mTORC2 controls the activity of PKC and Akt by phosphorylating a conserved TOR interaction motif

Area of Science:

• Molecular Biology
• Cell Signaling
• Biochemistry

Background:

• The mechanistic target of rapamycin complex 2 (mTORC2) is known to regulate AGC kinases, but its direct role in phosphorylating the hydrophobic motif remains debated.
• AGC kinases play critical roles in various cellular processes, and their regulation is essential for maintaining cellular homeostasis.

Purpose of the Study:

• To identify and characterize a novel mTOR-mediated phosphorylation site regulating AGC kinase activity.
• To elucidate the mechanism by which mTORC2 controls the phosphorylation of the hydrophobic motif in kinases like Protein Kinase C (PKC) and Akt.

Main Methods:

• Identification of a novel phosphorylation site, the TOR interaction motif (TIM).
• Site-directed mutagenesis to assess the functional role of TIM in Akt1 and PKCβII.
• In vitro kinase assays using purified components.
• Biophysical proximity assays and structural analysis to investigate PKC dimerization.
• Experiments in mTORC2-deficient cells and overexpression studies.

Main Results:

• A conserved mTOR-mediated phosphorylation site, TIM (F-x3-F-pT), was identified, which is invariant in mTORC2-dependent AGC kinases.
• Mutation of TIM abolished kinase activity by impairing activation loop and hydrophobic motif phosphorylation.
• mTORC2 directly phosphorylates the PKC TIM in vitro, a process observed in mouse brain.
• mTORC2 facilitates PDK1-mediated phosphorylation, enabling autophosphorylation, and relieves nascent PKC dimerization via TIM phosphorylation.

Conclusions:

• The discovery of TIM phosphorylation provides a new mechanism for AGC kinase regulation by mTORC2.
• mTORC2-mediated TIM phosphorylation is essential for recruiting PDK1 and initiating autophosphorylation, thereby controlling kinase activity.
• This finding offers insights into the regulation of PKC and Akt, with implications for understanding cellular signaling pathways.