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Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism
1Diabetes Unit and Medical Services, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129, USA.
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.
Insights
Mammalian cells use amino acid levels to control protein synthesis initiation via a pathway involving p70 S6 kinase and eIF-4E BP1. This signaling pathway, dependent on mTOR, links nutrient availability to cell growth regulation.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Cellular processes like protein synthesis are tightly regulated by nutrient availability.
- Amino acid sufficiency is a critical factor influencing cell growth and proliferation.
- Signal transduction pathways mediate the communication between nutrient status and cellular machinery.
Purpose of the Study:
- To identify the signal transduction pathway linking amino acid sufficiency to peptide chain initiation control in mammalian cells.
- To elucidate the role of p70 S6 kinase and eIF-4E BP1 in this regulatory mechanism.
- To investigate the involvement of mTOR in sensing amino acid levels and controlling protein synthesis.
Main Methods:
- Utilized Chinese Hamster Ovary-Insulin Receptor (CHO-IR) cells.
- Manipulated amino acid concentrations in the cell culture medium.
- Assessed the phosphorylation status and activity of key signaling proteins, including p70 S6 kinase and eIF-4E BP1.
- Examined the effects of amino acid withdrawal/readdition on insulin signaling pathways.
- Employed a rapamycin-resistant p70 mutant to probe the role of mTOR.
Main Results:
- Amino acid withdrawal rapidly deactivated p70 S6 kinase and dephosphorylated eIF-4E BP1, halting peptide chain initiation.
- Readdition of amino acids restored protein phosphorylation and responsiveness to insulin.
- Elevated amino acid levels increased basal p70 activity, overriding insulin stimulation.
- Selective inhibition of p70 and eIF-4E BP1 by amino acid withdrawal mimicked rapamycin's effects, implicating mTOR.
- A rapamycin-resistant p70 mutant was also resistant to amino acid withdrawal inhibition.
Conclusions:
- Amino acid sufficiency acts as a checkpoint for controlling peptide chain initiation in mammalian cells.
- The mTOR pathway is essential for sensing amino acid levels and regulating p70 S6 kinase and eIF-4E BP1.
- Amino acid sufficiency and mTOR signal to p70 through a common effector, potentially mTOR itself or a downstream phosphatase.