G.P.160: Cysteine mutations cause defective tyrosine phosphorylation in MEGF10 myopathy

Abstract

MEGF10 is a single transmembrane protein that is expressed in satellite cells of skeletal muscle, with 17 EGF-like domains in the extracellular region and 13 tyrosine residues in the cytoplasmic domain. Recessive mutations in MEGF10 are known to cause a congenital myopathy in humans, including the syndrome of early onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD). Previously we reported a family with a milder phenotype who harbored the compound heterozygous missense mutations C326R and C774R. A heterozygous C774R mutation was also found in a patient with EMARDD, paired with a heterozygous nonsense mutation in MEGF10, suggesting that C774R may be more damaging than C326R. Both mutations reside in the extracellular EGF-like domains of MEGF10. We found that these mutations were associated with decreased tyrosine phosphorylation of MEGF10 in vitro, and that C774R caused a greater impairment of tyrosine phosphorylation than C326R. We also found that Y1030 is the major tyrosine phosphorylation site in MEGF10 and is phosphorylated at least in part by c-Src. Co-expression of dominant-negative c-Src with MEGF10 shows decreased tyrosine phosphorylation of MEGF10, suggesting that MEGF10 is phosphorylated by endogenous c-Src. Overexpression of wild-type MEGF10 enhanced C2C12 myoblast proliferation, while overexpression of Y1030F mutated MEGF10 did not. In addition, overexpression of either wild type MEGF10 or Y1030D caused mild activation of ERK1/2. These findings indicate that MEGF10-mediated signaling via tyrosine phosphorylation plays an important role in myoblast proliferation, possibly via activation of ERK1/2. We conclude that MEGF10-mediated signaling via tyrosine phosphorylation helps to regulate myoblast proliferation. Our findings suggest that defects in this signaling pathway may contribute to the disease mechanism of MEGF10 myopathy.