SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• Mammalian target of rapamycin (mTOR) regulates cell growth and proliferation through TORC1 and TORC2 complexes.
• TORC2 is implicated as PDK2, phosphorylating Akt/PKB at Ser473, a site crucial for full Akt activation.
• The precise roles of Akt phosphorylation sites and their regulatory mechanisms require further elucidation.

Purpose of the Study:

• To identify essential subunits of TORC2 involved in Akt phosphorylation.
• To investigate the role of SIN1/MIP1 in the context of TORC2 function and Akt activation.
• To determine the physiological consequences of impaired Akt Ser473 phosphorylation on downstream targets and cell survival.

Main Methods:

• Genetic ablation of the SIN1/MIP1 gene in mammalian cells.
• Biochemical analysis of protein-protein interactions within mTOR complexes.
• Assessment of Akt/PKB phosphorylation at Ser473 and Thr308.
• Evaluation of phosphorylation status of known Akt and TORC1 targets (FoxO1/3a, TSC2, GSK3, S6K, 4E-BP1).

Main Results:

• SIN1/MIP1 was identified as an essential subunit of TORC2, critical for rictor-mTOR interaction.
• Genetic ablation of SIN1 abolished Akt Ser473 phosphorylation while preserving Thr308 phosphorylation.
• Impaired Ser473 phosphorylation selectively affected Akt targets like FoxO1/3a, leaving TSC2, GSK3, and TORC1 effectors (S6K, 4E-BP1) unaffected.
• TORC2's role in Akt Ser473 phosphorylation, mediated by SIN1, is vital for cell survival.

Conclusions:

• SIN1 is indispensable for TORC2-mediated Akt Ser473 phosphorylation.
• This specific phosphorylation event is crucial for TORC2's function in cell survival.
• TORC2's role in Akt Ser473 phosphorylation is distinct from and dispensable for TORC1 activity.