MuSK sumoylation and neuromuscular junction development

Posttranslational modifications are important regulatory events that greatly influence protein function, among them phosphorylation and ubiquitination are the best known. Sumoylation has recently been added to this list. Addition of SUMO is mechanistically similar to ubiquitination. But whereas ubiquitination is functionally restricted to internalization or degradation of target proteins, sumoylation has a more diverse role. It can regulate activity, stability, localization and interactions of substrates. Posttranslational modifications like phosphorylation and glycosylation have been shown to regulate the function of the receptor tyrosine kinase MuSK. MuSK is the key signaling molecule at the neuromuscular junction whose activity is crucial for the development of a mature and functional neuromuscular junction. The critical events during synapse formation include agrin-mediated activation of MuSK via Lrp4, concentration of MuSK at the postsynaptic muscle membrane, tyrosine phosphorylation and initiation of a downstream signaling cascade. We have preliminary evidence that MuSK is also modified by sumoylation. We now intend to examine the molecular nature of MuSK sumoylation and its role during MuSK function. In particular, we want to understand whether MuSK sumoylation plays a role during the formation and maintenance of the neuromuscular junction.

Neuromuscular synapses 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 synapse. Neuromuscular synapses regulate any skeletal movement including respiratory function within an organism. In addition, due to their size and simplicity neuromuscular synapses represent a good model system to study the process of synapse formation. The postsynaptic kinase MuSK is the key player in neuromuscular synapse formation. Signal transduction downstream of MuSK regulates pre- as well as postsynaptic differentiation including acetylcholine receptor clustering. Posttranslational modifications greatly regulate the activity and functions of proteins. Likewise we have been aiming at understanding how posttranslational modifications influence MuSK activity. 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 studied the effects of phosphorylation and have been able to identify a novel phosphorylation site. Further we examined the role of sumoylation in MuSK-dependent signaling. Sumoylation is a posttranslational modification that is characterized by the attachment of SUMO protein to specific lysines within a protein. Sumoylation has diverse effects on protein function modulating localization, transport or degradation. Our data suggest that sumoylation does not play a critical role during MuSK activation and signaling. In a collaborative study we have examined the function of the RNA kinase CLP1 during neuromuscular synapse formation. CLP1 phosphorylates tRNAs, which are important components of the machinery that translates genes into proteins. Expression of inactive CLP1 in mice results in a progressive loss of motor neurons. Motor neurons express an aberrant tRNA form and are susceptible to oxidative stress and apoptosis via p53. Depending on the mouse strain either Clp1 mutant mice die at birth or develop motor problems leading to premature death early during adulthood. Mice show a severe denervation of muscle tissues and neuromuscular synapse degeneration, which result in respiratory problems and paralysis.