CaMKII regulates the depalmitoylation and synaptic removal of the scaffold protein AKAP79/150 to mediate structural long-term depression

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.


CaMKII inhibition blocks cLTD-induced synaptic removal of AKAP79
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). As the anchored 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). Indeed, CaMKII inhibition with the highly selective and cell-penetrating peptide inhibitor tatCN21 (23) completely blocked cLTD-induced translocation of endogenous AKAP150 in hippocampal cultures (Fig. 1, B-D), as assessed by analysis of line scans of fluorescence intensity through spines and neighboring dendrites (Fig. 1C) and spine to dendritic shaft mean fluorescence ratios (Fig. 1D) of AKAP150 immunostained neurons. CaMKII inhibition by a mechanistically different inhibitor, KN93 (24 -26), had a similar effect of preventing cLTD-induced translocation (Fig. 1D). In contrast to AKAP79, the postsynaptic marker Shank1 was not significantly removed from spines in response to the cLTD stimuli (Fig. 1E). Thus, our quantification of the AKAP150 shows a characteristic fluorescent profile with a high concentration in the spine head compared with shaft. This profile is disrupted by cLTD, indicating translocation, but is preserved in the presence of tatCN21 during cLTD. D, quantification of spine to dendritic shaft ratio (normalized to control) from experiments from three individual neuron preparations as determined by 10 masks per neuron over areas of puncta co-localization of AKAP150 and PSD95 (data not shown), and nearby dendritic shafts. Mean AKAP150 spine to shaft ratio (2.5 Ϯ 0.14, n ϭ 19 neurons) was significantly decreased after cLTD (1.0 Ϯ 0.1, n ϭ 19 neurons; p Ͻ 0.001, one-way ANOVA, Newman-Keuls post hoc analysis) compared with all other conditions: tatCN21 (2.6 Ϯ 0.13, n ϭ 19 neurons); tatCN21 ϩ cLTD (2.6 Ϯ 0.2, n ϭ 18 neurons); KN93 (10 M) (2.0 Ϯ 0.15, n ϭ 15 neurons); KN93 ϩ cLTD (2.6 Ϯ 0.25, n ϭ 18 neurons). E, mean spine to shaft ratio of endogenous SHANK (2.36 Ϯ 0.18, n ϭ 16) revealed no translocation of SHANK with cLTD (2.64 Ϯ 0.2, n ϭ 13 neurons; p ϭ 0.9885, two-tailed t test). Data shown in graphs are normalized to control.

CaMKII regulates AKAP79/150 function in LTD
AKAP79 removal was not affected by the spine shrinkage caused by cLTD stimuli (see also Fig. 7). The requirement of CaMKII for cLTD-induced synaptic removal of AKAP79/150 provides a potentially new mechanism for the recently described CaMKII function in NMDA-receptor-dependent LTD (7).

CaMKII inhibition does not significantly interfere with cLTD-induced F-actin re-organization
The AKAP79/150-targeting domain (see Fig. 1A) binds to F-actin (14) and the acidic membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ) (17), and AKAP79/150 removal from synapses requires cLTD-induced actin reorganization that is controlled by CaN signaling and phospholipase C (PLC)-mediated hydrolysis of PIP 2 (14,16). As the CaMKII␤ isoform binds and bundles F-actin only in its inactive state but not after Ca 2ϩ /CaM stimulation (27), CaMKII inhibition could directly or indirectly affect cLTD-induced actin dynamics. The cLTD stimuli caused a marked redistribution of F-actin from enrichment in spines to localization more in longer fibers along the dendrites ( Fig. 2A), as expected from previous work (14,16,28). Although tatCN21 (but not KN93) increased the basal spine localization of F-actin, neither of the CaMKII inhibitors blocked the pronounced cLTD-induced F-actin redistribution (Fig. 2B). CaMKII␤ has been shown to be able to affect actin dynamics in principle (27, 29 -34); however, the lack of an effect of CaMKII activity inhibition in cLTD-induced actin dynamics is consistent with our previous observation that LTD specifically requires the CaMKII ␣ rather than the ␤ isoform (7).

AKAP79/150-targeting domain is a high-autonomy CaMKII substrate
As CaMKII inhibition had no detectable effect on cLTDinduced F-actin re-organization that is required for synaptic removal of AKAP79/150, we next tested for direct phosphorylation of the AKAP79/150-targeting domain by CaMKII as a possible underlying mechanism for the CaMKII requirement in the cLTD-induced removal of AKAP79/150 from synapses. LTD specifically requires the autonomous (Ca 2ϩ -independent) activity of CaMKII that is induced by Thr-286 autophosphorylation (7); as expected, this Thr-286 autophosphorylation is  ). B, percent of F-actin synaptic localization from experiments from three individual neuron preparations, as determined by defining synaptic puncta using threshold masks as described under the "Experimental procedures." cLTD-induced F-actin removal from spines as indicated by decreased percentage of F-actin in puncta was significant for control (before 1 Ϯ 0.17 and after 0.5 Ϯ 0.08, n ϭ 19 neurons, ***, p Ͻ 0.001), tatCN21-(before 1.4 Ϯ 0.17 and after 0.8 Ϯ 0.07, n ϭ 17 neurons, *, p Ͻ 0.05), and KN93 (before 1.1 Ϯ 0.13 and after 0.6 Ϯ 0.1, n ϭ 19 neurons, ***, p Ͻ 0.001)-treated neurons as assessed by one-way ANOVA, Newman-Keuls post hoc analysis. Basally, tatCN21 and KN93 both stabilized F-actin in spines compared with control (##, p Ͻ 0.01; #, p Ͻ 0.05, respectively, one-way ANOVA, Newman-Keuls post hoc analysis). C, induction of the CaMKII Thr-286 autophosphorylation that generates autonomous activity after cLTP or cLTD stimuli of hippocampal cultures, assessed by Western blot analysis. The arrowhead marks 50 kDa. D, in contrast to regular substrates like MAP2, AKAP79/150 is phosphorylated more efficiently by autonomous versus Ca 2ϩ /CaM-stimulated activity. Both MAP2 and the AKAP79-targeting domain were present in the same radioactive phosphorylation assays; shown are autoradiographs after SDS-PAGE (left) and quantification of three experiments (right; mean Ϯ S.E.). E, phosphorylation of the AKAP79-targeting domain by autonomous CaMKII reduces its interaction with F-actin in vitro, as assessed by a co-sedimentation assay. Shown is a Western blot of the supernatants (S) and pellets (P) after centrifugation at 100,000 ϫ g that sediments F-actin together with bound proteins.

