Phosphorylation of a -dystrobrevin is essential for a kap accumulation and acetylcholine receptor stability

The maintenance of a high density of the acetylcholine receptor (AChR) is the hallmark of the neuromuscular junction. Muscle-specific anchoring protein ( a kap) encoded within the calcium/calmodulin-dependent protein kinase II a (CAMK2A) gene is essential for the maintenance of AChR clusters both in vivo and in cultured muscle cells. The underlying mechanism by which a kap is maintained and regulated remains unknown. Here, using human cell lines, fluorescence microscopy, and pulldown and immunoblotting assays, we show that a -dystro-brevin ( a -dbn), an intracellular component of the dystrophin glycoprotein complex, directly and robustly promotes the stability of a kap in a concentration-dependent manner. Mechanis-tically, we found that the phosphorylatable tyrosine residues of a -dbn are essential for the stability of a -dbn itself and its interaction with a kap, with substitution of three tyrosine residues in the a -dbn C terminus with phenylalanine compromising the a kap – a -dbn interaction and significantly reducing both a kap and a -dbn accumulation. Moreover, the a kap – a -dbn interaction was critical for a kap accumulation and stability. We also found that the absence of either a kap or a -dbn markedly reduces AChR a accumulation and that overexpression

The maintenance of a high density of the acetylcholine receptor (AChR) is the hallmark of the neuromuscular junction. Muscle-specific anchoring protein (akap) encoded within the calcium/calmodulin-dependent protein kinase IIa (CAMK2A) gene is essential for the maintenance of AChR clusters both in vivo and in cultured muscle cells. The underlying mechanism by which akap is maintained and regulated remains unknown.
Here, using human cell lines, fluorescence microscopy, and pulldown and immunoblotting assays, we show that a-dystrobrevin (a-dbn), an intracellular component of the dystrophin glycoprotein complex, directly and robustly promotes the stability of akap in a concentration-dependent manner. Mechanistically, we found that the phosphorylatable tyrosine residues of a-dbn are essential for the stability of a-dbn itself and its interaction with akap, with substitution of three tyrosine residues in the a-dbn C terminus with phenylalanine compromising the akap-a-dbn interaction and significantly reducing both akap and a-dbn accumulation. Moreover, the akap-a-dbn interaction was critical for akap accumulation and stability. We also found that the absence of either akap or a-dbn markedly reduces AChRa accumulation and that overexpression of a-dbn or akap in cultured muscle cells promotes the formation of large agrin-induced AChR clusters. Collectively, these results indicate that the stability of akap and a-dbn complex plays an important role in the maintenance of high-level expression of AChRs.
The maintenance of AChR clusters at high density in the postsynaptic membrane is a hallmark of the neuromuscular junction (NMJ) (1). Core proteins such as MuSK (muscle-specific tyrosine kinase), LRP4 (low-density lipoprotein receptorrelated protein 4), rapsyn, and Dok7 (dedicator of cytokinesis family member 7) (2-7) are required for initial steps in the formation of AChR clusters, whereas auxiliary proteins such as the dystrophin glycoprotein complex (DGC), neuregulin signaling molecules, and Wnt proteins are essential for the maturation and stability of AChR clusters in the postsynaptic apparatus (8)(9)(10)(11)(12)(13).
Recent work has shown that akap, a non-kinase muscle-specific anchoring protein encoded within the Camk2a gene that contains a putative transmembrane domain and an association domain but lacks the catalytic domain (14)(15)(16), is also involved in promoting the stability of AChRs (16)(17)(18). Knockdown of akap gene expression with shRNA in cultured myotubes or in mouse sternomastoid muscles resulted in a significant reduction of the postsynaptic receptor density, an alteration in the metabolic stability of AChR, and an impairment of the structural integrity of the synapse (17,18). Earlier work also reported that akap plays a role in targeting multiple calcium/calmodulin kinase II isoforms to specific subcellular locations where they can perform their function through its unique hydrophobic Nterminal domain that tightly associates with membranes (16). However, the mechanistic link between akap and AChR stability remains unknown.
On the other hand, a-dystrobrevin, a component of the DGC that links the intracellular cytoskeleton to the extracellular basal lamina (19), is also essential for the maintenance of the muscle integrity and the maturation and stability of the NMJ (20,21). NMJs of mice deficient in a-dystrobrevin (a-dbn) bear few receptors and exhibit an abnormal pattern of AChR distribution and a higher turnover rate of AChR. In muscle cells, at least two predominant isoforms of a-dbn (a-dbn 1 and 2) are generated by alterative splicing of the a-dbn gene (22). Full-length a-dbn 1 has a unique C-terminal tyrosine kinase substrate domain and is mainly concentrated at the NMJ, whereas a-dbn 2, which lacks a C terminus, is mainly localized on the sarcolemmal membrane but also present at the synapse (23). It was reported that the phosphorylatable tyrosine residues of a-dystrobrevin are essential for the stability and normal distribution pattern of postsynaptic AChRs (8,24,25). a-dbn1, a substrate for tyrosine kinases in vivo phosphorylated by neuregulin/ErbB receptor signaling pathway in muscles (8,26,27), plays a critical role in the maintenance and the stability of the NMJ, whereas the nonphosphorylated a-dbn 2 is involved in the maintenance of the structural integrity of the muscle (25). Because both akap and a-dbn are involved in the stability of AChRs, we sought to investigate the interplay between these proteins and their effect on the stability of AChR.
