Acetylcholine Receptor (AChR) Clustering Is Regulated Both by Glycogen Synthase Kinase 3β (GSK3β)-dependent Phosphorylation and the Level of CLIP-associated Protein 2 (CLASP2) Mediating the Capture of Microtubule Plus-ends

• DOI: 10.1074/jbc.m114.589457
• Publication Date: 2014-09-18T06:27:47Z
• Authors: Sreya Basu; Stefan Sladecek; Hayley Pemble; Torsten Wittmann; Johan A. Slotman; Wiggert van Cappe...; Hans-Rudolf Brenner; Niels Galjart
• Keywords: 570; Nicotinic Acetylcholine Receptors; Biochemistry & Molecular Biology; Biomedical and clinical sciences; 1.1 Normal biological development and functioning; Knockout; Muscle Fibers, Skeletal; Primary Cell Culture; Microtubule; Microtubule-associated Protein; Inbred C57BL; Muscle Fibers; Medical and Health Sciences; Microtubules; Mice; Glycogen Synthase Kinase 3; 03 medical and health sciences; Underpinning research; Receptors; Chlorocebus aethiops; Animals; Humans; Receptors, Cholinergic; Agrin; Phosphorylation; Protein Processing; Cholinergic; Mice, Knockout; 0303 health sciences; Glycogen Synthase Kinase 3 beta; Neurosciences; Post-Translational; EMC MGC-02-13-02; Skeletal; Biological Sciences; Synapse; Mice, Inbred C57BL; Biological sciences; Protein Transport; HEK293 Cells; Chemical sciences; Chemical Sciences; COS Cells; Biochemistry and Cell Biology; Microtubule-Associated Proteins; Protein Processing, Post-Translational

The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2-9XS/9XA) and are resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2-8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends.