spine shrinkage associated with LTD indeed requires synaptic AKAP79/150 removal, which in turn requires CaMKII activity. In contrast to normal CaMKII substrates, the substrate sites within the AKAP79/150 N-terminal polybasic membrane-cytoskeletal targeting domain were phosphorylated more efficiently by autonomous compared with Ca2+/CaM-stimulated CaMKII activity. This unusual regulation was mediated by Ca2+/CaM binding to the substrate sites resulting in protection from phosphorylation in the presence of Ca2+/CaM, a mechanism that favors phosphorylation by prolonged, weak LTD stimuli versus brief, strong LTP stimuli. Phosphorylation by CaMKII inhibited AKAP79/150 association with F-actin; it also facilitated AKAP79/150 removal from spines but was not required for it. By contrast, LTD-induced spine removal of AKAP79/150 required its depalmitoylation on two Cys residues within the N-terminal targeting domain. Notably, such LTD-induced depalmitoylation was also blocked by CaMKII inhibition. These results provide a mechanism how CaMKII can indeed mediate not only LTP but also LTD through regulated substrate selection; however, in the case of AKAP79/150, indirect CaMKII effects on palmitoylation are more important than the effects of direct phosphorylation. Additionally, our results provide the first direct evidence for a function of the well-described AKAP79/150 trafficking in regulating LTD-induced spine shrinkage.
Highlights
Both long-term potentiation (LTP) and depression (LTD) of excitatory synapse strength require the Ca2؉/calmodulin (CaM)-dependent protein kinase II (CaMKII) and its autonomous activity generated by Thr-286 autophosphorylation
As the anchored protein kinase A (PKA) is removed together with AKAP79/150, this removal is thought to aid LTD by preventing re-phosphorylation of the PKA site GluA1 Ser-845 after dephosphorylation by anchored CaN [12, 15]. For quantification of this AKAP79/150 movement, we utilized the spine to shaft ratio, a measurement that was validated in our previous studies [15, 16]
We decided to test whether the LTD-induced synaptic removal of AKAP79/ 150 is dependent on CaMKII, a kinase recently shown to be required for NMDA-receptor-dependent LTD [7]
Summary
In agreement with previous studies [12, 15, 16], AKAP79/150 (Fig. 1A) was removed from dendritic spine synapses in cultured hippocampal neurons within minutes after cLTD stimuli by 30 M NMDA application for 3 min (Fig. 1, B–D). CaMKII activity was required for cLTD-induced synaptic removal of AKAP79/150 in neurons (see Fig. 1), and CaMKIImediated phosphorylation of the AKAP79/150-targeting domain inhibited its binding to F-actin in vitro (see Fig. 2F). F-actin co-localization of the phosphomimetic AKAP79-YFP EE mutant was statistically undistinguishable from the negative control with YFP alone (Fig. 4B) These results indicate that phosphorylation of the Thr-87/ Ser-92 CaMKII sites in the targeting domain can disrupt the F-actin association of AKAP79/150 within cells. Combining the C36S/C129S and 9D mutations was sufficient to fully mimic the cLTD-induced synaptic removal of AKAP79 such that the 9DCS combination mutant was localized predominantly in the dendritic shaft cytosol even under basal control conditions without any stimulation (Fig. 6, A and B). When mCherry localization is assessed in the same manner as the AKAP79/150 localization, no movement was detected (Fig. 7I)
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