In the present study, we have used muscle cells and heterologous systems to investigate the interplay between a-dbn, akap, and AChRs accumulation. We found that akap expression levels, which are critical for promoting accumulation of AChRs, are regulated by a-dbn in a dose-dependent manner. Furthermore, we discovered that the ability of the three tyrosine residues of a-dbn1 to be phosphorylated are critical for the stability of a-dbn and its interaction with akap. This interaction is essential for akap accumulation and AChR stability.

Results akap forms complexes with a-dystrobrevin and AChRs
Previous studies have shown that akap forms complexes with AChRs and promotes its stability (17,18). Because a-dbn is also essential for the stability of AChRs (24), we sought to investigate whether there was a molecular link between akap, a-dbn, and AChRs. As a first test, we examined whether these proteins are present within the same complex and whether the stability of AChRs requires the presence of both akap and a-dbn. Lysates from cultured C2C12 myotubes were incubated with BTX-biotin to label AChRs, after which AChR-BTX-biotin complexes were isolated with NeutrAvidin-coated beads. The eluates were subjected to one blotting using anti-AChRa, anti-akap, and anti-adbn antibodies. Fig. 1A shows that akap and adbn were pulled down with AChRs. This indicates that AChRs are present in the same complex as akap and a-dbn adbn. It should be noted that neither a-dbn nor akap have AChR binding sites, suggesting that the pulldown by Neutr Avidin beads is indirect.
Similar results were observed when akap and AChRa constructs were co-transfected in HEK cells (express endogenous a-dbn) and subjected to HA immunoprecipitation (Fig. 1B). Altogether, these results indicate that AChRs, akap, and a-dbn are present within the same complex.
Because akap and a-dbn are present within the same complex, we asked whether the promoting effect of akap on the accumulation of AChRs requires the presence of a-dbn. To investigate this, we used HeLa cells (cells that do not express a-dbn, AChR, or akap; Fig. 2A) that were co-transfected with constructs of AChRa (1 mg) and HA (0.4 mg), with constructs of AChRa (1 mg/ml) and akap-GFP (0.4 mg/ml), with AChRa (1 mg) and a-dbn-GFP (0.4 mg), or with AChRa (1 mg) and both akap-GFP and a-dbn-GFP at 0.4 mg, respectively. Twentyfour hours later, lysates from co-transfected cells were immunoblotted with anti-AChRa and anti-GFP antibodies (to probe for akap-GFP and a-dbn-GFP). Quantification of the intensity bands on Western blots shows that the amount of AChRa expression levels is not significantly different between cells transfected with (1.12, n = 4, p = 0.91) and without akap-GFP (set at 100%). In the presence of a-dbn-GFP alone, AChRa lev-els were slightly increased (153%, n = 4, p , 0.186) compared with cells transfected with (112%) or without akap-GFP (100%). However, in the presence of both akap-GFP and a-dbn-GFP (0.4 mg of plasmids), AChRa levels were significantly increased (240%, n = 4) compared with cells transfected with akap-GFP alone (112%, n = 4) or a-dbn-GFP alone (153%, n = 4) (Fig. 2, B and C). These results were further confirmed in HEK cells (cells that express endogenous a-dbn, but not akap and AChRa; Fig. 2A) that were co-transfected with an increasing concentration of akap construct (0, 0.2, 0.4, or 0.8 mg) and a fixed concentration of AChRa plasmid (1 mg) (Fig.  2D). Collectively, these results suggest that high AChRs expression levels require the presence of both akap and a-dbn.
