The Phosphoproteome during Neuromuscular Synapse Formation

The reversible phosphorylation of proteins is one of the most abundant posttranslational modifications and a biochemical process of highest biological relevance. It regulates enzyme activities, substrate specificity, protein localization, degradation and complex formation and therefore plays a role in almost every cellular process including cell proliferation, differentiation and survival. Likewise, the development of the neuromuscular synapse depends on signaling processes, which involve protein phosphorylation as crucial regulatory event. The receptor tyrosine kinase MuSK is the key signaling molecule at the neuromuscular synapse whose activity is required for the formation of a mature and functional neuromuscular synapse. However, the signaling cascade downstream of MuSK and the regulation of the different components is still poorly understood. Within this project we propose to study the phosphorylation events and phosphotargets downstream of MuSK. We intend to use a mass spectrometry approach, which has become the method of choice to identify and characterize phosphorylation events. Using different methods of enrichment and quantitative mass spectrometry tools we will be able to identify novel targets and to analyze the complex interplay within the signaling cascade. The role of the identified phosphotargets during neuromuscular synapse formation will be characterized using in vitro and in vivo approaches. Thereby we will focus on how protein phosphorylation influences protein function and on how the novel phosphotargets fit into the MuSK-dependent signaling cascade. This multidisciplinary approach to study the signaling processes induced by MuSK action will provide a better understanding of the molecular mechanisms, which control the formation of a functional neuromuscular synapse.

Neuromuscular junctions form when a motor axon reaches a muscle fiber. Acetylcholine receptors become concentrated at the site of innervation and processes at the molecular and cellular level lead to the development of a mature and functional neuromuscular junction. Neuromuscular junctions regulate any skeletal movement including respiratory function within an organism. Impaired neuromuscular function results in acute neuromuscular deficiencies including muscle weakness and respiratory distress. The postsynaptic kinase MuSK is the key player in neuromuscular junction formation. Impaired MuSK signaling causes neuromuscular disorders or, even more severely, respiratory failure and perinatal death in newborn mice lacking MuSK. Signal transduction events downstream of MuSK activation induce pre- as well as postsynaptic differentiation, which, most prominently, includes the clustering of acetylcholine receptors at synaptic sites. The crucial events regulating these processes are the phosphorylation and subsequent activation of MuSK. Posttranslational modifications greatly regulate the activity and functions of proteins. Phosphorylation is the best-known form of posttranslational modification. MuSK activity is critically regulated by phosphorylation: blocked phosphorylation inhibits MuSK activation and downstream signaling. We have used a quantitative phosphoproteomics screen to identify phosphotargets of MuSK signaling. We have been able to show that transcriptional as well as cytoskeletal protein networks are activated. We have been analyzing proteins within these networks as well as novel MuSK phosphorylation events to better understand the complex interplay between molecular and cellular determinants of neuromuscular junction development. Our studies have focused on the cytoskeletal regulator protein palladin. Palladin exists in multiple isoforms that are specifically up- or downregulated during muscle differentiation. Using doxycycline-inducible shRNA expression to silence palladin in differentiated myotubes we find changes in AChR clustering implicating palladin as modulator of postsynaptic differentiation. In further studies, we characterized a novel phosphoserine site, S751, which is upregulated during late agrin stimulation and lies in the activation loop of the MuSK kinase domain. A phosphomimetic mutant of S751 increased basal MuSK kinase activity, AChR phosphorylation and AChR cluster size suggesting a mechanism to relief the autoinhibition of the MuSK activation loop, which could foster or stabilize MuSK kinase activity. Therefore, phosphorylation of S751 might represent a novel mechanism to modulate MuSK kinase activity during prepatterning or neuromuscular junction maintenance. We expect that these studies will provide detailed mechanistic insights into the complex signaling network downstream of MuSK.