Abstract

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During skeletal muscle differentiation several growth factor mediated signaling pathways must shut off to allow cycling myoblasts to permanently withdraw from cell cycle and fuse into multinucleated myotubes. The level of eukaryotic initiation factor 2 (eIF2)-associated glycoprotein p67 gradually increases when C2C12 myoblasts are differentiating into myotubes. At the same time, the level of lipid induced expression and activity of protein kinase C (cPKCalpha) gradually decreases. cPKCalpha is activated in response to growth factors and sends signal to downstream effectors for cell growth and proliferation and differentiation. We therefore examined whether constitutive expression of rat p67 has any effect on expression of PKC both in C2C12 myoblasts and myotubes. We found ~10-fold increase in cPKCalpha level in p67-overexpressing myoblasts and p67’s N-terminal lysine-rich domains I & II and in some extend its acidic residue-rich domain are essential for this activity. Although, p67’s conserved D251 residue alone has no effect of cPKCalpha level, this residue is however needed to cooperate with the function(s) of lysine-rich domains I & II. The later cooperation was prominent in differentiated myoblasts where cPKCalpha level was down in p67-overexpressing myotubes. In addition, p67’s another conserved residue H331 also cooperates with the functions lysine-rich domains I & II in lowering the level of cPKCalpha in C2C12 differentiated myotubes. Altogether, our data suggest that increased p67 level is involved in increasing the level of cPKCalpha in C2C12 myoblasts and decreasing in the 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; cPKCalpha, Protein kinase C; and V, vector expressing enhanced green fluorescence protein (EGFP).

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