a-Dystrobrevin promotes the expression levels of akap in a concentration-dependent manner Given the fact that akap, a-dbn, and AChRa are present within the same complex and that the promoting effect of akap on AChRs necessitates the presence of a-dbn, we asked whether expression levels of akap are regulated by a-dbn. In the first set of experiments, we examined the effect of a-dbn on akap expression levels. For this, we used three independent cell lines: nonmuscle HeLa cells (no endogenous expression of both a-dbn and akap), HEK cells (endogenous expression of a-dbn only), and muscle C2C12 myoblasts (endogenous expression of both a-dbn1 and akap). All of these cells were co-transfected with akap-GFP and a-dbn-GFP. Twenty-four hours later, cell lysates were immunoblotted with anti-akap and anti-a-dbn antibodies to probe for the endogenous proteins and anti-GFP antibody to probe for the exogenous expression of akap-GFP and a-dbn-GFP. We found that akap levels were significantly higher in all three cell lines where a-dbn is exogenously and/or endogenously expressed compared with cells transfected with akap alone (Fig. 3, A-G). In C2C12 muscle cells, the overexpression of a-dbn-GFP also enhanced expression levels of endogenous akap (Fig. 3, F and G). We also asked whether a-dbn and akap are present in the same intracellular organelles. To examine this, HeLa cells were co-transfected with a-dbn-GFP (green) and akap-mCherry (red) constructs, and 24 h later, the cells were fixed and imaged with the confocal microscope. As shown in Fig. 3C, we found that ;50%, green and red punctas co-localized in the same vesicles (n = 250 vesicles analyzed, number of cells = 25). Interestingly, it appears that the size of the intracellular vesicles in which both green and red co-localized are larger than vesicles in cells transfected by akap-mCherry only (Fig. 3C).
We next asked whether the expression level of akap depends on a-dystrobrevin expression levels. To examine this, HeLa cells (neither akap nor a-dbn are expressed) were co-transfected with the same amount (1 mg) of akap-GFP construct and increasing a-dbn-GFP construct doses (0, 0.2, and 0.4 mg). The HA control plasmid was co-transfected with akap and a-dbn plasmids, so that all transfections contained the same amount of plasmid DNA. We found that in HeLa cells co-transfected with 0.2 mg of a-dbn-GFP and 1 mg of akap plasmid, the expression level of akap was increased ;2-fold compared with akap alone (in absence of a-dbn-GFP). In cells co-transfected Figure 1. akap forms complexes with a-dbn and AChR in muscle cells and HEK cells. A, lysates from C2C12 myotubes were incubated BTX-biotin, and then biotin-containing complexes were isolated with NeutrAvidin beads. Pulldown proteins (PD) were probed with anti-akap, anti-AChRa, and anti-adbn antibodies. B, lysates from HEK293T cells co-transfected with AChRa and akap-HA (hemagglutinin) were immunoprecipitated (IP) with anti-HA followed by protein A/G-agarose beads and blotted (IB) with anti-akap, anti-AChRa, and anti-a-dbn anti-antibodies. Western blotting analysis of cell lysate inputs is shown (left panels).
with 0.4 mg of a-dbn-GFP plasmid, the expression levels of akap were increased 1.5-fold compared with 0.2 mg of a-dbn-GFP and ;3-fold compared with akap alone (Fig. 4, A and B). In C2C12 muscle cells (in which both akap and a-dbn are endogenously expressed) that were co-transfected with 1 mg of akap-GFP and a-dbn-GFP constructs (0 and 0.2 mg), we found that both exogenous and endogenous akap expression levels were also increased ( Fig. 4, C and D). These results strongly suggest that a-dbn is involved in promoting the expression of akap in a concentration-dependent manner. Conversely, when HeLa cells were co-transfected with the same amount (1 mg) of a-dbn and increasing akap constructs doses (0, 0.2, and 0.4 mg), the expression levels of a-dbn remained unchanged (Fig. 4, E and F), indicating that expression of a-dbn is not controlled by akap. Altogether, these results indicate that akap expression depends on a-dbn expression, whereas a-dbn expression is independent of akap.
Effect of phosphorylatable tyrosine residues of a-dystrobrevin on the expression of a-dystrobrevin and akap We wanted to know the mechanism by which a-dbn controls akap accumulation. First, we confirmed that the fused a-dbn1-GFP protein underwent tyrosine phosphorylation. HEK cells were transfected with either WT a-dbn1-GFP or the nonphosphorylatable a-dbn-GFP mutant (the three tyrosine residues were changed to phenylalanine; 3Y/F) and treated with peroxivanadate before lysis to inhibit tyrosine phosphatases. Lysates were then incubated with anti-GFP antibodies to immunoprecipitate a-dbn, and eluates were subjected to Figure 2. AChRa expression is enhanced in presence of a-dystrobrevin and akap. A, Western blots of akap, a-dbn, and AChRa expression in C2C12 myoblasts, myotubes, HEK cells, and HeLa cells. Note that HeLa cells do not express these proteins, whereas HEK cells express only a-dbn1. C2C12 myotubes express all these proteins (AChR, a-dbn (1 and 2), and akap), whereas myoblasts express only a-dbn and akap. Tubulin was used as a loading control. B, HeLa cells were transfected with the same amount of AChRa (1 mg) construct and either individual akap-GFP (0.4 mg), a-dbn-GFP (0.4 mg), or both (0.4 mg of each) constructs. mCherry was used as a control for the transfection efficiency and for the loading of total proteins. C, scatter plot summarizing quantification from four independent blots (data are presented as means 6 S.D.). Statistical analysis was done by one-way ANOVA (F = 22.30) followed by Tukey's post hoc multiple comparisons test. ****, P , 0.0001; ***, P , 0.001; **, P , 0.01; *, P , 0.05. n.s, not significant. D, HEK cells were transfected with the same amount of AChRa (1 mg) and with increasing amounts of akap-GFP (0, 0.2, 0.4, and 0.8 mg). Enhanced GFP (eGFP) was used as a control for the transfection efficiency and for the loading of total proteins with tubulin. Note that in the presence of adbn, the expression levels of AChRa subunit depend on akap concentration.
