Structure of UBE2K-Ub/E3/polyUb reveals mechanisms of K48-linked Ub chain extension.

Ubiquitin (Ub) chain types govern distinct biological processes. K48-linked polyUb chains target substrates for proteasomal degradation, but the mechanism of Ub chain synthesis remains elusive due to the transient nature of Ub handover. Here, we present the structure of a chemically trapped complex of the E2 UBE2K covalently linked to donor Ub and acceptor K48-linked di-Ub, primed for K48-linked Ub chain synthesis by a RING E3.

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.

Structural insights into ADP-ribosylation of ubiquitin by Deltex family E3 ubiquitin ligases.

Cellular cross-talk between ubiquitination and other posttranslational modifications contributes to the regulation of numerous processes. One example is ADP-ribosylation of the carboxyl terminus of ubiquitin by the E3 DTX3L/ADP-ribosyltransferase PARP9 heterodimer, but the mechanism remains elusive. Here, we show that independently of PARP9, the conserved carboxyl-terminal RING and DTC (Deltex carboxyl-terminal) domains of DTX3L and other human Deltex proteins (DTX1 to DTX4) catalyze ADP-ribosylation of ubiquitin's Gly Structural studies reveal a hitherto unknown function of the DTC domain in binding NAD Deltex RING domain recruits E2 thioesterified with ubiquitin and juxtaposes it with NAD bound to the DTC domain to facilitate ADP-ribosylation of ubiquitin.

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.

Structural basis for DNA damage-induced phosphoregulation of MDM2 RING domain.

Phosphorylation of MDM2 by ATM upon DNA damage is an important mechanism for deregulating MDM2, thereby leading to p53 activation. ATM phosphorylates multiple residues near the RING domain of MDM2, but the underlying molecular basis for deregulation remains elusive. Here we show that Ser429 phosphorylation selectively enhances the ubiquitin ligase activity of MDM2 homodimer but not MDM2-MDMX heterodimer.

Structural insights into non-covalent ubiquitin activation of the cIAP1-UbcH5B∼ubiquitin complex.

Ubiquitin (Ub)-conjugating enzymes and Ub ligases control protein degradation and regulate many cellular processes in eukaryotes. Cellular inhibitor of apoptosis protein-1 (cIAP1) plays a central role in apoptosis and tumor necrosis factor signaling. It harbors a C-terminal RING domain that homodimerizes to recruit E2∼Ub (where ∼ denotes a thioester bond) complex to catalyze Ub transfer.

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.

Structural analysis of MDM2 RING separates degradation from regulation of p53 transcription activity.

MDM2-MDMX complexes bind the p53 tumor-suppressor protein, inhibiting p53's transcriptional activity and targeting p53 for proteasomal degradation. Inhibitors that disrupt binding between p53 and MDM2 efficiently activate a p53 response, but their use in the treatment of cancers that retain wild-type p53 may be limited by on-target toxicities due to p53 activation in normal tissue. Guided by a novel crystal structure of the MDM2-MDMX-E2(UbcH5B)-ubiquitin complex, we designed MDM2 mutants that prevent E2-ubiquitin binding without altering the RING-domain structure.

Mutational activation of BRAF confers sensitivity to transforming growth factor beta inhibitors in human cancer cells.

Recent data implicate elevated transforming growth factor-β (TGFβ) signalling in BRAF inhibitor drug-resistance mechanisms, but the potential for targeting TGFβ signalling in cases of advanced melanoma has not been investigated. We show that mutant BRAFV600E confers an intrinsic dependence on TGFβ/TGFβ receptor 1 (TGFBR1) signalling for clonogenicity of murine melanocytes. Pharmacological inhibition of the TGFBR1 blocked the clonogenicity of human mutant BRAF melanoma cells through SMAD4-independent inhibition of mitosis, and also inhibited metastasis in xenografted zebrafish.

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.

Activation of a primed RING E3-E2-ubiquitin complex by non-covalent ubiquitin.

RING ubiquitin ligases (E3) recruit ubiquitin-conjugate enzymes (E2) charged with ubiquitin (Ub) to catalyze ubiquitination. Non-covalent Ub binding to the backside of certain E2s promotes processive polyUb formation, but the mechanism remains elusive. Here, we show that backside bound Ub (Ub(B)) enhances both RING-independent and RING-dependent UbcH5B-catalyzed donor Ub (Ub(D)) transfer, but with a more prominent effect in RING-dependent transfer.

Essentiality of a non-RING element in priming donor ubiquitin for catalysis by a monomeric E3.

RING E3 ligases catalyze the transfer of ubiquitin (Ub) from E2 ubiquitin-conjugating enzyme thioesterified with Ub (E2~Ub) to substrate. For RING E3 dimers, the RING domain of one subunit and tail of the second cooperate to prime Ub, but how this is accomplished by monomeric RING E3s in the absence of a tail-like component is currently unknown. Here, we present a crystal structure of a monomeric RING E3, Tyr363-phosphorylated human CBL-B, bound to a stabilized Ub-linked E2, revealing a similar mechanism in activating E2~Ub.

