Receptor tyrosine kinases (RTKs) control stem cell maintenance vs. differentiation decisions. Casitas B-lineage lymphoma (CBL) family ubiquitin ligases are negative regulators of RTKs, but their stem cell regulatory roles remain unclear. Here, we show that Lgr5+ intestinal stem cell (ISC)-specific inducible Cbl-knockout (KO) on a Cblb null mouse background (iDKO) induced rapid loss of the Lgr5 Hi ISCs with transient expansion of the Lgr5 Lo transit-amplifying population. LacZ-based lineage tracing revealed increased ISC commitment toward enterocyte and goblet cell fate at the expense of Paneth cells. Functionally, Cbl/Cblb iDKO impaired the recovery from radiation-induced intestinal epithelial injury. In vitro, Cbl/Cblb iDKO led to inability to maintain intestinal organoids. Single-cell RNA sequencing in organoids identified Akt-mTOR (mammalian target of rapamycin) pathway hyperactivation upon iDKO, and pharmacological Akt-mTOR axis inhibition rescued the iDKO defects. Our results demonstrate a requirement for Cbl/Cblb in the maintenance of ISCs by fine-tuning the Akt-mTOR axis to balance stem cell maintenance vs. commitment to differentiation.
© 2024 The Author(s).

Highly homologous E3 ubiquitin ligases, Cbl and Cbl-b, mediate ubiquitination of EGF receptor (EGFR), leading to its endocytosis and lysosomal degradation. Cbl and Cbl-b, are thought to function in a redundant manner by binding directly to phosphorylated Y1045 (pY1045) of EGFR and indirectly via the Grb2 adaptor. Unexpectedly, we found that inducible expression of Cbl or Cbl-b mutants lacking the E3 ligase activity but fully capable of EGFR binding does not significantly affect EGFR ubiquitination and endocytosis in human oral squamous cell carcinoma (HSC3) cells which endogenously express Cbl-b at a relatively high level. Each endogenous Cbl species remained associated with ligand-activated EGFR in the presence of an overexpressed counterpart species or its mutant, although Cbl-b overexpression partially decreased Cbl association with EGFR. Binding to pY1045 was the preferential mode for Cbl-b:EGFR interaction, whereas Cbl relied mainly on the Grb2-dependent mechanism. Overexpression of the E3-dead mutant of Cbl-b slowed down EGF-induced degradation of active EGFR, while this mutant and a similar mutant of Cbl did not significantly affect MAPK/ERK1/2 activity. EGF-guided chemotaxis migration of HSC3 cells was diminished by overexpression of the E3-dead Cbl-b mutant but was not significantly affected by the E3-dead Cbl mutant. By contrast, the inhibitory effect of the same Cbl mutant on the migration of OSC-19 cells expressing low Cbl-b levels was substantially stronger than that of the Cbl-b mutant. Altogether, our data demonstrate that Cbl and Cbl-b may operate independently through different modes of EGFR binding to jointly control receptor ubiquitination, endocytic trafficking, and signaling.

Deregulated epidermal growth factor receptor (EGFR) signaling is a key feature in different stages of oncogenesis. One important mechanism whereby cancer cells achieve increased and uncontrolled EGFR signaling is escaping down-modulation of the receptor. Ubiquitylation of the EGFR plays a decisive role in this process, as it regulates receptor internalization, trafficking and degradation. Deubiquitinating enzymes (DUBs) may oppose the ubiquitylation process, antagonizing or even promoting receptor degradation. Here, we use qualitative and quantitative assays to measure EGFR internalization and degradation after Ubiquitin Specific Peptidase 25 (USP25) depletion. We show that, by acting at the early steps of EGFR internalization, USP25 restrains the degradation of the EGFR by assisting in the association of the E3 ubiquitin ligase c-Cbl with EGFR, thereby modulating the amplitude of ubiquitylation on the receptor. This study establishes USP25 as a negative regulator of the EGFR down-modulation process and suggests that it is a promising target for pharmacological intervention to hamper oncogenic growth signals in tumors that depend on the EGFR.

Activation of both the DNA damage response (DDR) and transforming growth factor β (TGF-β) signaling induces growth arrest of most cell types. However, it is unclear whether the DDR activates TGF-β signaling that in turn contributes to cell growth arrest. Here, we show that in response to DNA damage, ataxia telangiectasia mutated (ATM) stabilizes the TGF-β type II receptor (TβRII) and thus enhancement of TGF-β signaling. Mechanistically, ATM phosphorylates and stabilizes c-Cbl, which promotes TβRII neddylation and prevents its ubiquitination-dependent degradation. Consistently, DNA damage enhances the interaction among ATM, c-Cbl, and TβRII. The ATM-c-Cbl-TβRII axis plays a pivotal role in intestinal regeneration after X-ray-induced DNA damage in mouse models. Therefore, ATM not only mediates the canonical DDR pathway but also activates TGF-β signaling by stabilizing TβRII. The double brake system ensures full cell-cycle arrest, allowing efficient DNA damage repair and avoiding passage of the damaged genome to the daughter cells.
Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.

To investigate the effect of ascorbic acid (AA) on hemostatic function during living donor liver transplantation (LDLT).
Blood samples from 21 LDLT recipients were taken within 30 minutes after induction and at 120 minutes after reperfusion. Rotational thromboelastography (TEG) and western blot analysis were used to analyze for fibrinolysis and functional changes in c-Cbl and Cbl-b, respectively. TEG test samples were prepared as one of three groups: C group (0.36 mL of blood), N group (0.324 mL of blood + 0.036 mL of 0.9% normal saline), and A group (0.324 mL of blood + 0.036 mL of 200 µmol/L-AA dissolved in 0.9% normal saline).
AA decreased fibrinolysis and increased clot rigidity at baseline and 120 minutes after reperfusion. Cbl-b expression was significantly increased at baseline and 120 minutes after reperfusion in the A group compared with the C and N groups. However, c-Cbl phosphorylation was most significantly decreased in the A group at baseline and 120 minutes after reperfusion.
AA can significantly decrease fibrinolysis and improve clot rigidity in LT recipients during LDLT, and functional changes in Cbl-b and c-Cbl might represent the underlying mechanism. AA may be considered for use during LDLT to decrease hyperfibrinolysis.