Western blotting with anti-a-dbn antibody, anti-phosphotyrosine antibody, and anti-GFP antibodies. As shown in Fig. 5A, the a-dbn1-GFP WT showed the presence of tyrosine phosphorylation sites but not the mutant one.
We next examined whether the phosphorylatability of the three tyrosine residues of a-dbn is essential for a-dbn stability. HeLa cells were transfected with either WT GFP-a-dbn or mu-tant GFP-a-dbn (3Y/F) and then harvested 18 and 24 h later. Western blotting analysis of proteins with anti-GFP or anti-adbn antibodies showed that the expression levels of a-dbn mutant (a-dbn-3Y/F) is significantly reduced compared with WT ( Fig. 5B). To determine whether the reduction in the amount of a-dbn1 mutant was due to a decrease in its transcript, we analyzed RT-PCR products specific to a-dbn of both WT and mutant. Fig. 5C showed that there was no significant difference in the amount of a-dbn mRNA products between cells transfected with the phosphorylatable and nonphosphorylatable a-dbn constructs, suggesting that post-translational modifications of a-dbn by the phosphorylation of the three tyrosine residues play an important role in its accumulation/stability. Because a significant amount of a-dbn mutant proteins are degraded, we wanted to know whether the degradation of a-dbn mutant is mediated by the proteasome. The cells were transfected with the phosphorylatable tyrosine WT or nonphosphorylatable the mutant a-dbn and treated for 6 h with MG132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal), a widely used proteasome inhibitor that can prevent degradation of many ER-associated degradation substrates (28). Lysates from transfected cells were subjected to Western blots using anti-GFP antibodies, and a-dbn expression levels of WT and mutant were analyzed. As shown in Fig. 5 (D and E), we found that in the presence of MG132, the expression levels of the mutant were significantly increased (;93.1% of WT, S.D. 0.09 n = 4, p = 0.163), comparable with the phosphorylatable WT a-dbn, which suggests that the degradation of mutant a-dbn is mediated by the proteasome. Fig. 4 showed that a-dbn promotes akap accumulation in a concentration-dependent manner. Thus, we wanted to know the mechanism by which a-dbn regulates akap expression lev-els. We hypothesized that the three tyrosine phosphorylatable sites of a-dbn are essential for the interaction with akap and that interaction is critical for akap accumulation/stability. To test this, HeLa cells were co-transfected with akap-HA construct and either the 1) phosphorylatable tyrosine residues a-dbn1-GFP (WT) or 2) a-dbn-GFP mutant and then treated with MG132, so the same amount of expression levels of WT and mutant a-dbn1 would be used for immunoprecipitation. Lysates were incubated with anti-GFP antibody to immunoprecipitate a-dbn-GFP, and eluates were subjected to Western blotting analysis with anti-akap, anti-a-dbn, and anti-HA antibodies. Fig. 6A shows that akap was pulled down successively and specifically enriched in lysates transfected with the phosphorylatable a-dbn1. However, in lysates from cells co-transfected with nonphosphorylated a-dbn-GFP mutant, akap was barely visible (or not detectable), indicating that the interaction between akap and nonphosphorylated a-dbn was impaired. These results suggest that the phosphorylatability of a-dbn1 is essential for the interaction with akap.