CaMKII regulates AKAP79/150 function in LTD
induced by our cLTD stimuli in hippocampal neurons (Fig. 2C). Indeed, autonomous CaMKII efficiently phosphorylated the AKAP79-targeting domain (residues 1-153) in a biochemical assay with purified protein (Fig. 2D). In fact, phosphate incorporation into AKAP79 was more efficient even when compared with the classical CaMKII substrate protein MAP2 that was present within the same reaction mix (Fig. 2D). However, although further stimulation of the autonomous CaMKII by addition of Ca 2ϩ /CaM significantly enhanced MAP2 phosphorylation (as expected for traditional CaMKII substrates (21)), phosphorylation of the AKAP79-targeting domain was instead suppressed (Fig. 2D). Thus, the CaMKII autonomy (i.e. the ratio of autonomous over maximal stimulated activity) was around the traditional level of ϳ20% for MAP2 phosphorylation but well over 300% for AKAP79 phosphorylation (Fig.  2D). Importantly, phosphorylation of the AKAP79-targeting domain by autonomous CaMKII decreased its binding to F-actin, as assessed by an in vitro co-sedimentation assay that showed more free AKAP79 that is not bound to F-actin in the supernatant after phosphorylation ( Fig. 2E; a finding later corroborated also by imaging studies within cells, see Fig. 4).

Molecular mechanism for high-autonomy phosphorylation of AKAP79/150
Notably, LTP stimuli favor CaMKII-mediated phosphorylation of traditional low-autonomy substrates such as GluA1 whereas LTD stimuli favor phosphorylation of the high-autonomy substrate GluA1 Ser-567 (7). The novel high-autonomy substrate AKAP79/150 identified here exhibited a substantially further elevated level of autonomy compared with GluA1 Ser-567 (i.e. Ͼ300% versus ϳ100%), further strengthening possible mechanistic links between AKAP79/150 phosphorylation by CaMKII and LTD.
Sequence analysis showed that the polybasic region B of the AKAP79-targeting domain (AKAP-B) contains three Ser/Thr residues that meet the basic criterion for CaMKII consensus sites, an Arg residue at position Ϫ3 (Fig. 3A; compare also Fig.  1A). Additionally, AKAP-B contains a predicted CaM-binding site, as determined by sequence analysis using the calmodulin target database (University of Toronto, Toronto, Ontario, Canada; http://calcium.uhnres.utoronto.ca/ctdb/ctdb/sequence.html) 8 and as shown in previous studies (35,36). Thus, addition of Ca 2ϩ / CaM may further stimulate the activity of autonomous CaMKII in principle, but may additionally specifically protect the AKAP79 phosphorylation sites from phosphorylation by binding to the overlapping binding site, thereby resulting in the apparent autonomy of over 300%. This hypothesis was tested, and it is indeed supported by the following findings.
Two of the predicted AKAP-B residues, Thr-87 and Ser-92, were phosphorylated by autonomous CaMKII, as determined by in vitro phosphorylation assays of the AKAP-B peptide and its various site-specific phosphorylation-incompetent mutants (Fig. 3B). Ca 2ϩ /CaM indeed bound to AKAP-B in an overlay assay with biotinylated CaM, and two mutations predicted to interfere with the CaM-binding site (np, W79N/L82P; ⌬CaM, W79N/L82N) indeed disrupted or abolished the CaM binding (Fig. 3C). The hypothesized mechanism for high CaMKII autonomy by CaM-mediated substrate protection requires that CaM must bind to AKAP-B only when stimulated by Ca 2ϩ but not in basal conditions without Ca 2ϩ . Such Ca 2ϩ -dependent binding to AKAP-B was indeed observed using an IAEDANSlabeled CaM (CaM-I) that increases in fluorescence upon bind- . AKAP79 is protected from phosphorylation by a substrate-directed Ca 2؉ /CaM mechanism. A, sequence of the AKAP-B peptide, which is derived from the AKAP79 polybasic region B, with mutations and potential phosphorylation sites indicated; the CaMKII phosphorylation consensus sequence is shown for comparison. B, Thr-87 and Ser-92 are substrate sites for autonomous CaMKII, as indicated by reduced in vitro phosphorylation of their respective alanine-mutant peptides. C, ⌬CaM mutation (see A) prevents Ca 2ϩ /CaM binding to AKAP-B, as detected by blot-overlay with biotinylated CaM. D, rat AKAP-B region also binds CaM in a Ca 2ϩ -dependent manner, as detected using the labeled CaM-I that increases in fluorescence upon binding to Ca 2ϩ and protein. Binding to rat AKAP-B was detected only in the presence of Ca 2ϩ . E, ⌬CaM mutation abolishes the substrate protection by Ca 2ϩ /CaM and results in phosphorylation like a regular substrate, with more phosphorylation in the presence of Ca 2ϩ /CaM, resulting in ϳ20% autonomy. F, high CaMKII autonomy for the phosphorylation site in the GluA1 loop1 is not due to substrate protection by Ca 2ϩ /CaM, as no binding of CaM-I was detected in the fluorescence assay. In the same assay, clear CaM-I binding to rat AKAP-B (but not its ⌬CaM mutant) was detected.