BIRC7-E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer.

Certain RING ubiquitin ligases (E3s) dimerize to facilitate ubiquitin (Ub) transfer from ubiquitin-conjugating enzyme (E2) to substrate, but structural evidence on how this process promotes Ub transfer is lacking. Here we report the structure of the human dimeric RING domain from BIRC7 in complex with the E2 UbcH5B covalently linked to Ub (UbcH5B∼Ub). The structure reveals extensive noncovalent donor Ub interactions with UbcH5B and both subunits of the RING domain dimer that stabilize the globular body and C-terminal tail of Ub.

Structural basis for autoinhibition and phosphorylation-dependent activation of c-Cbl.

Cbls are RING ubiquitin ligases that attenuate receptor tyrosine kinase (RTK) signal transduction. Cbl ubiquitination activity is stimulated by phosphorylation of a linker helix region (LHR) tyrosine residue. To elucidate the mechanism of activation, we determined the structures of human CBL, a CBL-substrate peptide complex and a phosphorylated-Tyr371-CBL-E2-substrate peptide complex, and we compared them with the known structure of a CBL-E2-substrate peptide complex.

Deep evolutionary conservation of an intramolecular protein kinase activation mechanism.

DYRK-family kinases employ an intramolecular mechanism to autophosphorylate a critical tyrosine residue in the activation loop. Once phosphorylated, DYRKs lose tyrosine kinase activity and function as serine/threonine kinases. DYRKs have been characterized in organisms from yeast to human; however, all entities belong to the Unikont supergroup, only one of five eukaryotic supergroups.

Characterization of a domain that transiently converts class 2 DYRKs into intramolecular tyrosine kinases.

Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) autophosphorylate an essential tyrosine residue in their activation loop and phosphorylate their substrates on serine and threonine residues. Phosphorylation of the activation loop tyrosine occurs intramolecularly, is mediated by a short-lived transitional intermediate during protein maturation, and is required for functional serine-threonine kinase activity of DYRKs. The DYRK family is separated into two subclasses.

A chaperone-dependent GSK3beta transitional intermediate mediates activation-loop autophosphorylation.

Glycogen synthase kinase 3 (GSK3), a key component of the insulin and wnt signaling pathways, is unusual, as it is constitutively active and is inhibited in response to upstream signals. Kinase activity is thought to be increased by intramolecular phosphorylation of a tyrosine in the activation loop (Y216 in GSK3beta), whose timing and mechanism is undefined. We show that GSK3beta autophosphorylates Y216 as a chaperone-dependent transitional intermediate possessing intramolecular tyrosine kinase activity and displaying different sensitivity to small-molecule inhibitors compared to mature GSK3beta.

dDYRK2 and Minibrain interact with the chromatin remodelling factors SNR1 and TRX.

The DYRKs (dual specificity tyrosine phosphorylation-regulated kinases) are a conserved family of protein kinases that autophosphorylate a tyrosine residue in their activation loop by an intra-molecular mechanism and phosphorylate exogenous substrates on serine/threonine residues. Little is known about the identity of true substrates for DYRK family members and their binding partners. To address this question, we used full-length dDYRK2 (Drosophila DYRK2) as bait in a yeast two-hybrid screen of a Drosophila embryo cDNA library.

Activation-loop autophosphorylation is mediated by a novel transitional intermediate form of DYRKs.

Autophosphorylation of a critical residue in the activation loop of several protein kinases is an essential maturation event required for full enzyme activity. However, the molecular mechanism by which this happens is unknown. We addressed this question for two dual-specificity tyrosine-phosphorylation-regulated protein kinases (DYRKs), as they autophosphorylate their activation loop on an essential tyrosine but phosphorylate their substrates on serine and threonine.

dDYRK2: a novel dual-specificity tyrosine-phosphorylation-regulated kinase in Drosophila.

Dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs) are an emerging family of protein kinases that have been identified in all eukaryotic organisms examined to date. DYRK family members are involved in regulating key developmental and cellular processes such as neurogenesis, cell proliferation, cytokinesis and cellular differentiation. Two distinct subgroups exist, nuclear and cytosolic.

Down-regulation of MHC class I by bovine papillomavirus E5 oncoproteins.

The papillomavirus E5 protein is localized in the endoplasmic reticulum (ER) and Golgi apparatus (GA) of the host cell. Transformed bovine fibroblasts expressing bovine papillomavirus (BPV) E5 are highly vacuolated and have a much enlarged, distorted and fragmented GA. Major histocompatibility complex class I (MHC I) is processed and transported to the cell surface through the GA.