We next wanted to know whether the interaction between akap and phosphorylatable a-dbn1 leads to akap accumulation. Lysates from HeLa cells co-transfected with a fixed amount of akap and WT a-dbn-GFP or a-dbn-3Y/F-GFP mutant constructs were subjected to Western blotting analysis with anti-akap and anti-a-dbn antibodies. As shown in Fig. 6  (B and C), the amount of akap was significantly higher in cells co-transfected with the phosphorylatable a-dbn1 isoform compared with cells co-transfected with nonphosphorylated a-dbn mutant. These results were further confirmed in cells that were co-transfected with a fixed amount of akap, and an increasing amount of WT and mutant a-dbn. Fig. 6 (D and E) showed that akap expression levels increased with WT a-dbn concentration as expected, whereas an increase in mutant a-dbn has no significant effect on akap accumulation. These results indicate that the phosphorylation of the three tyrosine residues of a-dbn is essential for the interaction and the stability of akap in HeLa cells.
akap and a-dystrobrevin promote the formation of agrininduced AChR clusters Because a-dbn promotes akap accumulation, which in turn enhances AChR stability, we asked whether overexpressing a-dbn and akap in muscle cells would increase the size of AChR clusters. C2C12 muscle cells were transfected with either akap-mCherry (red) or a-dbn-GFP (green) or co-transfected with both akap-mCherry and a-dbn-GFP, and differentiated myotubes were treated with agrin overnight, fixed, and bathed with fluorescently tagged BTX (Alexa 647, Alexa 488, or Alexa 594) to label only surface AChRs. Because transfection efficiency was only ;50%, we were able to use the same culture dish and quantify the area and length of AChR clusters on both transfected and nontransfected myotubes. Fig. 7 shows that both the area and length of agrin-induced AChR clusters were significantly increased on muscle transfected with either akap (area, 35.1 6 2.8 mm 2 ; length, 10.4 6 0.6 mm), or a-dbn1 (area, 31.9 6 1.9 mm 2 ; length, 9.9 6 0.4 mm) compared with nontransfected cells with akap (area, 19.4 6 2.8 mm 2 ; length, 7.5 6 0.4 mm) or a-dbn clusters (area, 17.5 6 1.7 mm 2 ; length, 6.5 6 0.4 mm). However, the averages of area (41.2 6 3.9 mm 2 ) and length (11.2 6 0.7 mm) of AChR clusters in muscles overexpressing both a-dbn and akap were not significantly different from myotubes expressing either a-dbn or akap ( Fig. 7; see "Discussion"). However, because most of the myotubes do not form clusters in the absence of agrin (at least in our hand), the effect Figure 5. Phosphorylatable tyrosine residues of a-dystrobrevin are critical for a-dystrobrevin accumulation levels. A, immunoblot (IB) of HEK cells transfected with WT a-dbn1-GFP, mutant a-dbn (3Y/F)-GFP, or GFP constructs (1 mg). 18 h later and 15 min prior to lysis, the cells were treated with peroxivanadate to inhibit tyrosine phosphatases. Lysates were then incubated with GFP antibody, and immunoprecipitate (IP) proteins were probed with anti-GFP, anti-adbn or an anti-phosphotyrosine antibody. B, representative Western blots of a-dbn1-GFP and Ponceau stain of protein extracts from transfected HeLa cells. Lanes 1, control plasmid; lanes 2, a-dbn-WT-GFP; lanes 3, a-dbn-3Y/F-GFP. Note that expression levels of a-dbn-WT-GFP were significantly increased compared with nonphosphorylated mutant a-dbn-3Y/F-GFP. C, graph shows that real-time RT-PCR of WT and mutant a-dbn1 mRNA expression levels are similar. D, HeLa cells transfected with phosphorylated WT and nonphosphorylated mutant a-dbn were treated with the proteasome inhibitor MG132. Lysates were immunoblotted with anti-GFP antibody. mCherry was used as a control for the transfection efficiency and as a loading control with Ponceau S. E, quantification of four independent experiments as in D (data are presented as means 6 S.D.). The a-dbn signals were normalized to Ponceau S signals. Note that in the presence of the proteasome inhibitor MG132, the expression level of nonphosphorylated a-dbn mutant was significantly increased (93.1%, comparable with WT (100%)). In the absence of MG132 treatment, however, the expression level of a-dbn mutant was significantly low (37.3% of WT (100%)). Statistical analysis was done by two-way ANOVA (interaction P value = 0.0120; F = 8.747) followed by Tukey's post hoc multiple comparisons test. ****, P , 0.0001; ***, P , 0.001; **, P , 0.01; *, P , 0.05. n.s, not significant. of a-dbn or akap overexpression on AChR clusters size and length was not assessed.

Discussion
This work demonstrates that a-dystrobrevin plays an important role in maintaining high expression of akap and subsequently AChRs' accumulation. The major findings are: 1) a-dbn, akap, and AChRa are present within the same complex; 2) a-dbn promotes the expression levels of akap in a concentration-dependent manner, whereas akap has no regulatory effect on a-dbn; 3) the promoting effect of akap on AChRa accumulation requires the presence of a-dbn; 4) the tyrosine phosphorylation of a-dbn is critical for its stability and its interaction with akap; 5) a stable interaction of phosphorylated a-dbn isoform with akap is essential for akap accumulation; Regulation of akap by a-dystrobrevin and 6) high expression of a-dbn and akap significantly enhances the formation of large agrin-induced AChR clusters. Altogether, these results demonstrate that a-dystrobrevin is essential not only for akap expression levels but also for the formation of large AChR clusters.
