Phosphorylation of α-dystrobrevin is essential for αkap accumulation and acetylcholine receptor stability

Abstract

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

A link between agrin signalling and Cav3.2 at the neuromuscular junction in spinal muscular atrophy

“…An increase of Dyn2 and GAPDH pro-tein levels was shown with flunarizine. No drug effects were observed on CaMKII protein levels or phosphorylation, ruling out a possible involvement of CaMKII, which also regulates the AChR expression and clustering 54,55 . SMN and Unrip (another SMN-complex component) protein levels were unchanged, as expected 56 .…”