CaMKII regulates AKAP79/150 function in LTD
ing to Ca 2ϩ and protein or peptide (37,38). Fluorescence of CaM-I increased after addition of Ca 2ϩ and then increased further after addition of AKAP-B (Fig. 3D). By contrast, adding AKAP-B first did not cause any fluorescence increase, whereas subsequent addition of Ca 2ϩ again increased fluorescence to the maximal level (Fig. 3D).
For a final test of the mechanism underlying the high autonomy of CaMKII toward AKAP79/150, the phosphorylation of AKAP-B wild-type (WT) and its CaM-binding-deficient mutant (⌬CaM) was compared in biochemical kinase assays in vitro. Both AKAP-B WT and ⌬CaM were equally efficiently phosphorylated by autonomous CaMKII in the absence of Ca 2ϩ . However, although addition of Ca 2ϩ /CaM to naive CaMKII resulted in only negligible phosphorylation of AKAP-B WT, phosphorylation of AKAP-B ⌬CaM was instead even 4 -5fold greater compared with the autonomous phosphorylation (Fig. 3E). Thus, abolishing the CaM-binding site transformed AKAP-B into a regular CaMKII substrate, demonstrating that the Ca 2ϩ /CaM binding is indeed the mechanism underlying suppression of phosphorylation during stimulation, thereby causing the unusually high autonomy. In the initial experiments ( Fig. 3E, left panel), concentrations of substrate and CaM were used that are typical for CaMKII assays. However, these typical assay conditions contain an excess of substrate (75 M AKAP-B) over CaM (2 M); the observed results are likely due to sequestration of CaM by AKAP-B (which would in turn dramatically reduces stimulation of activity for the non-phosphorylated naive CaMKII), rather than to substrate protection by CaM binding. However, the same results were also observed when CaM (10 M) was instead in excess over AKAP-B (7.5 M), albeit at lower total phosphorylation rates (Fig. 3E, right panel), as expected at lower substrate concentration. Under these conditions, even full occupancy of AKAP-B with CaM still results in sufficient free Ca 2ϩ /CaM (2.5 M) for maximal CaMKII stimulation. Thus, substrate-directed Ca 2ϩ /CaM binding is indeed a mechanism underlying high CaMKII autonomy and suppression of phosphorylation during Ca 2ϩ stimulation.

AKAP79/150 and GluA1 Ser-567 belong to different LTD-related CaMKII substrate classes
As mentioned above, the other known LTD-related highautonomy substrate of CaMKII is GluA1 Ser-567 (7), but the mechanism underlying the high autonomy is unknown in this case. Although CaMKII autonomy for the AKAP79/150-targeting domain (Ͼ300%) is significantly greater than for GluA1 Ser-567 (ϳ100%), we argued that this autonomy for GluA1 Ser-567 could also be caused by overlapping Ca 2ϩ /CaM binding, but perhaps with lower affinity. Indeed, the GluA1 loop1 region that contains Ser-567 contains a sequence with similarity to the "basic 1-5-10 motif" for Ca 2ϩ /CaM binding, albeit with a one amino acid insertion compared with the consensus sequence. However, in an overlay assay with biotinylated CaM, no CaM binding to the GluA1 loop1 was detected. This result could still be consistent with a lower affinity binding. However, even in the more sensitive binding assay that utilizes CaM-I fluorescence, no CaM binding to GluA1 loop1 was detectable (Fig. 3F). Thus, GluA1 Ser-567 and the AKAP79/150-targeting domain represent two distinct classes of LTD-related high-autonomy CaMKII substrates. Although the mechanism underlying high autonomy for AKAP79/150 was demonstrated here (see Fig. 3E), the mechanism for GluA1 Ser-567 remains to be elucidated.

Phosphomimetic Thr-87/Ser-92 mutations in AKAP79/150 decrease F-actin co-localization in COS7 cells but only mildly decrease synaptic localization
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). Thus, we decided to test the effect of mutating the CaMKII phosphorylation sites on the co-localization of AKAP79/150 with F-actin in heterologous cells. AKAP79 tagged with YFP on its C terminus was expressed in COS7 cells, and F-actin was stained with phalloidin-Texas Red (Fig. 4A). Mutating the CaMKII phosphorylation sites Thr-87 and Ser-92 to Ala (AA) had no effect on F-actin co-localization, as expected. By contrast, corresponding phospho-mimetic mutations to Glu (EE) still exhibited prominent localization in membrane structures but significantly reduced AKAP79 co-localization with F-actin ( Fig. 4B). In fact, 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. To examine the effects of the CaMKII phosphorylation sites on AKAP79/ 150 in neurons, AKAP79-YFP and its Thr-87/Ser-92 mutants were expressed in hippocampal cultures (Fig. 4C). Although the phosphorylation-incompetent AA mutant localized to dendritic spines significantly more than the phospho-mimetic EE mutant, this difference was rather mild (Fig. 4D). The spine localization of mTurquois2 used as a cell fill was unaffected by the co-expressed AKAP79-YFP construct (Fig. 4E). No significant differences in spine size were detected (data not shown). Thus, although the CaMKII-mediated phosphorylation of Thr-87/Ser-92 can inhibit targeting domain interaction with F-actin, it does not appear to be sufficient to induce the removal of AKAP79/150 from dendritic spines.

Combined phosphorylation and depalmitoylation removes AKAP79/150 from synapses
Next, we decided to test whether additional phosphorylation sites in the AKAP79/150 polybasic targeting domain might be responsible for the LTD-induced CaMKII-dependent removal of AKAP79/150 from spines. Mutating all five potential Ser/Thr phosphorylation sites in the B region (in the 5A mutant) reduced CaMKII-mediated phosphorylation of the AKAP79/150-targeting domain; however, mutating additional sites in the C region (in the 9A mutant) reduced phosphorylation even further (Fig. 5, A and B). More importantly, phosphorylation of both AKAP79/150 WT and 5A (but not 9A) by autonomous CaMKII was suppressed by addition of Ca 2ϩ /CaM (Fig. 5B), indicating that the additional sites in the C region are also LTD-related high autonomy sites that are protected by Ca 2ϩ /CaM, whereas the remaining phosphorylation sites in the 9A mutant, which are