The current experiments show that akap is present in the same complex with a-dbn and AChRs. However, at AChR clusters, in which a-dbn is concentrated, akap is not present with AChR/a-dbn. This suggests the possibility that the interaction between akap and a-dbn may occur in a vesicular complex during their intracellular trafficking, specifically in the ER where akap has been found to be highly expressed in skeletal and cardiac muscles (18,29,30). Interestingly, in the absence of either akap or a-dbn, AChR accumulation was reduced (Fig.  2). For instance, in cells that do not express endogenous a-dbn, such as HeLa cells, when akap and AChRa were overexpressed, akap has little to no effect on AChR accumulation/stability. Similarly, in HEK cells that express endogenous a-dbn but not akap, when AChRa was overexpressed, a-dbn has little effect on AChR accumulation. One caveat of this study is that the cotransfection efficiency of multiple plasmids (four AChR subunits (AChRa, b, g, and d), akap, a-dbn, and empty mCherry constructs) into HeLa cells was difficult to obtain, which hinders the analysis of the effect of akap and a-dbn on the assembly and delivery of the pentameric AChR channel. Of note, in heterologous cells transfected with all receptor subunits, the assembly of AChR subunits into a pentameric complex occurs in the endoplasmic reticulum (35,37), which is essential for the stability and delivery of functional AChR subunits to the cell surface (36,38). However, the absence of one or more AChR subunits blocks the transport of other subunits to the cell surface, and the unassembled AChR subunits are presumably targeted to degradation (37). In the ER, AChRa undergoes a conformational change in protein structure that enables it to bind bungarotoxin before assembly with other subunits and that toxin-binding activity increased when all four subunits together were expressed (37). In the present study, we showed that nAChRs associate with akap and adbn, perhaps directly or indirectly as part of a complex and that the presence of akap-a dbn complex substantially increased the amount of unassembled AChRa subunit. Although we have not examined where akap-adbn are acting/interacting and stabilizing the AChRa subunit, we think it is likely that increased accumulation of AChRa occurs in the ER/Golgi compartments, because the assembly of AChR and localization of akap occurs there (18,29,35,37). However, it remains unknown whether the stabilizing effect of the akap-adbn complex on nAChRa expression has any impact on the regulation/stability and/or the delivery of the fully assembled pentameric receptor to the cell surface. It is worth mentioning that in our previous studies, we have shown that akap is present with AChRs in the same complex in cultured myotubes and muscle tissues of mice and that the knockdown of akap endogenous expression in either cultured muscle cells or in the sternomastoid muscle of living mice with shRNA caused a significant reduction in the number of AChR clusters on cultured myotubes and the postsynaptic receptor density at the neuromuscular junction, in addition to its structural changes (17,18). On the other hand, NMJs of mice deficient in a-dystrobrevin (a component of the DGC involved in the anchoring of the AChRs in the synaptic membrane) are structurally aberrant and have a reduced level of AChRs and a lower stability of receptors in the postsynaptic membrane (20,24). Because the abnormal NMJ phenotype of mice deficient in a-dystrobrevin resembles, in some aspects, that in muscles electroporated with shRNA akap and that akap and a-dbn are within the same complex, it is possible that akap and a-dbn interaction is essential for promoting AChR stability and the maintenance of the NMJ.
Interestingly, previous studies have shown that akap in cultured muscle cells or heterologous system promotes the accumulation of AChRs in a ubiquitin-dependent proteasome mechanism. However, akap does not share any similarities with known deubiquitinase, suggesting that akap through an association with deubiquitinases may control the stability of AChRs. Interestingly, it has been reported that a-dbn also forms complex with the deubiquitinases Usp9x (31), and because the complex a-dbn and akap is indispensable for AChR accumulation, it is conceivable that a-dbn1, through its association with Usp9x, may protect AChRs from degradation.
Several studies have reported that phosphorylation may serve to recruit adaptor/signaling proteins. Along these lines, in the absence of the phosphorylatable tyrosine residues, a-dbn1 expression levels were significantly reduced (Fig. 5). In cells transfected with WT phosphorylatable a-dbn1 and mutant nonphosphorylated a-dbn, expression of mutant isoform was significantly reduced compared with WT, suggesting that phosphorylatable tyrosine residues in the C terminus is essential for the stability of a-dbn1. One plausible possibility is that the three site of tyrosine phosphorylation may affect the folding of a-dbn1, leading to its instability/degradation. Indeed, when cells were treated with a proteasome inhibitor (MG132), there was no significant difference between phosphorylatable WT and nonphosphorylated mutant a-dbn. This suggests that ubiquitination and degradation of a-dbn1 by the ER-associated degradation pathway are likely accelerated in the absence of the phosphorylatable sites in the C terminus of a-dbn1 (32). It is also possible that the interaction of phosphorylated a-dbn with partners may enhance its stability. A recent study identified several WT a-dbn-binding partners that associate with a-dbn through phosphorylation-dependent mechanisms (32). This include scaffold proteins Grb2 and SH3BP2, Rho guanine nucleotide exchange factor Arhgef5, phosphoinositide 3-kinase, and the nonreceptor tyrosine-protein kinase Fyn. Thus, it is possible that when tyrosine phosphorylation residues are mutated, the interaction of a-dbn with its partners is reduced, leading to its instability.
