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Phosphorylation of TSC2 by PKC-δ reveals a novel signaling pathway that couples protein synthesis to mTORC1 activity

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Abstract

Downstream of insulin-like growth factor receptor, the TSC1/2/ TCB1D7 (tuberous sclerosis complex) and mTOR (mechanistic target of rapamycin) pathways are implicated in many human diseases, including cancer and diabetes. Targeting this pathway is currently an important approach for palliating or eradicating cancer. Downstream of mTOR, translational machinery targeting holds great promise for anticancer drug development. Therefore, we investigated whether the protein synthesis machinery that is regulated by mTORC1 (mTOR complex 1) signaling can in turn regulate mTORC1 activity. We found that inhibition of protein synthesis results in rapid activation of mTORC1 signaling, thereby uncovering a feedback loop between mTOR and the translation machinery. This mTORC1 activation requires tuberous sclerosis complex (TSC) but is independent of AKT. In addition, by using a PKC-δ (protein kinase c delta)-specific inhibitor and PKC-δ siRNA knockdown, we found that PKC-δ kinase activity is required for mTORC1 activation in response to translation inhibitors. Furthermore, translation inhibition activates PKC-δ. Subsequently, we investigated whether PKC-δ can phosphorylate and inactivate TSC1/2, leading to mTORC1 activation. In vitro kinase assays showed direct phosphorylation of TSC2 (S932 and S939) by PKC-δ, which was confirmed by mass spectrometry. In vivo kinase analysis further indicated that both S932 and S939 are phosphorylated in response to translation inhibitors. Finally, phosphorylation defective TSC2 mutants (S932A and S939A single mutants and a S932A/S939A double mutant) failed to upregulate mTORC1 activity in the presence of translation inhibitors, suggesting that activation of mTORC1 by translation inhibitors is mediated by PKC-δ phosphorylation of TSC2 at S932/S939, which inactivates TSC.

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Acknowledgements

We thank Peter Houghton for his initial support and advice. We thank Nisha Badders for scientific editing of the manuscript.

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Authors and Affiliations

  1. Department of Genetics, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA

    Jun Zhan, Frank C. Harwood & Gerard C. Grosveld

  2. Hartwell Center, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA

    Raghu K. Chitta

  3. Bayer Pharmaceuticals, 800 Dwight Way, Berkeley, CA, 94710, USA

    Raghu K. Chitta

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  1. Jun Zhan
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  2. Raghu K. Chitta
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JZ designed and performed the experiments; FH assisted the experiments; RKC performed mass-spectrometry; and GG and JZ wrote the manuscript.

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Correspondence to Jun Zhan.

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Zhan, J., Chitta, R.K., Harwood, F.C. et al. Phosphorylation of TSC2 by PKC-δ reveals a novel signaling pathway that couples protein synthesis to mTORC1 activity. Mol Cell Biochem 456, 123–134 (2019). https://doi.org/10.1007/s11010-019-03498-8

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