Abstract
The present study identifies the operation of a signal tranduction pathway in mammalian cells that provides a checkpoint control, linking amino acid sufficiency to the control of peptide chain initiation. Withdrawal of amino acids from the nutrient medium of CHO-IR cells results in a rapid deactivation of p70 S6 kinase and dephosphorylation of eIF-4E BP1, which become unresponsive to all agonists. Readdition of the amino acid mixture quickly restores the phosphorylation and responsiveness of p70 and eIF-4E BP1 to insulin. Increasing the ambient amino acids to twice that usually employed increases basal p70 activity to the maximal level otherwise attained in the presence of insulin and abrogates further stimulation by insulin. Withdrawal of most individual amino acids also inhibits p70, although with differing potency. Amino acid withdrawal from CHO-IR cells does not significantly alter insulin stimulation of tyrosine phosphorylation, phosphotyrosine-associated phosphatidylinositol 3-kinase activity, c-Akt/protein kinase B activity, or mitogen-activated protein kinase activity. The selective inhibition of p70 and eIF-4E BP1 phosphorylation by amino acid withdrawal resembles the response to rapamycin, which prevents p70 reactivation by amino acids, indicating that mTOR is required for the response to amino acids. A p70 deletion mutant, p70Delta2-46/DeltaCT104, that is resistant to inhibition by rapamycin (but sensitive to wortmannin) is also resistant to inhibition by amino acid withdrawal, indicating that amino acid sufficiency and mTOR signal to p70 through a common effector, which could be mTOR itself, or an mTOR-controlled downstream element, such as a protein phosphatase.
Highlights
Brief starvation engenders a decrease in protein synthesis, in skeletal muscle, which is rapidly reversed on refeeding [1]
Synthesis in skeletal muscle; much evidence indicates that a major portion of the increase in the skeletal muscle protein synthesis in vivo seen on refeeding is independent of changes in insulin and perhaps attributable to the nutrients themselves [2]
Amino Acids Do Not Regulate Insulin Receptor Kinase, PI 3-Kinase, c-Akt/PKB, or MAPK Activities—Inasmuch as the withdrawal of amino acids from CHO-IR cells reversibly blocks the ability of insulin to stimulate the phosphorylation and activation of p70 S6K and the eIF-4E-BPs, we examined the effects of amino acid withdrawal on several steps in insulin signal transduction upstream of, or parallel with the activation of the p70 S6 kinase (Fig. 5, A–D)
Summary
Brief starvation engenders a decrease in protein synthesis, in skeletal muscle, which is rapidly reversed on refeeding [1]. The eIF-4E component binds the 5Ј mRNA cap; 4A/4B unwind the secondary structure in the 5Ј-untranslated segment, thereby enabling the 40 S-Met-tRNAi complex, aided by eIF-3, to efficiently scan the mRNA 5Ј-untranslated segment to the AUG start site Both the assembly and activity of the 4F complex are regulated by phosphorylation. PHAS1 was purified as a heat-and acid-soluble polypeptide that exhibited rapid and multiple phosphorylation in vivo in response to insulin and mitogens [15], and was independently isolated as an eIF-4E-binding protein by interaction cloning [16]. The 4E-BP1/PHAS1 polypeptide binds to 4E in a manner that is competitive with 4G [17]; the insulin/ mitogen-stimulated phosphorylation of 4E-BP1, which inhibits 4E-BP binding to 4E [16, 18], makes 4E available for incorporation into the 4F complex. 4E and 4E-BPs are coordinately regulated by insulin and mitogens, examples of discordant regulation are common [13, 19], indicating that the phosphorylation of the two polypeptides is independently mediated
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