One unexpected result from this work is that a-dbn promotes akap accumulation in both heterologous nonmuscle cells and cultured muscle cells in a concentration-dependent manner, whereas akap has no effect on a-dbn accumulation (Figs. 3 and 4). This raises the question of how might a-dbn promote akap accumulation. The present data show that the phosphorylatable tyrosine residues of a-dystrobrevin are essential for interacting with akap. In contrast to WT phosphorylatable a-dbn, which forms a complex with akap, there was little pulldown of akap when akap was expressed with the nonphosphorylated mutant a-dbn (3Y/F) in HeLa cells. It is possible that phosphorylation of a-dbn could lead to its conformational changes, leading to enhancing strength of the interaction with akap. Such strong interactions may enhance the accumulation and stability of akap. However, when a-dbn is not phosphorylated, the interaction with akap is weakened, which leads to its destabilization.
The tyrosine phosphorylation of a-dbn1 has been shown to play an important role in the maintenance of the structural integrity of the synapse (25). Similarly, the knockdown of akap both in culture muscle cells and in vivo (17,18) dramatically reduced AChR expression levels and altered the structural integrity of the postsynaptic apparatus of the NMJs. The current experiments show that the overexpression of either akap or a-dbn in C2C12 muscle cells significantly increased the number and size of AChR clusters (Fig. 7). However, when both Regulation of akap by a-dystrobrevin akap and a-dbn are co-expressed in cultured muscle cells, the size of AChR clusters is not significantly different from AChR clusters on muscle cells overexpressing either akap or a-dbn (Fig. 7). One possible explanation is that when a-dbn is overexpressed, the endogenous expression of akap is also significantly increased (Fig. 3). However, it must be acknowledged that, despite the fact that the C2C12 differentiated myotube is a widely used experimental model for studying the underlying mechanism for the formation of receptor clusters, it does have several limitations compared with innervated muscle cells, in which nerve impulses are critical for the stability and the formation of the neuromuscular in a timely manner. Thus, further in vivo studies are warranted.
In summary, the present work suggests a model by which the complex a-dbn-akap controls AChR stability during intracellular trafficking. Phosphorylated a-dbn recruits a deubiquitinase Usp9x and/or chaperon proteins. The complex interacts and stabilizes akap. akap, through its unique hydrophobic Nterminal domain, targets the complex to the ER (where akap has been found to be highly expressed) where it promotes AChR stability by a ubiquitin-dependent mechanism. In the absence of phosphorylated a-dbn and akap interaction, most of AChRa subunits are targeted to degradation in a proteasomedependent mechanism.

Materials and methods Plasmid constructs and reagents
The plasmids of the mouse AChR subunit a was a generous gifts from Dr. William Green at the University of Chicago. Plasmids of akap-GFP and a-dystrobrevin-GFP were described in our previous work (17,18). a-Dystrobrevin-1-GFP is the fulllength phosphorylatable isoform; a-dystrobrevin-3Y/F-GFP is the mutated isoform in which the C-terminal phosphorylatable three tyrosine residues were changed to phenylalanine (Y705F, Y713F, and Y730F). akap-mCherry was cloned by swapping GFP (from the akap-GFP) to mCherry (from pmCherry-N3) with 59 EcoRI and 39 KpnI sites. Both a-dystrobrevin-3Y/F-GFP and akap-mCherry were verified by sequencing, transfection, and Western blots. The proteasome inhibitor MG132 (Enzo Life Sciences) was used at 2.5 mM as described by Mouslim et al. (18) to determine whether the degradation of a-dbn-GFP mutant is mediated by the proteasome.
To examine the effect of overexpression of akap and a-dbn on the formation of AChR clusters, cells (at 80% confluency) were transfected with appropriate plasmids using Lipofectamine 2000 (1467572; Invitrogen) according to the manufac-turer's instructions. Differentiated myotubes were treated with C-terminal agrin (550-AG; R&D Systems) at a concentration of 500 ng/ml overnight. The cells were then fixed by 2% paraformaldehyde in PBS for 20 min and then washed with PBS three times. The cells were incubated with fluorescent BTX-Alexa (Molecular Probes) for 2 h at room temperature, followed by four washes in PBS and 4[prime],6[prime]-diamino-2-phenylindole Fluoromount-G mounting medium (0100-20; Southern Biotech). The cells were then imaged using the Olympus spinning-disk confocal microscope (Andor/Olympus IX81). The zstacks were then collapsed, and the contrast was adjusted with Photoshop (Adobe Systems).