CaMKII regulates AKAP79/150 function in LTD
located outside the AKAP-targeting domain, are not protected by Ca 2ϩ /CaM. (Please also note that at least some of these remaining phosphorylation sites may actually be located in the vector-encoded linker regions flanking the C-terminal His 6 epitope tag, see under "Experimental procedures"). Indeed, experiments with IAEDANS-labeled CaM showed that all of the three AKAP79/150 polybasic regions bind Ca 2ϩ /CaM, with region B showing the highest affinity, followed by regions C and A (Fig. 5C). Specificity of CaM binding was examined further by single point mutations of the predicted binding site of each region; in each case, Ca 2ϩ / CaM binding was significantly reduced, although not completely abolished (Fig. 5D). Although Ca 2ϩ /CaM binding to the A and B regions has been indicated by previous studies (35,36,39), this is the first report of Ca 2ϩ /CaM binding to the C region, as well as the first direct comparison of all three binding sites.
Similar to the T87E/S92E mutation, the full phospho-mimetic 9D mutation reduced basal synaptic localization of AKAP79 only mildly. However, the 9D mutation significantly facilitated the cLTD-induced synaptic removal of AKAP79. Although full removal of AKAP79 wild-type required stimulation with 30 M NMDA, 10 M NMDA was sufficient

CaMKII regulates AKAP79/150 function in LTD
for full removal of the 9D mutant (Fig. 6, A and B). The corresponding phospho-incompetent 9A mutant was still fully removed from spines by 30 M NMDA; however, in contrast to AKAP79 wild type (or 9D), it did not significantly move in response to 10 M NMDA (Fig. 6, A and B). Thus, the AKAP79-targeting domain phosphorylation facilitates the cLTD-induced removal from spines, but is neither absolutely necessary nor sufficient. . Compared with 79WT, 9D basal spine localization was slightly reduced (#, p Ͻ 0.05 by unpaired t test to 79WT control), and 9SDCS basal spine localization was strongly reduced (control 0.59 Ϯ 0.05; ###, p Ͻ 0.0001 by unpaired t test to 79WT control). C, [ 3 H]palmitate labeling in HEK-293 cells confirms complete loss of palmitoylation of 9DCS but lacks the resolution necessary to quantitatively compare the other AKAP mutant species. D, APEGS assay of AKAP palmitoylation mutants expressed in HEK cells reveals three bands for AKAP79WT-GFP. The highest molecular weight band corresponds to a dual palmitoylation species; the middle band represents mono-palmitoylated species, and the bottom band reveals unpalmitoylated species. Two bands are present for the C36S and C129S mutants, and a single band is present in the C36S/C129S and WT (HϪ) conditions. E, APEGS assay comparing palmitoylation states among the indicated AKAP79-GFP mutants. The proportion of each mutant that is palmitoylated was quantitated as a ratio of the higher molecular weight band intensities relative to the lowest molecular weight band.

CaMKII regulates AKAP79/150 function in LTD
A similarly facilitated spine removal with minimal effect on basal spine localization has been observed for the palmitoylationincompetent AKAP79 C36S/C129S (CS) mutant (18), indicating that AKAP79 depalmitoylation also facilitates spine removal without being sufficient. Thus, the effect of the 9D mutant on facilitating synaptic AKAP79 removal could be mediated by preventing AKAP79 palmitoylation. However, AKAP79 WT, 9D, or 9A all appeared to be palmitoylated as assessed by [ 3 H]palmitate labeling in HEK-293 cells (Fig. 6C). Nonetheless, we further investigated the effect of AKAP79 phosphorylation on its palmitoylation state using a recently developed acyl-PEGyl exchange gel shift (APEGS) assay that allows palmitoylation stoichiometry to be determined more directly (40). By labeling palmitoylated cysteines with bulky polyethylene glycol moieties and subsequent SDS-PAGE/Western blotting, palmitoylation species can be visualized as higher molecular weight (MW) bands. Consistent with the two known palmitoylation sites, APEGS analysis of AKAP79-GFP WT in HEK-293 cells revealed two bands corresponding to mono-and di-palmitoylated AKAP79 that migrated at higher MW than the unmodified/depalmitoylated species (Fig. 6C). Importantly, neither of these higher MW bands was visible for the C36S/ C129S double mutant, and only one of these bands was visible for each of the C36S and C129S single mutants (Fig. 6C). Using this APEGS approach, we found a significant reduction in palmitoylation for the 9D mutant, with no changes detected for either the 9A or EE mutants (Fig. 6, E and F). Thus, although mimicking AKAP79 phosphorylation reduces palmitoylation, it does not eliminate it, indicating that phosphorylation and depalmitoylation must act in concert to mediate synaptic removal of AKAP79/150. Indeed, 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).

CaMKII inhibition blocks cLTD-induced AKAP150 depalmitoylation
Our results show that combined AKAP79/150 phosphorylation and depalmitoylation is sufficient to remove the AKAP from dendritic spines, although individually phosphorylation or depalmitoylation are not. In agreement with our prior studies, using two complementary biotin-switch assays for the AKAP150 palmitoylation state (18,19), we found that cLTD stimulation that removes AKAP150 from dendritic spines also caused robust AKAP150 depalmitoylation in cultured hippocampal neurons (ABE assay, Fig. 7, A and B; BMCC assay, Fig.  7, D and E). As AKAP79/150 phosphorylation facilitated its LTD-induced spine removal but was not strictly required for it, we tested whether the CaMKII dependence of this spine removal could be explained instead by CaMKII dependence of the AKAP150 depalmitoylation. Indeed, CaMKII inhibition with tatCN21 blocked the cLTD-induced AKAP150 depalmitoylation (Fig. 7, A-E). By contrast, the basal AKAP150 palmitoylation state in the absence of any cLTD stimulus was unaffected by the tatCN21 treatment. Finally, we found no effect of cLTD or CaMKII inhibition on the GluA1 palmitoylation state (Fig. 7, A and C), supporting the notion that AKAP79/150 depalmitoylation is specifically regulated and does not reflect a global depalmitoylation of synaptic proteins.

