Abstract
Modification of the transforming growth factor β (TGF-β) signaling components by (de)ubiquitination is emerging as a key regulatory mechanism that controls cell signaling responses in health and disease. Here, we show that the deubiquitinating enzyme UBH-1 in Caenorhabditis elegans and its human homolog, ubiquitin C-terminal hydrolase-L1 (UCH-L1), stimulate DAF-7/TGF-β signaling, suggesting that this mode of regulation of TGF-β signaling is conserved across animal species. The dauer larva-constitutive C. elegans phenotype caused by defective DAF-7/TGF-β signaling was enhanced and suppressed, respectively, by ubh-1 deletion and overexpression in the loss-of-function genetic backgrounds of daf7, daf-1/TGF-βRI, and daf4/R-SMAD, but not of daf-8/R-SMAD. This suggested that UBH-1 may stimulate DAF-7/TGF-β signaling via DAF-8/R-SMAD. Therefore, we investigated the effect of UCH-L1 on TGF-β signaling via its intracellular effectors, i.e. SMAD2 and SMAD3, in mammalian cells. Overexpression of UCH-L1, but not of UCH-L3 (the other human homolog of UBH1) or of the catalytic mutant UCH-L1C90A, enhanced TGF-β/SMAD-induced transcriptional activity, indicating that the deubiquitination activity of UCH-L1 is indispensable for enhancing TGF-β/SMAD signaling. We also found that UCH-L1 interacts, deubiquitinates, and stabilizes SMAD2 and SMAD3. Under hypoxia, UCH-L1 expression increased and TGF-β/SMAD signaling was potentiated in the A549 human lung adenocarcinoma cell line. Notably, UCH-L1-deficient A549 cells were impaired in tumorigenesis, and, unlike WT UCH-L1, a UCH-L1 variant lacking deubiquitinating activity was unable to restore tumorigenesis in these cells. These results indicate that UCH-L1 activity supports DAF-7/TGF-β signaling and suggest that UCH-L1's deubiquitination activity is a potential therapeutic target for managing lung cancer.
Highlights
The ubiquitin (Ub) system is involved in numerous cellular processes including protein quality control, cell proliferation, apoptosis, signal transduction, and membrane protein internalization [1, 2]
We found that C. elegans UBH-1 and its mammalian homolog ubiquitin C-terminal hydrolase-L1 (UCH-L1), but not UCH-L3, enhance DAF-7/transforming growth factor b (TGF-b) signaling by binding to TGF-b–regulated RSmads to promote their deubiquitination and increase stability
Western blotting analysis of the tumor tissue showed a decrease in Smad2, but not in Smad3, protein (Fig. S7), to our previous findings in in vitro. These results suggest that UCH-L1, HIF-1a, and TGF-b signaling cooperate in tumorigenesis
Summary
The ubiquitin (Ub) system is involved in numerous cellular processes including protein quality control, cell proliferation, apoptosis, signal transduction, and membrane protein internalization [1, 2]. The deubiquitinase UBH1/UCH-L1 enhances DAF7/TGF-b signaling neurons known to express daf-7. UCH-L1 expression is up-regulated in hypoxia and enhances TGF-b/Smad signaling. The daf-14 gene encodes a Smad-related protein that is predicted to function as a transducer of the DAF-7/TGF-b–mediated signal that negatively regulates dauer formation and promotes reproductive growth [19, 21].
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