Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR
Hongbing Zhang, Gregor Cicchetti, Hiroaki Onda, Henry B. Koon, Kirsten Asrican, Natalia Bajraszewski, Francisca Vazquez, Christopher L. Carpenter, David J. Kwiatkowski
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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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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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.