Depalmitoylation is required for both AKAP79 removal and structural LTD
To mimic persistent palmitoylation of AKAP79, we added a consensus sequence for N-terminal myristoylation, a related lipid modification that is irreversible (19). In addition, APEGS analysis of this myr-AKAP79-GFP mutant expressed in HEK-293 cells revealed significantly increased palmitoylation compared with WT (Fig. 6, E and F). Importantly, preventing delipidation in this myr-AKAP79 mutant completely blocked the cLTD-induced AKAP79 movement in response to 30 M NMDA (Fig. 7, F and G). Thus, depalmitoylation is required for cLTD-induced synaptic AKAP removal, whereas phosphorylation facilitates this movement without being required. Notably, the myr-AKAP79 mutant also enabled us to directly address the question whether or not removal of AKAP79/150 from spines is also required for structural LTD, i.e. the spine shrinkage that occurs in response to LTD stimuli. Although expression of AKAP79-GFP wild-type allowed normal spine shrinkage in response to cLTD stimulation with 30 M NMDA, expression of the myr-AKAP79 mutant completely blocked such cLTDinduced spine shrinkage (Fig. 7H). Note that spine size was determined by the area occupied by a fluorescent spine filler (mCherry), whereas AKAP79 spine localization was determined by point masks within the spines, which instead reflects its concentration. Thus, the noted movement of AKAP79/150 out of spines is not simply a direct consequence of the reduced spine size. Indeed, when mCherry localization is assessed in the same manner as the AKAP79/150 localization, no movement was detected (Fig. 7I).

Discussion
We show here that the well-studied removal of the synaptic anchoring protein AKAP79/150 from dendritic spines in response to LTD stimuli is functionally required for structural LTD, and like LTD expression, it also requires CaMKII activity. Direct phosphorylation by CaMKII facilitated the removal of AKAP79/150 from spines, but it was not required; instead, the essential function of CaMKII was additional regulation of AKAP79/150 depalmitoylation (shown schematically in Fig. 8).
AKAP79/150 targeting to dendritic spines is mediated at least in part by its interaction with the F-actin cytoskeleton (14,16), and CaMKII-mediated phosphorylation of AKAP79/150 disrupted this interaction. Although previous studies found that PKC phosphorylation of the AKAP79-targeting domain could also inhibit its interaction with F-actin in vitro (14), inhibition of PKC did not prevent LTD-induced AKAP translocation from spines (16), as we observed here for inhibition of CaMKII (Fig. 1). Thus, although both CaMKII and PKC can phosphorylate the AKAP79-targeting domain and regulate F-actin binding in vitro, only CaMKII activity appears to be important for control of AKAP79/150 targeting in neurons. A possible explanation could be that the persistent autonomous activity of CaMKII generated by LTD stimuli (Fig. 2C)

CaMKII regulates AKAP79/150 function in LTD
domain when Ca 2ϩ levels fall low enough to reverse the Ca 2ϩ / CaM protection that we observed for these substrate sites (Fig.  8). Although low Ca 2ϩ would also allow PKC access to these previously Ca 2ϩ /CaM-protected substrate sites (14), low Ca 2ϩ levels likely would be insufficient to maintain PKC activation. In addition, CaMKII could be promoting AKAP depalmitoylation through additional mechanisms (discussed below) as well as inhibiting AKAP interactions with MAGUK scaffold proteins to further promote spine loss (42).
Related to this Ca 2ϩ /CaM protection of targeting domain phosphorylation, we found that AKAP79/150 represents a novel class of LTD-related high-autonomy CaMKII substrates that is only appreciably phosphorylated by autonomous CaMKII activity. One other LTD-related high-autonomy CaMKII substrate identified in our previous studies is GluA1 Ser-567 (7), a site that inhibits AMPAR synaptic localization (20). However, GluA1 Ser-567 was phosphorylated equally well by autonomous or Ca 2ϩ /CaM-stimulated CaMKII activity, and it does Note: this assay involves immunoprecipitating the protein of interest, followed by labeling the palmitoylated fraction of the protein with biotin, processing via SDS-PAGE, and Western blotting first with streptavidin-conjugated HRP and then antibody toward the protein of interest. Irreversible lipidation of AKAP79 blocks its NMDA-induced spine-to-shaft translocation and spine shrinkage. F, 12 DIV hippocampal neurons transfected with AKAP79WT-GFP or myr-AKAP79-GFP along with an mCh cell fill were imaged before and 10 min following 30 M NMDA application. G, NMDA treatment reduced AKAP79WT spine-to-shaft ratio but had no effect on myr-AKAP79 (AKAP79WT, 0.81 Ϯ 0.04; myr-AKAP79, 1.02 Ϯ 0.04, p Ͻ 0.001 by unpaired t test). ***, p Ͻ 0.001. H, NMDA application resulted in a signification reduction in mean spine cross-sectional area in AKAP79WT-expressing cells but not in those expressing myr-AKAP79 (fold change relative to pretreatment; AKAP79WT, 0.83 Ϯ 0.03; myr-AKAP79, 1.08 Ϯ 0.09, p Ͻ 0.01 by unpaired t test). *, p Ͻ 0.05. I, there was no significant NMDA-induced change in the spine-to-shaft ratio of the mCh cell fill in either condition. Scale bar, 5 m. NS, not significant.

