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// Katsuyuki Nagashima 1, 2, 6 , Hidefumi Fukushima 1 , Kouhei Shimizu 3 , Aya Yamada 4 , Masumi Hidaka 2 , Hisashi Hasumi 5 , Tetsuro Ikebe 6 , Satoshi Fukumoto 1, 4 , Koji Okabe 2 , Hiroyuki Inuzuka 1, 3 1 Center for Advanced Stem Cell and Regenerative Research, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan 2 Department of Physiological Sciences and Molecular Biology, Fukuoka Dental College, Fukuoka 814-0193, Japan 3 Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA 4 Division of Pediatric Dentistry, Department of Oral Health and Development Sciences, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan 5 Department of Urology and Molecular Genetics, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan 6 Department of Oral and Maxillofacial Surgery, Fukuoka Dental College, Fukuoka 814-0193, Japan Correspondence to: Hiroyuki Inuzuka, email: hinuzuka@bidmc.harvard.edu Hidefumi Fukushima, email: hidefumi.fukushima.b7@tohoku.ac.jp Keywords: FNIP, FLCN, tumor suppressor, β-TRCP, renal cancer Received: October 11, 2016 Accepted: November 22, 2016 Published: December 25, 2016 ABSTRACT Folliculin-interacting protein 1 and 2 (FNIP1 and FNIP2) play critical roles in preventing renal malignancy through their association with the tumor suppressor FLCN. Mutations in FLCN are associated with Birt-Hogg-Dube (BHD) syndrome, a rare disorder with increased risk of renal cancer. Recent studies indicated that FNIP1/FNIP2 double knockout mice display enlarged polycystic kidneys and renal carcinoma, which phenocopies FLCN knockout mice, suggesting that these two proteins function together to suppress renal cancer. However, the molecular mechanism functionally linking FNIP1/FNIP2 and FLCN remains largely elusive. Here, we demonstrated that FNIP2 protein is unstable and subjected to proteasome-dependent degradation via β-TRCP and Casein Kinase 1 (CK1)-directed ubiquitination in a nutrition-dependent manner. Degradation of FNIP2 leads to lysosomal dissociation of FLCN and subsequent lysosomal association of mTOR, which in turn promotes the proliferation of renal cancer cells. These results indicate that SCF β-TRCP negatively regulates the FLCN complex by promoting FNIP degradation and provide molecular insight into the pathogenesis of BHD-associated renal cancer.

Hajdu-Cheney syndrome (HCS), a rare autosomal disorder caused by heterozygous mutations in NOTCH2, is clinically characterized by acro-osteolysis, severe osteoporosis, short stature, neurological symptoms, cardiovascular defects, and polycystic kidneys. Recent studies identified that aberrant NOTCH2 signaling and consequent osteoclast hyperactivity are closely associated with the bone-related disorder pathogenesis, but the exact molecular mechanisms remain unclear. Here, we demonstrate that sustained osteoclast activity is largely due to accumulation of NOTCH2 carrying a truncated C terminus that escapes FBW7-mediated ubiquitination and degradation. Mice with osteoclast-specific Fbw7 ablation revealed osteoporotic phenotypes reminiscent of HCS, due to elevated Notch2 signaling. Importantly, administration of Notch inhibitors in Fbw7 conditional knockout mice alleviated progressive bone resorption. These findings highlight the molecular basis of HCS pathogenesis and provide clinical insights into potential targeted therapeutic strategies for skeletal disorders associated with the aberrant FBW7/NOTCH2 pathway as observed in patients with HCS.

The SCFβ-TRCP E3 ubiquitin ligase complex plays pivotal roles in normal cellular physiology and in pathophysiological conditions. Identification of β-transducin repeat-containing protein (β-TRCP) substrates is therefore critical to understand SCFβ-TRCP biology and function. Here, we used a β-TRCP-phosphodegron-motif specific antibody in a β-TRCP substrate screen coupled with tandem mass spectrometry and identified multiple β-TRCP substrates. One of these substrates was Lipin1, an enzyme and suppressor of the family of sterol regulatory element-binding protein (SREBP) transcription factors, which activate genes encoding lipogenic factors. We showed that SCFβ-TRCP specifically interacted with and promoted the polyubiquitination of Lipin1 in a manner that required phosphorylation of Lipin1 by mechanistic target of rapamycin 1 (mTORC1) and casein kinase I (CKI). β-TRCP depletion in HepG2 hepatocellular carcinoma cells resulted in increased Lipin1 protein abundance, suppression of SREBP-dependent gene expression, and attenuation of triglyceride synthesis. Moreover, β-TRCP1 knockout mice showed increased Lipin1 protein abundance and were protected from hepatic steatosis induced by a high-fat diet. Together, these data reveal a critical physiological function of β-TRCP in regulating hepatic lipid metabolic homeostasis in part through modulating Lipin1 stability.