Failure to ubiquitinate c-Met leads to hyperactivation of mTOR signaling in a mouse model of autosomal dominant polycystic kidney disease
Shan Qin, … , Jing Zhou, Jordan A. Kreidberg
Shan Qin, … , Jing Zhou, Jordan A. Kreidberg
Published September 13, 2010
Citation Information: J Clin Invest. 2010;120(10):3617-3628. https://doi.org/10.1172/JCI41531.
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Research Article

Failure to ubiquitinate c-Met leads to hyperactivation of mTOR signaling in a mouse model of autosomal dominant polycystic kidney disease

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder that is caused by mutations at two loci, polycystin 1 (PKD1) and polycystin 2 (PKD2). It is characterized by the formation of multiple cysts in the kidneys that can lead to chronic renal failure. Previous studies have suggested a role for hyperactivation of mammalian target of rapamycin (mTOR) in cystogenesis, but the etiology of mTOR hyperactivation has not been fully elucidated. In this report we have shown that mTOR is hyperactivated in Pkd1-null mouse cells due to failure of the HGF receptor c-Met to be properly ubiquitinated and subsequently degraded after stimulation by HGF. In Pkd1-null cells, Casitas B-lineage lymphoma (c-Cbl), an E3-ubiquitin ligase for c-Met, was sequestered in the Golgi apparatus with α3β1 integrin, resulting in the inability to ubiquitinate c-Met. Treatment of mouse Pkd1-null cystic kidneys in organ culture with a c-Met pharmacological inhibitor resulted in inhibition of mTOR activity and blocked cystogenesis in this mouse model of ADPKD. We therefore suggest that blockade of c-Met is a potential novel therapeutic approach to the treatment of ADPKD.

Authors

Shan Qin, Mary Taglienti, Surya M. Nauli, Leah Contrino, Ayumi Takakura, Jing Zhou, Jordan A. Kreidberg

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Figure 2

Increased expression and impaired degradation of c-Met in Pkd1–/– cells.

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Increased expression and impaired degradation of c-Met in Pkd1–/– cells....
(A) Western blot for phospho–c-Met (Tyr1234/1235) and c-Met in Pkd1+/+ and Pkd1–/– E17.5 kidneys. Quantification is shown on the right. (B) Western blot of c-Met in Pkd1+/+, Pkd1–/–, Itga3+/+, and Itga3–/– cells, with or without HGF stimulation (50 ng/ml HGF for 30 minutes). Densitometric quantification is shown on the right. c-Met was more abundant before stimulation and failed to be degraded in Pkd1–/– cells. Degradation was also reduced in Itga3–/– cells. GAPDH is shown as a loading control. (C) Western blot of c-Met in extract of human non-cystic and PKD kidneys. Higher levels of c-Met and an additional higher-molecular-weight species are present in the PKD sample. Densitometric quantification is shown on right, each bar represents the average of the 3 samples on left. (D) Western blot for c-Met after shRNA knockdown of Pkd1 (KD4 cells), also showing increased levels of c-Met in nonstimulated cells and decreased degradation. (E and F) Reverse transcription quantitative PCR (RT-qPCR) for c-Met in Pkd1+/+ and Pkd1–/– cells (E), or Pkd1+/+ cells and KD4 cells (F). In E and F, the level of c-Met mRNA in Pkd1–/– cells is shown relative to the amount in Pkd1+/+ or Ctrl cells. 18S RNA was used as an input control (D) and for normalization of RT-qPCR (E and F).