Casitas B-lineage lymphoma linker helix mutations found in myeloproliferative neoplasms affect conformation

Background: Casitas B-lineage lymphoma (Cbl or c-Cbl) is a RING ubiquitin ligase that negatively regulates protein tyrosine kinase (PTK) signalling. Phosphorylation of a conserved residue (Tyr371) on the linker helix region (LHR) between the substrate-binding and RING domains is required to ubiquitinate PTKs, thereby flagging them for degradation. This conserved Tyr is a mutational hotspot in myeloproliferative neoplasms. Previous studies have revealed that select point mutations in Tyr371 can potentiate transformation in cells and mice but not all possible mutations do so. To trigger oncogenic potential, Cbl Tyr371 mutants must perturb the LHR-substrate-binding domain interaction and eliminate PTK ubiquitination. Although structures of native and pTyr371-Cbl are available, they do not reveal how Tyr371 mutations affect Cbl’s conformation. Here, we investigate how Tyr371 mutations affect Cbl’s conformation in solution and how this relates to Cbl’s ability to potentiate transformation in cells. Results: To explore how Tyr371 mutations affect Cbl’s properties, we used surface plasmon resonance to measure Cbl mutant binding affinities for E2 conjugated with ubiquitin (E2–Ub), small angle X-ray scattering studies to investigate Cbl mutant conformation in solution and focus formation assays to assay Cbl mutant transformation potential in cells. Cbl Tyr371 mutants enhance E2–Ub binding and cause Cbl to adopt extended conformations in solution. LHR flexibility, RING domain accessibility and transformation potential are associated with the extent of LHR-substrate-binding domain perturbation affected by the chemical nature of the mutation. More disruptive mutants like Cbl Y371D or Y371S are more extended and the RING domain is more accessible, whereas Cbl Y371F mimics native Cbl in solution. Correspondingly, the only Tyr371 mutants that potentiate transformation in cells are those that perturb the LHR-substrate-binding domain interaction. Conclusions: c-Cbl’s LHR mutations are only oncogenic when they disrupt the native state and fail to ubiquitinate PTKs. These findings provide new insights into how LHR mutations deregulate c-Cbl

A general strategy for discovery of inhibitors and activators of RING and U-box E3 ligases with ubiquitin variants

RING and U-box E3 ubiquitin ligases regulate diverse eukaryotic processes and have been implicated in numerous diseases, but targeting these enzymes remains a major challenge. We report the development of three ubiquitin variants (UbVs), each binding selectively to the RING or U-box domain of a distinct E3 ligase: monomeric UBE4B, phosphorylated active CBL, or dimeric XIAP. Structural and biochemical analyses revealed that UbVs specifically inhibited the activity of UBE4B or phosphorylated CBL by blocking the E2∼Ub binding site. Surprisingly, the UbV selective for dimeric XIAP formed a dimer to stimulate E3 activity by stabilizing the closed E2∼Ub conformation. We further verified the inhibitory and stimulatory functions of UbVs in cells. Our work provides a general strategy to inhibit or activate RING/U-box E3 ligases and provides a resource for the research community to modulate these enzymes

DELTEX2 C-terminal domain recognizes and recruits ADP-ribosylated proteins for ubiquitination

Cross-talk between ubiquitination and ADP-ribosylation regulates spatiotemporal recruitment of key players in many signaling pathways. The DELTEX family ubiquitin ligases (DTX1 to DTX4 and DTX3L) are characterized by a RING domain followed by a C-terminal domain (DTC) of hitherto unknown function. Here, we use two label-free mass spectrometry techniques to investigate the interactome and ubiquitinated substrates of human DTX2 and identify a large proportion of proteins associated with the DNA damage repair pathway. We show that DTX2-catalyzed ubiquitination of these interacting proteins requires PARP1/2-mediated ADP-ribosylation and depends on the DTC domain. Using a combination of structural, biochemical, and cell-based techniques, we show that the DTX2 DTC domain harbors an ADP-ribose–binding pocket and recruits poly-ADP-ribose (PAR)–modified proteins for ubiquitination. This PAR-binding property of DTC domain is conserved across the DELTEX family E3s. These findings uncover a new ADP-ribose–binding domain that facilitates PAR-dependent ubiquitination

