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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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 3

c-Met is localized at the plasma membrane in WT, Pkd1–/–, and Pkd1-knockdown cells.

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c-Met is localized at the plasma membrane in WT, Pkd1–/–, and Pkd1-knock...
Cells were labeled with membrane-impermeable sulfo-NHS-biotin; after extraction, labeled proteins were pulled down by avidin beads. Residual c-Met, representing unlabeled protein, was detected by c-Met immunoprecipitation. (A and B) Most c-Met is localized at the plasma membrane in WT, Pkd1–/–, and KD4 cells. Little c-Met is detected in the post-immunoprecipitation fraction (after IP). (C and D) Failure to ubiquitinate c-Met in Pkd1–/– or KD4 cells. Pkd1–/– and Pkd1+/+ cells (C) or KD4 and control cells (D) were stimulated with HGF, and cell lysates were immunoprecipitated with c-Met antibody and blotted with anti-ubiquitin. Nonstimulated cells showed little ubiquitination of c-Met. The immunoprecipitation was validated by a re-blot for c-Met (lower panels). (E) c-Cbl phosphorylation after HGF stimulation is decreased in Pkd1–/– cells. Control and mutant cells were incubated with HGF (50 ng/ml, 10 minutes). Phospho–c-Cbl and total c-Cbl were detected by Western blot. c-Cbl phosphorylation after HGF stimulation is weaker in both Pkd1–/– and Itga3–/– cells, compared with their WT controls. (F) c-Cbl binds α3β1 integrin in both Pkd1+/+ and Pkd1–/– cells. Pairs of lanes are designated as starting lysate, anti–α3 integrin or non-immune IgG control immunoprecipitated material, and residual non-immunoprecipitated material. The membrane was reblotted with anti–α3 integrin antibody to validate the immunoprecipitation. c-Cbl partially coimmunoprecipitated with α3β1 integrin in Pkd1+/+ and completely in Pkd1–/– cells. (G) α3β1 integrin and c-Cbl are not localized to the membrane in Pkd1–/– cells. Cells were labeled with sulfo-NHS-biotin, labeled proteins were pulled down with avidin-coupled beads, and nonlabeled protein immunoprecipitated with anti–α3 integrin. Western blots for α3 integrin (upper panel) and c-Cbl (lower panel). In Pkd1–/– cells, little α3β1 integrin or c-Cbl is localized at the plasma membrane.