CaMKII regulates AKAP79/150 function in LTD
not exhibit high autonomy due to Ca 2ϩ /CaM-mediated substrate protection as seen for AKAP79/150. A similar negative regulatory mechanism has been described previously for the CaMKII auto-phosphorylation sites Thr-305/Thr-306, which prevent subsequent stimulation of CaMKII by Ca 2ϩ /CaM (43)(44)(45) and may thereby promote LTD by suppressing phosphorylation of LTP-related regular substrates (7,22,46,47). Overall, these mechanisms together would favor phosphorylation of high autonomy substrates, such as AKAP79/150 and GluA1 Ser-567, by prolonged, weak LTD stimuli, whereas phosphorylation of regular substrates such as GluA1 Ser-831 are favored by brief, strong LTP stimuli (7).
Whereas mimicking AKAP79-targeting domain phosphorylation in the 9D mutant facilitated its LTD-induced removal from spines (Fig. 6, A and B) and reduced its palmitoylation (Fig. 7, E and F), this mutation was not sufficient to disrupt AKAP spine localization. Likewise our previous studies found that mimicking AKAP79 depalmitoylation in the CS mutant also facilitated LTD-induced removal from spines but was not sufficient to disrupt basal spine localization (18). However, the 9DCS mutant that mimics both phosphorylation and depalmitoylation was able to disrupt AKAP79 basal spine targeting (Fig.  6, A and B). Yet the 9A phospho-deficient mutant did not prevent LTD-induced translocation from spines, indicating that although CaMKII phosphorylation of the targeting domain may promote AKAP depalmitoylation and LTD-induced translocation from spines, it is not strictly required. In contrast, through use of the constitutively lipidated myr-AKAP79 mutant we were able to determine that AKAP79 depalmitoylation is indeed required for LTD-induced removal from spines. Importantly, LTD-induced depalmitoylation of endogenous AKAP150 required CaMKII activity (Fig. 7, A, B, D, and E). Overall, these analyses indicate that CaMKII mediates AKAP79/150 removal from spines during LTD in part through direct phosphorylation of the targeting domain but more prominently through promoting its depalmitoylation (Fig. 8).
Protein palmitoylation is controlled by the opposing enzymatic activities of DHHC palmitoyl acyltransferases (PATs), which are integral membrane proteins that catalyze the addition of palmitate to free cysteine residues, and a subset of metabolic serine hydrolases known as protein-palmitoyl thioesterases (PPTs), which are peripheral membrane proteins that reverse this modification (40,48,49). Thus, CaMKII activity could either be promoting depalmitoylation by PPTs or inhibiting palmitoylation by PATs. This regulation by CaMKII may involve direct enhancement of PPT or inhibition of PAT enzymatic activities. AKAP79/150 is known to be palmitoylated by the PAT DHHC2 (19), but the PPTs that control its depalmitoylation have yet to be determined, and the signaling mechanisms regulating the enzymatic activities of DHHC2 (and other PATs) remain uncharacterized. In addition, as suggested by the reduced basal palmitoylation of the 9D phosphomimetic mutant, CaMKII phosphorylation of the AKAP-targeting domain can also indirectly regulate palmitoylation/depalmitoylation by controlling the access of PATs and/or PPTs to the embedded Cys-36/129 substrate sites. However, although the experiments with the 9A and 9D mutants suggest that phosphorylation contributes to facilitating AKAP depalmitoylation and removal from the synapse, they also indicate that phosphorylation is neither necessary nor sufficient for this regulation. Thus, future work exploring how CaMKII might suppress PAT activity and/or promote PPT activity will be highly informative.
Palmitoylation of postsynaptic scaffold proteins, including PSD-95, GRIP1, ␦-catenin, and AKAP79/150, is emerging as a key signaling mechanism regulating excitatory synaptic plasticity, including both LTP/LTD and homeostatic plasticity (18, 19, 50 -54). Synaptic protein palmitoylation is also affected by seizures and anticonvulsants (18,55,56), and several DHHC PAT mutations and substrates have been linked to neurological and neuropsychiatric disorders, including Huntington's, schizophrenia, and X-linked intellectual disability, that are characterized by altered synaptic plasticity (48,57,58). Our findings here reveal novel cross-talk between postsynaptic Ca 2ϩ signaling, palmitoylation, and phosphorylation pathways that are required for synaptic plasticity and thus have implications for understanding these diseases.
Our present findings for CaMKII inhibition, along with our past findings for CaN (12,14) and PLC inhibition (16), show that blocking the signaling pathways that mediate AKAP79/150 synaptic removal also block structural and functional aspects of LTD. Yet such correlations do not show causation, as these treatments, including inhibition of CaMKII, PLC, CaN, or stabilization of F-actin, could also have global effects that prevent structural LTD independently from their effects on AKAP79/ 150 movement. However, the myr-AKAP79 mutant utilized here provides the most direct evidence to date that AKAP79/ 150 removal from spines is indeed required for postsynaptic modifications underlying LTD. AKAP79/150 palmitoylation promotes its inclusion in lipid rafts and controls not only its localization to the plasma membrane but also to recycling endosomes that are important for surface delivery of GluA1-AMPARs that support both LTP and LTD (13,18,19,59). Indeed, LTP stimuli promote AKAP palmitoylation and its recycling endosome-dependent recruitment to spines, and the palmitoylation-deficient AKAP79CS mutant interferes with both this spine recruitment and LTP-associated spine enlargement (18). In contrast, LTD stimuli decrease AKAP palmitoylation and promote its removal from spines along with spine shrinkage; as shown here, these postsynaptic changes are prevented by both the constitutively lipidated myr-AKAP79 mutant and CaMKII inhibition.
Finally, if AKAP removal from spines is a key step in LTD as our present findings indicate, then how is it mediating LTD? Many of AKAP79/150's effects on synaptic function are derived from its bound pools of PKA and CaN (11-13, 60, 61). In particular, phosphorylation/dephosphorylation of GluA1 Ser-845 is critical for AMPAR regulation in LTD (62)(63)(64)(65)(66), and recently, we described roles for AKAP79/150-anchored PKA and CaN in controlling GluA1 Ser-845 phosphorylation and the recruitment to and subsequent removal of GluA1-AMPARs from synapses during LTD (12,13). CaN control of the actin-severing protein cofilin (through the phosphatase Slingshot-1L) is also thought to play a critical role in F-actin remodeling and spine shrinkage during LTD (67, 68). However, both spine F-actin depolymerization and AKAP removal from spines during LTD

