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

ABSTRACT: Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed two decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, MuSK agonist antibodies function independently of its co-receptor Lrp4. Recent reports suggest that MuSK agonist antibodies can delay the onset of muscle denervation in mouse models of amyotrophic lateral sclerosis (ALS). To get a deeper understanding of MuSK signaling, and potentially identify Lrp4-independent signaling downstream of MuSK activation, we performed phosphoproteome profiling of myotubes treated with each agonist. Our approach involved quantifying changes in site-specific phosphorylation in orthogonal dose response and time course experiments with each agonist using isobaric mass tags. We observed that both agonists elicited remarkably similar intracellular responses, as evidenced by highly correlating tyrosine phosphorylation on known MuSK signaling components and newly identified cytoskeletal and intracellular trafficking nodes. Among these was inducible phosphorylation on a conserved N-terminal phosphorylation site present on a family of endosomal Rab GTPases. We confirmed that suppression of MuSK signaling by a small molecule reduces Rab10 tyrosine phosphorylation. Importantly, Rab10 mutation at this site disrupts its association with adaptor molecules Mical1 and Mical3. Together these data provide an in depth characterization of the MuSK signaling pathway, describe two small molecule kinase inhibitors, and reveal a novel regulatory mechanism of small Rab GTPases that is poised to regulate intracellular trafficking downstream of receptor tyrosine kinase activation.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Hanna Budayeva   Donald Kirkpatrick  

PROVIDER: MSV000085547 | MassIVE | Thu Jun 04 14:27:00 BST 2020

REPOSITORIES: MassIVE