Post translational Modulations of PTEN and Their Implication in Cancer

PTEN since its identification has been in large associated with the control of human malignancies. The dominance of PTEN over the PI3K signaling cascade is mainly responsible for rendering it the tumor suppressive role that results in the control of a wide array of physiological processes; growth, apoptosis, proliferation and migration. However, in recent years, PTEN has expanded its TENtacles in regulating processes like stem cell population maintenance, genomic stability regulation and activation of ovarian follicle. Meanwhile, PTEN protein phosphatase activity has gained significant importance and is even capable of inhibiting cell migration irrespective of the Akt pathway. As a protein phosphatase, it targets among others SRC kinases, CREB in the nucleus thereby regulating migration of glioma cells. Crucial for PTEN to be able to render its functions properly is the ability to translocate to different sub-cellular compartments of the cell and even out of it through exosomes while retaining its phosphatase activity in the cells where it is internalized. Justifiably, PTEN has fast attained the stature of one of the prime regulators of the cell and therefore it needs to be subjected to intricate surveillance. Although, PTEN like other major players of the cell shows regulation at the transcriptional level as well as through epigenetic silencing, post-translational modifications of PTEN and the associated localization based alterations in its activities have been the focal point of research on PTEN regulatory events. There have been reports that suggest for negligible PTEN expression although the transcript levels seemed to be good enough. Thus, this study aims to delineate the post-translational modulations of PTEN and their implication in cancer. The first two parts of the work provide insights into the post-translational regulation of PTEN mainly by ubiquitination and ubiquitination-phosphorylation crosstalk. While in the third part, a strategy to stabilize PTEN has been established through an exosomes-mediated delivery of the intrinsic C-terminus domain of PTEN that causes tumor inhibition. In the first part of the study, we show that CHIP, the chaperone-associated E3 ligase induces ubiquitination and proteasomal degradation of PTEN as well as regulates the turnover of the protein. It was apparent from our findings that PTEN transiently associates with the molecular chaperones and thereby gets diverted to the degradation pathway through its interaction with CHIP. The TPR domain of CHIP and parts of the N-terminal domain of PTEN are required for their interaction. Overexpression of CHIP leads to elevated ubiquitination and a shortened half-life of endogenous PTEN. On the other hand, depletion of endogenous CHIP stabilizes PTEN. CHIP is also shown to regulate PTENdependent transcription presumably through its downregulation. PTEN shared an inverse correlation with CHIP in human prostate cancer patient samples thereby triggering the prospects of a more complex mode of PTEN regulation in cancer.PTEN mutation is a frequent feature across a plethora of human cancers, the hot-spot being its C-terminus (CT) regulatory domain resulting in a much diminished level of protein expression. In the second part of the work, the presence of C-terminus mutations was confirmed through sequencing of different human tumor samples. CKII-mediated phosphorylation of PTEN at these sites makes it a loopy structure competing with the E3 ligases for binding to its lipid anchoring C2 domain. Accordingly, it was found that PTEN-CT expressing stable cell lines could inhibit tumorigenesis in syngenic breast tumor mice models. The third part shows the strategy to design a novel exosome-mediated delivery of the intrinsic PTEN domain, PTEN-CT into different cancer cells and observed reduced proliferation, migration and colony forming ability. The delivery of exosome containing PTEN-CT to breast tumor mice model was found to result in significant regression in tumor size with the tumor sections showing increased apoptosis. Here, we also report for the first time an active PTEN when its C2 domain is bound by PTEN-CT, probably rendering its antitumorigenic activities through the protein phosphatase activity. Therefore, therapeutic interventions that focuses on PTEN E3 ligase inhibition through exosome-mediated PTENCT delivery can be a probable route in the treatment of cancers with low PTEN expression. Altogether, the findings emanating from this research work could be useful in designing therapeutic strategies against cancers showing low or negligible PTEN expression

E3 ligase-inactivation rewires CBL interactome to elicit oncogenesis by hijacking RTK–CBL–CIN85 axis

Casitas B-lineage lymphoma (CBL) is a ubiquitin ligase (E3) that becomes activated upon Tyr371-phosphorylation and targets receptor protein tyrosine kinases for ubiquitin-mediated degradation. Deregulation of CBL and its E3 activity is observed in myeloproliferative neoplasms and other cancers, including breast, colon, and prostate cancer. Here, we explore the oncogenic mechanism of E3-inactive CBL mutants identified in myeloproliferative neoplasms. We show that these mutants bind strongly to CIN85 under normal growth conditions and alter the CBL interactome. Lack of E3 activity deregulates CIN85 endosomal trafficking, leading to an altered transcriptome that amplifies signaling events to promote oncogenesis. Disruption of CBL mutant interactions with EGFR or CIN85 reduces oncogenic transformation. Given the importance of the CBL–CIN85 interaction in breast cancers, we examined the expression levels of CIN85, CBL, and the status of Tyr371-phosphorylated CBL (pCBL) in human breast cancer tissue microarrays. Interestingly, pCBL shows an inverse correlation with both CIN85 and CBL, suggesting that high expression of inactivated CBL could coordinate with CIN85 for breast cancer progression. Inhibition of the CBL–CIN85 interaction with a proline-rich peptide of CBL that binds CIN85 reduced the proliferation of MDA-MB-231 cells. Together, these results provide a rationale for exploring the potential of targeting the EGFR–CBL–CIN85 axis in CBL-inactivated mutant cancers