Ubiquitination and degradation of the hominoid- specific oncoprotein TBC1D3 Is mediated by CUL7 E3 ligase

Expression of the hominoid-specific TBC1D3 oncoprotein enhances growth factor receptor signaling and subsequently promotes cellular proliferation and survival. Here we report that TBC1D3 is degraded in response to growth factor signaling, suggesting that TBC1D3 expression is regulated by a growth factor-driven negative feedback loop. To gain a better understanding of how TBC1D3 is regulated, we studied the effects of growth factor receptor signaling on TBC1D3 post-translational processing and turnover. Using a yeast two-hybrid screen, we identified CUL7, the scaffolding subunit of the CUL7 E3 ligase complex, as a TBC1D3-interacting protein. We show that CUL7 E3 ligase ubiquitinates TBC1D3 in response to serum stimulation. Moreover, TBC1D3 recruits F-box 8 (Fbw8), the substrate recognition domain of CUL7 E3 ligase, in pull-down experiments and in an in vitro assay. Importantly, alkaline phosphatase treatment of TBC1D3 suppresses its ability to recruit Fbw8, indicating that TBC1D3 phosphorylation is critical for its ubiquitination and degradation. We conclude that serum- and growth factor-stimulated TBC1D3 ubiquitination and degradation are regulated by its interaction with CUL7-Fbw8

CD36 maintains the gastric mucosa and associates with gastric disease

The gastric epithelium is often exposed to injurious elements and failure of appropriate healing predisposes to ulcers, hemorrhage, and ultimately cancer. We examined the gastric function of CD36, a protein linked to disease and homeostasis. We used the tamoxifen model of gastric injury in mice null for Cd36 (Cd3

CD36 maintains the gastric mucosa and associates with gastric disease.

The gastric epithelium is often exposed to injurious elements and failure of appropriate healing predisposes to ulcers, hemorrhage, and ultimately cancer. We examined the gastric function of CD36, a protein linked to disease and homeostasis. We used the tamoxifen model of gastric injury in mice null for Cd36 (Cd36-/-), with Cd36 deletion in parietal cells (PC-Cd36-/-) or in endothelial cells (EC-Cd36-/-). CD36 expresses on corpus ECs, on PC basolateral membranes, and in gastrin and ghrelin cells. Stomachs of Cd36-/- mice have altered gland organization and secretion, more fibronectin, and inflammation. Tissue respiration and mitochondrial efficiency are reduced. Phospholipids increased and triglycerides decreased. Mucosal repair after injury is impaired in Cd36-/- and EC-Cd36-/-, not in PC-Cd36-/- mice, and is due to defect of progenitor differentiation to PCs, not of progenitor proliferation or mature PC dysfunction. Relevance to humans is explored in the Vanderbilt BioVu using PrediXcan that links genetically-determined gene expression to clinical phenotypes, which associates low CD36 mRNA with gastritis, gastric ulcer, and gastro-intestinal hemorrhage. A CD36 variant predicted to disrupt an enhancer site associates (p < 10-17) to death from gastro-intestinal hemorrhage in the UK Biobank. The findings support role of CD36 in gastric tissue repair, and its deletion associated with chronic diseases that can predispose to malignancy

Metabolically normal obese people are protected from adverse effects following weight gain