To examine the co-localization between a-dbn and akap, HeLa cells were co-transfected with a-dbn-GFP (green) and akap-mCherry (red) constructs, and 24 h later, the cells were fixed and imaged with the confocal microscope. The z-stacks were then collapsed, and the contrast was adjusted with Photoshop (Adobe Systems).

Quantification of AChR clusters
Spinning-disk images of myotubes were processed and analyzed by using ImageJ (National Institutes of Health, version 1.49). Labeled clusters of AChRs with fluorescent BTX-Alexa (either 647, 594 or, 488) on transfected and nontransfected myotubes were quantified using the following procedure: images were set to 8-bit binary, and a threshold that clearly identified clusters and excluded background was used. The area (mm 2 ) of AChR clusters and their lengths (mm) were measured. At least 30 myotubes from 3 independent plates were quantified from each experimental condition.
In another series of experiments, HeLa and HEK cells at 90-100% confluence were washed with cold PBS, lysed with lysis buffer (100 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM EDTA, 5 mM NaF, 5 mM Na 3 VO 4 , 2.5 mM Na 4 P 2 O 7 , 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM phenylmethanesulfonyl fluoride and protease inhibitor mixture), and centrifuged for 10 min at 10,000 [times] g. The lysates were resuspended in LDS reducing sample buffer and boiled for 5 min prior to Western blotting. The proteins were separated by SDS-PAGE (10%) and transferred to PVDF membranes. The membranes were stained with Ponceau S for 1 h, imaged, washed three times with PBST (PBS with 0.05% Tween 20), and then bathed in a blocking solution (5% dry milk and 0.05% Tween 20 in TBS, 103 TBS: 170-6435; Bio-Rad) for 1-3 h. The membranes were incubated with primary antibodies in blocking solution overnight at 4°C. After three washes with PBST, the membranes were incubated with secondary horseradish peroxidase-conjugated antibodies and developed using SuperSignalWest Pico solutions (34077; Thermo Fisher Scientific) or Clarity Western ECL substrate (1705060; Bio-Rad). Mouse anti-HA (clone 16B12, MMS-101R; Covance) and mouse anti-akap (calcium/calmodulin kinase IIa A-1, sc-13141; Santa Cruz Biotechnology) were diluted at 1:2000; mouse anti-a-dystrobrevin (610766; BD Biosciences) was diluted at 1:4000; rabbit anti-GFP (A6455; Invitrogen), rat anti-AChRa (MAB210; Covance), and mouse anti-tubulin (AA4.3-s; Developmental Studies Hybridoma Bank) were diluted at 1: 5000. All horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) were diluted at 1:10,000. Western blotting band signals were captured using either the Bio-Rad ChemiDoc MP imaging system or FluorChem M FM0467. We first used the auto-exposure mode to determine the optimum exposure time to avoid pixels' saturation (according to the manufacturers' handbook). Within in that time range, we captured at least two images to use for quantification. Only the nonsaturated bands in the images were quantified. Between the two images, the samples were assigned proportional values to ensure that the signals fell within the ECL linear exposure range. Because of inconsistencies and variations in signal intensity between the same protein bands in different gel lanes, we normalized the proteins bands to its transfection efficiency indicator, housekeeping proteins, or total protein identified by Ponceau S. In this way, errors related to variations in sample loading and transfers were corrected. Western blotting bands were quantified with ImageJ (National Institutes of Health, version 1.49). The intensity of bands was quantified by using the rectangular selection tool and keeping the area uniform, which covered each band's active area, where a straight line was used to cut off nonspecific bands, and the wand tool selected the target peak. These were compared with equal area baseline selections of an empty lane nearby (no protein loaded) to subtract background. If the background was not uniform across the blot, the background subtraction would be individually selected for each lane, for which the criteria is a no-signal, slightly upper or lower active band area. For all data presented as histograms, results from several blots were averaged, with the number indicated in the figure legends.

Statistical analysis
Statistical analysis was performed with GraphPad Prism (version 8.2.0). The experimental results were analyzed by Student's t test (compare differences between two groups) and either by one-way ANOVA or two-way ANOVA followed by Tukey's or Dunnett's post hoc comparison tests where appropriate. The data are expressed as means 6 S.D. Significance was defined as p , 0.05 for all analyses.

Data availability
All the data for this study are available within the article.