CaMKII regulates AKAP79/150 function in LTD
additionally require PLC-dependent PIP 2 hydrolysis, which may both weaken AKAP membrane targeting interactions, as discussed above, and facilitate cofilin activation (16). In addition, although it is known that disruption of CaN-anchoring to AKAP79/150 inhibits its removal from spines and that inhibition of CaN phosphatase activity prevents both LTD-induced F-actin reorganization and AKAP removal, it is not known if AKAP-anchored CaN specifically controls F-actin reorganization during LTD (14,68).
Thus, taken together, the model that emerges is that LTD induction results in activation of multiple signaling pathways involving AKAP-bound CaN, PLC, F-actin depolymerization, autonomous CaMKII, and AKAP depalmitoylation that all converge to disrupt AKAP79/150-targeting domain interactions with F-actin and lipid membranes. In turn, this inhibition of AKAP79/150 targeting interactions promotes its removal from the plasma membrane, as well as endosomes, to prevent re-phosphorylation of postsynaptic CaN substrates such as GluA1 Ser-845 that are important for LTD. Overall, our findings reveal new cross-talk between two dynamic post-translational modifications, phosphorylation and palmitoylation, and two major postsynaptic kinase/phosphatase signaling nodes, CaMKII and AKAP-PKA/CaN, that cooperatively control LTD-associated postsynaptic modifications.
For experiments analyzing fixed cells, induction of cLTD was achieved by 30 M NMDA (Tocris) treatment for 3 min followed by a 10-min recovery (unless indicated otherwise) in conditioned media and subsequent fixation. The same cLTD stimulus was used for assessing biochemical effects on CaMKII phosphorylation; the cLTP stimulation in these experiments was a 10-min incubation in Tyrode's buffer without Mg 2ϩ and with 200 M glycine. In experiments assessing the effects of CaMKII inhibition, neurons were treated with tatCN21 (5 M) or KN93 (10 M) 20 min prior to induction of cLTD. In liveimaging experiments, cLTD was induced by 10 or 30 M NMDA for 10 min as described under the "Results" and in the figure legends.
Neurons were fixed in a 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), 4% sucrose/PBS, permeabilized in 0.1% Triton/PBS at room temperature, and blocked in 10% BSA overnight at 4°C. Fixed neurons were stained with primary antibodies in 5% BSA at room temperature, washed three times in PBS, and incubated in the indicated Alexa fluorophore or Texas Red-conjugated species-specific secondary antibodies. For additional F-actin staining, fixed neurons were stained with 0.165 M Texas Red phalloidin.

Imaging and quantification of live and fixed hippocampal neurons
Images of live and fixed cells were taken on a Zeiss Axiovert 200M system with ϫ63 oil immersion objective, CoolSnap HQ charge-coupled device camera (Roper Scientific), xenon lamp LB-LS/17 (Sutter Instruments), using Slidebook software (Intelligent Imaging Innovations). Additional live imaging in Fig. 7 was conducted at 37°C on a Zeiss Axio Observer microscope with a ϫ63 Plan-Apo/1.4 NA objective, using 488-and 561-nm laser excitation and a CSU-XI spinning disk confocal scan head (Yokogawa) coupled to an Evolve 512 EM-CCD camera (Photometrics) controlled by Slidebook software.
In experiments that assessed the effects of CaMKII inhibition on AKAP150 translocation following NMDA stimulation and those that assessed the synaptic localization of AKAP79 phospho-and palmitoyl-mutants, 15 images were taken at 0.5-m intervals in the z-plane, deconvolved, and maximum-projected. Ten point masks per neuron were drawn on dendritic spines. For each spine mask, a corresponding line mask was also drawn in the dendritic shaft, and the ratio of mean fluorescence of the spine masks to the mean fluorescence of shaft masks was calculated.
For experiments evaluating the re-organization of F-actin, the co-localization of F-actin with PSD-95 (not shown in images) was assessed. Sections of dendrite were masked by

CaMKII regulates AKAP79/150 function in LTD
hand. Dendritic spines within this mask were then defined by a PSD-95 channel threshold hold mask 1.5ϫ mean PSD-95 fluorescence. The sum intensity of the F-actin channel within this dendritic spine mask was divided by the total dendritic F-actin channel intensity to yield the normalized synaptic localization of F-actin.
In experiments determining the sensitivity of myr-AKAP79 mutant to NMDA-induced translocation, 10 images were acquired at 1-m intervals immediately preceding and 10 min following NMDA treatment. Maximum projection images were generated, and 10 -15 spines from secondary or tertiary dendrites were randomly selected for analysis. Regions of interest for spines and dendritic shafts were manually generated. The same spines and shaft regions were created in the posttreatment neurons, and the cross-sectional area of spines and the AKAP79-GFP or mCh spine-to-shaft intensity ratio was calculated before and after treatment. These data are represented as mean fold-change Ϯ S.E.

F-actin co-localization in COS7 cells
COS7 cells were cultured on glass bottom dishes (35 mm with 14 mm glass, MatTek Co, Ashland, MA) and transfected by calcium phosphate method as described previously (73,74) with plasmid cDNA as indicated. Cells were fixed and stained with 0.165 M Texas Red phalloidin (Invitrogen) as done previously (27). Images were taken on a Zeiss Axiovert 200M system with ϫ63 oil immersion objective, CoolSnap HQ chargecoupled device camera (Roper Scientific, Trenton NJ), xenon lamp LB-LS/17 (Sutter Instrument, Novato, CA), using Slidebook software (Intelligent Imaging Innovations, Fort Collins, CO). 15 images were taken 0.5 m apart in the z-plane per cell, deconvolved, and maximum-projected. A threshold mask (mean fluorescence plus 1 S.D.) in the CY3 channel was drawn to label F-actin. The sum fluorescence intensity of YFP within the threshold mask was determined and divided by the sum intensity of YFP within the whole cell to determine the percentage of YFP-AKAP co-localized with F-actin.