BACKGROUND. Obesity is associated with insulin resistance and increased intrahepatic triglyceride (IHTG) content, both of which are key risk factors for diabetes and cardiovascular disease. However, a subset of obese people does not develop these metabolic complications. Here, we tested the hypothesis that people defined by IHTG content and insulin sensitivity as “metabolically normal obese” (MNO), but not those defined as “metabolically abnormal obese” (MAO), are protected from the adverse metabolic effects of weight gain. METHODS. Body composition, multiorgan insulin sensitivity, VLDL apolipoprotein B100 (apoB100) kinetics, and global transcriptional profile in adipose tissue were evaluated before and after moderate (~6%) weight gain in MNO (n = 12) and MAO (n = 8) subjects with a mean BMI of 36 ± 4 kg/m(2) who were matched for BMI and fat mass. RESULTS. Although the increase in body weight and fat mass was the same in both groups, hepatic, skeletal muscle, and adipose tissue insulin sensitivity deteriorated, and VLDL apoB100 concentrations and secretion rates increased in MAO, but not MNO, subjects. Moreover, biological pathways and genes associated with adipose tissue lipogenesis increased in MNO, but not MAO, subjects. CONCLUSIONS. These data demonstrate that MNO people are resistant, whereas MAO people are predisposed, to the adverse metabolic effects of moderate weight gain and that increased adipose tissue capacity for lipogenesis might help protect MNO people from weight gain–induced metabolic dysfunction. TRIAL REGISTRATION. ClinicalTrials.gov NCT01184170. FUNDING. This work was supported by NIH grants UL1 RR024992 (Clinical Translational Science Award), DK 56341 (Nutrition and Obesity Research Center), DK 37948 and DK 20579 (Diabetes Center Grant), and UL1 TR000450 (KL2 Award); a Central Society for Clinical and Translational Research Early Career Development Award; and by grants from the Longer Life Foundation and the Kilo Foundation

TBC1D3, a Hominoid-Specific Gene, Delays IRS-1 Degradation and Promotes Insulin Signaling by Modulating p70 S6 Kinase Activity

Insulin/IGF-1 signaling plays a pivotal role in the regulation of cellular homeostasis through its control of glucose metabolism as well as due to its effects on cell proliferation. Aberrant regulation of insulin signaling has been repeatedly implicated in uncontrolled cell growth and malignant transformations. TBC1D3 is a hominoid specific gene previously identified as an oncogene in breast and prostate cancers. Our efforts to identify the molecular mechanisms of TBC1D3-induced oncogenesis revealed the role of TBC1D3 in insulin/IGF-1 signaling pathway. We document here that TBC1D3 intensifies insulin/IGF-1-induced signal transduction through intricate, yet elegant fine-tuning of signaling mechanisms. We show that TBC1D3 expression substantially delayed ubiquitination and degradation of insulin receptor substrate-1 (IRS-1). This effect is achieved through suppression of serine phosphorylation at S636/639, S307 and S312 of IRS-1, which are key phosphorylation sites required for IRS-1 degradation. Furthermore, we report that the effect of TBC1D3 on IRS-1:S636/639 phosphorylation is mediated through TBC1D3-induced activation of protein phosphatase 2A (PP2A), followed by suppression of T389 phosphorylation on p70 S6 kinase (S6K). TBC1D3 specifically interacts with PP2A regulatory subunit B56γ, indicating that TBC1D3 and PP2A B56γ operate jointly to promote S6K:T389 dephosphorylation. These findings suggest that TBC1D3 plays an unanticipated and potentially unique role in the fine-tuning of insulin/IGF-1 signaling, while providing novel insights into the regulation of tumorigenesis by a hominoid-specific protein

TBC1D3 reduces S6K activation, but does not affect mTOR-C1 pathway.

<p>(A) HepG2 cells transfected with myc-TBC1D3 or empty vector were serum-starved, and stimulated with insulin (10 nM) for 30 min. Phosphorylation and protein levels of S6K were analyzed by Western blotting. (<i>Right panel</i>) Quantification data of S6K:T389 phosphorylation normalized to S6K protein levels (** <i>p</i><0.01). (B) HepG2 cells transfected with myc-TBC1D3 or empty vector were serum-starved, pre-treated with Rapamycin (50 nM) for 2 h, and stimulated with insulin (10 nM) for 30 min. Phosphorylation and protein levels of 4EBP1 and S6K were analyzed by Western blotting. (<i>Right panel</i>) Quantification data of 4EBP1 phosphorylation normalized to GAPDH protein levels. The data are presented as means ± SD of three independent experiments.</p

TBC1D3 expression blocks IRS-1 degradation.

<p>IRS-1 degradation is delayed in cells expressing TBC1D3. DU145 cells transfected with myc-TBC1D3 or empty vector were serum-starved, and stimulated with insulin (10 nM) for the indicated times. Protein levels of IRS-1 were analyzed by Western blotting. (<i>Right panel</i>) Quantification data of IRS-1 normalized to GAPDH protein levels. The value of IRS-1 at time 0 was set at 1.0. The data are presented as means ± SD of three independent experiments.</p