Abstract

---

During skeletal muscle differentiation the level of eukaryotic initiation 2 (eIF2)-associated glycoprotein, p67 gradually increases and its level peaks at the time of fusion of myoblasts into multinucleated myotubes. During the course of differentiation of C2C12 myoblasts, p67 dissociates from eIF2 that leads to increased eIF2α phosphorylation and suppression of global protein synthesis. At the same time, it associates with extracellular signal-regulated kinases 1 and 2 (ERK1/2) to inhibit the activation and activity of the later kinases that ultimately inhibits growth promoting signal to myotubes. To examine whether p67 has any role(s) in actin cytoskeleton dynamic and migration of myoblasts when fusing into myotubes, we investigated the levels of Myristoylated-alanine rich C kinase substrate (MARCKS) in C2C12 myoblasts cell lines constitutively expressing rat p67 and some of its mutants. We found that p67 is involved in higher level expression of MARCKS in myoblasts and its N-terminal lysine-rich domains I & II are required for such activity. On the other hand, in myotubes constitutively expressing rat p67 the level of MARCKS is very low and p67’s N-terminal lysine-rich domains I & II along with conserved D251 and H331 residues play important roles in this process. In addition, MARCKS is degraded in rat p67-expressing C2C12 myoblasts and p67’s conserved D251 residue and lysine residue-rich domains I & II are involved in this degradation. Altogether, our data suggest that p67 is possibly involved in the inhibition of myobalsts’ migration while growing in growth medium whereas, it promotes myoblasts’ migration and motility when myoblasts are fusing into myotubes.
Abbreviations Used: p67, a 67 kDa glycoprotein that binds to both eukaryotic initiation factor 2 (eIF2) and extracellular signal-regulated kinases 1 & 2 (ERK1/2); D251A, a p67 point mutant, which has alanine substitution for aspartic acid at 251 amino acid residue; D6/2, a p67 block mutant, where a stretch of N-terminal acidic amino acid residues has been replaced with uncharged amino acids; K1K2, a p67 block mutant, where both of its N-terminal lysine/arginine-rich stretches of amino acid residues have been replaced with uncharged amino acids; D6/2+D251A, a D6/2 mutant containing a second site point mutation, where its 251 aspartic amino acid residue has been changed to alanine; D6/2+H331A, a D6/2 mutant containing a second site point mutation, where its histidine amino acid residue at position 331 has been changed to alanine; MARCKS, Myristoylated alanine-rich C kinase substrate; p-MARCKS, the phosphorylated form of MARCKS; and V, vector expressing enhanced green fluorescence protein (EGFP).

---