CaMKII activity assays in vitro
CaMKII autonomy was generated by pre-phosphorylating the kinase at Thr-286 by incubating 200 nM CaMKII␣ subunits in 50 mM PIPES, pH 7.0 -7.2, 1 nM BSA, 10 mM MgCl 2 , 100 M ATP, 1 mM CaCl 2 , and 2 M CaM and incubating on ice for 10 min (7,21,75,76). The reaction was stopped by the addition of PIPES dilution buffer containing 5 mM EGTA. The CaMKII phosphorylation of AKAP79 region B peptides (hAKb) was then induced using a standard CaMKII assay; 2.5 nM CaMKII subunits were reacted with 7.5 M hAKb at 30°C for 1 min in buffer containing 50 mM PIPES, pH 7.2, 0.1 mg/ml BSA, 10 mM MgCl 2 , 100 M [␥-32 P]ATP (1 Ci/mmol). Reactions were spotted onto Whatman P81 phosphocellulose paper rectangles (2 ϫ 2.5 cm). Reactions were stopped by placing the paper rectangles into 300 ml of 0.5% phosphoric acid, and then rinsed four times with water, washed for 30 min, and rinsed four more times. Radioactivity bound to peptides was quantified in a Beckman 6000TA scintillation counter by the Cherenkov method. For Fig. 5B, purified recombinant C-terminal His 6 -tagged AKAP79(1-153) was phosphorylated by autonomous (ϩEGTA) or Ca 2ϩ /CaMstimulated (ϪEGTA) CaMKII in the presence [␥-32 P]ATP as described above for 30 min at 30°C followed by separation by SDS-PAGE and visualization by autoradiography. For Fig. 2C, the reaction additionally contained purified MAP2 and was carried out for 5 min. Please note that in Fig. 5B, the AKAP79(1-153) WT, 5A, and 9A proteins were expressed and purified as C-terminal His 6 -tagged proteins in Escherichia coli using a pET30 vector that also adds extra linker sequences that together contain three additional Ser/Thr residues. Moreover, there are 11 additional Ser/Thr residues in the AKAP79(1-153) fragment located outside of the A, B, and C polybasic membrane targeting sub-domains. Although none of these additional AKAP79 or vector-encoded Ser/Thr residues are predicted to be optimal consensus phosphorylation sites for CaMKII, they could account for the residual level of CaMKII phosphorylation observed in the 9A mutant that is not sensitive to Ca 2ϩ -CaM protection.

CaM-binding assays in vitro
CaM binding was determined by two different methods. Blot overlay with biotinylated CaM (STI Signal Transduction Products) was performed as described previously (22), with bound CaM detected by the Vectastain ABC kit (Vectastain) followed by Western Lightning (PerkinElmer Life Sciences), and chemiluminescence visualized in a ChemiImager (Alpha Innotech). For fluorescence-based detection of CaM binding, CaM was IAEDANS-labeled at a K75C mutation as described (37); the increase in CaM-I fluorescence after binding to Ca 2ϩ and to peptide was measured in a Fluorolog2 spectrofluorometer (HORIBA Jobin Yvon) at excitation/emission wavelengths of 345/465 nm with a 16-nm bandwidth, in a stirred cuvette containing 50 mM Hepes, pH 7.4, 100 mM KCl, 10 mM MgCl 2 , 0.1 mg/ml BSA and CaM-I (1 M or as indicated). Ca 2ϩ , peptide, or EGTA was added at the times indicated, with fluorescence monitored continuously.

CaMKII regulates AKAP79/150 function in LTD
CaCl 2 , 1.25 NaH 2 PO 4 , 1 MgSO 4 , 26 NaHCO 3 , 10 glucose) and then lysed in PBS buffer containing 4% SDS and 5 mM EDTA. After a 10-min spin at 1000 ϫ g, the supernatant was tumbled with 20 mM tris(2-carboxyethyl)phosphine for 1 h at room temperature. Free thiols were then blocked by incubation with 50 mM N-ethylmaleimide (NEM) overnight at room temperature. Following a chloroform/methanol precipitation (CMP), pellets were resuspended in 4% SDS PBS buffer and incubated with 1 M hydroxylamine (HAM, Sigma) for 1 h at room temperature with end-overend rotation. Following another CMP, free thiols were labeled with 5-kDa polyethylene glycol moieties (SUNBRIGHT maleimide PEG, NOF America) for 1 h at room temperature with rotation. After a final CMP, samples were resuspended and boiled in sample buffer with 50 mM dithiothreitol and resolved via SDS-PAGE and Western blotting with GFP antibody. For ABE, neuronal cultures were rinsed with artificial CSF and then lysed in buffer containing (in mM) 150 NaCl, 50 Tris, pH 7.4, 5 EDTA, 10 NEM, and 1.7% Triton X-100. Following a 1-h rotation at 4°C, a CMP was performed, and the dried pellet was resuspended in 4% SDS buffer plus 50 mM Tris, pH 7.4, and 5 mM EDTA. Samples were then diluted in lysis buffer plus 50 mM NEM and tumbled overnight at 4°C to block free thiols. Following CMP, each protein sample was resuspended in SDS buffer, halved, and diluted in labeling buffer (1 mM HPDP-biotin, EZ-link, Thermo Fisher Scientific) with or without 1 M HAM (Sigma) and tumbled for 1 h at room temperature to cleave palmitoylation thioester linkages and to biotinylate the resultant free cysteines. Following another CMP and resuspension, biotinylated proteins were affinity-purified by tumbling with streptavidinagarose beads (Thermo Fisher Scientific) overnight at 4°C. Proteins were eluted with dithiothreitol and resolved via SDS-PAGE and Western blotting with AKAP150 or CaMKII␣ antibodies. For BMCC, neuronal cultures were rinsed with aCSF and then lysed in buffer containing (in mM) 150 NaCl, 50 Tris, pH 7.4, 10% glycerol, 1% IGEPAL CA-630 (Sigma, Nonidet P-40 analogue), and 50 NEM. AKAP150 was immunoprecipitated with AKAP150 antibody overnight at 4°C followed by incubation with agarose beads (Thermo Fisher Scientific) for 1 h. Samples were divided into HAM-negative and -positive conditions and tumbled with or without 1 M HAM for 1 h at room temperature. Samples were then tumbled at 4°C for 1 h in pH 6.2 buffer plus 10 M biotin-BMCC (Thermo Fisher Scientific). Finally, beads were washed two times with pH 7.4 lysis buffer and boiled in sample buffer for 10 min at 80°C. Samples were resolved by SDS-PAGE and Western blotting with streptavidin-HRP and AKAP150 antibody.

Experimental design and statistical analyses
Hippocampal neuron culture experiments were conducted using a minimum of three distinct culture preparations. The cultures used cells from both male and female animals. Tests for statistical significance were conducted using Prism (Graphpad). Independent sample means were compared using t-tests or one-way ANOVA as indicated in the figure legends. F values that were statistically significant were further probed by post hoc tests comparing experimental conditions to control (Dunnett's test) or among all conditions (Newman-Keuls) as indicated in the figure legends. Statistical non-significance (ns) for each condition is also reported in figure legends. Type-1 error rates for all tests were 0.05, but p values lower than 0.05 are indicated by **, p Ͻ 0.01, or ***, p Ͻ 0.001.