Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR
Hongbing Zhang, … , Christopher L. Carpenter, David J. Kwiatkowski
Hongbing Zhang, … , Christopher L. Carpenter, David J. Kwiatkowski
Published October 15, 2003
Citation Information: J Clin Invest. 2003;112(8):1223-1233. https://doi.org/10.1172/JCI17222.
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Article Oncology

Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR

Abstract

Tuberous sclerosis (TSC) is a familial tumor syndrome due to mutations in TSC1 or TSC2, in which progression to malignancy is rare. Primary Tsc2–/– murine embryo fibroblast cultures display early senescence with overexpression of p21CIP1/WAF1 that is rescued by loss of TP53. Tsc2–/–TP53–/– cells, as well as tumors from Tsc2+/– mice, display an mTOR-activation signature with constitutive activation of S6K, which is reverted by treatment with rapamycin. Rapamycin also reverts a growth advantage of Tsc2–/–TP53–/– cells. Tsc1/Tsc2 does not bind directly to mTOR, however, nor does it directly influence mTOR kinase activity or cellular phosphatase activity. There is a marked reduction in Akt activation in Tsc2–/–TP53–/– and Tsc1–/– cells in response to serum and PDGF, along with a reduction in cell ruffling. PDGFRα and PDGFRβ expression is markedly reduced in both the cell lines and Tsc mouse renal cystadenomas, and ectopic expression of PDGFRβ in Tsc2-null cells restores Akt phosphorylation in response to serum, PDGF, EGF, and insulin. This activation of mTOR along with downregulation of PDGFR PI3K-Akt signaling in cells lacking Tsc1 or Tsc2 may explain why these genes are rarely involved in human cancer. This is in contrast to PTEN, which is a negative upstream regulator of this pathway.

Authors

Hongbing Zhang, Gregor Cicchetti, Hiroaki Onda, Henry B. Koon, Kirsten Asrican, Natalia Bajraszewski, Francisca Vazquez, Christopher L. Carpenter, David J. Kwiatkowski

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Figure 4

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The mTOR functional analysis and purification of a Tsc1/Tsc2 complex. (a...
The mTOR functional analysis and purification of a Tsc1/Tsc2 complex. (a) Autoradiograph from an mTOR kinase reaction. Addition of mTOR and Tsc1/Tsc2 are indicated at the top. Equivalent amounts of mTOR autokinase activity and kinase activity on 4E-BP1 are seen whenever mTOR is included. C, anti-C20 tuberin Ab; N, anti-N19 tuberin Ab; E, eluate. (b) Autoradiograph of a phosphatase assay. 4E-BP1 was phosphorylated in vitro using γ32P-ATP and then was included as a substrate to assess phosphatase activity of two TP53–/–Tsc2–/– and two TP53–/– control cell line extracts. There is no difference in the level of phosphatase activity. (c) Coomassie blue–stained gel showing successive steps in the purification of TSC1/TSC2 from brain extracts. Material bound to an anti-TSC1 affinity (H2 Ab) column, residual on the column after elution with peptide, the eluate, and the material obtained from an anti-TSC2 (C20) Ab column are shown in successive lanes. The location of 14-3-3γ is indicated by an asterisk. (d) Immunoblot analysis of Tsc1/Tsc2–binding partners. IP was performed with the indicated Ab’s (Tsc2 N19, C20) followed by immunoblotting. Tsc2 row: + indicates extract from a control TP53–/– cell line; – indicates a Tsc2–/–TP53–/– cell line.