The tuberous sclerosis complex subunit TBC1D7 is stabilized by Akt phosphorylation-mediated 14-3-3 binding

Area of Science:

• Cellular signaling
• Molecular biology
• Biochemistry

Background:

• The tuberous sclerosis complex (TSC) is a critical negative regulator of mTOR complex 1, a key node in cellular growth pathways.
• While TSC signaling is well-studied, several regulatory components remain uncharacterized.
• TBC1D7 is a known component of the TSC complex, but its specific regulatory mechanisms are not fully understood.

Purpose of the Study:

• To identify novel regulators and characterize the function of TBC1D7 within the TSC signaling pathway.
• To elucidate the post-translational modifications and protein interactions governing TBC1D7 stability and function.
• To investigate the role of Akt kinase in modulating TBC1D7 activity and its impact on cellular growth regulation.

Main Methods:

• Pulldown assays and mass spectrometry (MS) to identify protein interactors of TBC1D7.
• Crystal structure determination of TBC1D7 and its complexes.
• Site-directed mutagenesis to investigate the role of specific phosphorylation sites (e.g., Ser-124).
• Biochemical assays to assess protein binding, phosphorylation, ubiquitination, and stability.

Main Results:

• TBC1D7 was identified as a binding partner of PHLPP1, a negative regulator of Akt signaling.
• Akt phosphorylates TBC1D7 at Ser-124, which stabilizes the protein without affecting TSC1 binding.
• Phosphorylated TBC1D7 binds to 14-3-3 proteins in a growth factor-dependent manner, further stabilizing TBC1D7.
• The E3 ubiquitin ligase beta-TrCP2 ubiquitinates TBC1D7 at a degron near Ser-124, leading to decreased protein stability.
• Akt activity dictates TBC1D7 phosphorylation status, controlling its interaction with either 14-3-3 or beta-TrCP2, thereby regulating TBC1D7 stability.

Conclusions:

• TBC1D7 stability is dynamically regulated by Akt-mediated phosphorylation at Ser-124.
• Phosphorylation at Ser-124 acts as a switch, directing TBC1D7 to either stabilizing 14-3-3 proteins or destabilizing beta-TrCP2-mediated ubiquitination.
• This regulatory mechanism provides a novel link between Akt signaling and TSC/mTOR pathway control, impacting cellular growth.