Differentiation-dependent phosphorylation of a 175,000 molecular weight protein in response to insulin and insulin-like growth factor-I in L6 skeletal muscle cells.
Insulin and insulin-like growth factor-I (IGF-I) effects on protein phosphorylation were investigated in intact skeletal muscle cells at different stages of differentiation. In undifferentiated L6 myoblasts, stimulation by either insulin or IGF-I, but not IGF-II, led to a 3- to 5-fold increase in phosphorylation of insulin and IGF receptor beta-subunits and the appearance of a 175,000 mol wt (Mr) phosphoprotein (pp175). These effects reached a maximum within 3 min, were maintained for 12 min, and then declined. Dose-response curves for pp175 phosphorylation in response to insulin (ED50 = 2 nM) and IGF-I (ED50 = 0.2 nM) were consistent with occupancy and stimulation of each receptor kinase by its specific hormone. The 175,000 Mr phosphoprotein was not precipitated by antireceptor antibodies, and the phosphoamino acid composition differed markedly from that of insulin and IGF-I receptors, with a 10-fold lower phosphotyrosine/phosphoserine ratio after insulin stimulation. In contrast to insulin and IGF-I receptors, pp175 was not extracted by the nonionic detergent Triton X-100, but required sodium dodecyl sulfate for solubilization. When experiments were carried out with L6 cells after differentiation into skeletal muscle myotubes, hormone-induced phosphorylation of pp175 was almost undetectable. We conclude that pp175 is a phosphoprotein distinct from insulin and IGF-I receptors that is involved in the early phosphorylation events that follow the activation of the insulin and IGF-I receptor kinases. Its disappearance after terminal differentiation of the L6 cells is consistent with a role in hormonal stimulation of cell proliferation.
Beguinot
F
F
Joslin Research Laboratory, Boston, Massachusetts 02215.
Kahn
C R
CR
Moses
A C
AC
White
M F
MF
Smith
R J
RJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D007328:Insulin / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification*; D010766:Phosphorylation; D051381:Rats; D013002:Somatomedins / Q000494:pharmacology*
D000596:Amino Acids; D007328:Insulin; D010750:Phosphoproteins; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I