Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor–related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose–response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes. [Display omitted] •Different agonists of muscle-specific kinase (MuSK) elicit similar phosphoprofiles.•MuSK activation induces tyrosine phosphorylation of several Rab GTPases.•MuSK inhibitors diminish receptor signaling, including phosphorylation on Rab10 Y6.•Mutation of Rab10 Y6 disrupts its association with Mical adaptor proteins. We elucidated phosphosignaling network downstream of muscle-specific receptor tyrosine kinase (MuSK) activated by natural agonist agrin or agonist antibody. Dose–response and time-course experiments showed that both agonists elicited similar intracellular responses. We also characterized two small-molecule inhibitors of MuSK that effectively disrupted MuSK signaling pathway. We further studied the functions of MuSK-responsive tyrosine phosphorylation sites localized on several Rab GTPases. We showed that Rab10 Y6F mutation disrupts association with its adaptor molecules Micals.