Journal of Molecular Biology
E2–c-Cbl Recognition Is Necessary but not Sufficient for Ubiquitination Activity
Introduction
Posttranslational modification by ubiquitin is a major mechanism for regulating protein function in eukaryotes. The general enzymatic cascade of this pathway comprises three enzymes known as E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin-protein ligase enzyme). The first step in this cascade is the ATP-dependent formation of a thioester bond between ubiquitin and E1, after which ubiquitin is transferred to the catalytic cysteine of an E2 enzyme forming an E2∼Ub thioester. Next, the E3 catalyzes the transfer of the ubiquitin from the E2 to a lysine residue of the substrate, with which it forms an isopeptide bond. While HECT (homologous to the E6AP C-terminus) domain E3 ligases mediate this step by formation of a HECT-ubiquitin thioester intermediate, RING (really interesting new gene) domain E3 ligases facilitate a direct transfer of ubiquitin from E2 to the substrate via noncovalent interactions with the E2∼Ub and the substate.1 In yeast, the substrate can be tagged by a single ubiquitin or ubiquitin chains, which can vary in length and linkage specificity. The functions of chains linked through K48 and K63 of ubiquitin have been established. The K48-linked chains are shown to target proteins for degradation via the 26S proteasome, while K63-linked chains recruit binding partners during inflammation or DNA repair.2
The E2 enzymes are the key enzymes in the ubiquitin and ubiquitin-like pathways. More than 30 structures of E2s from various organisms are available, and all these E2 proteins share a topologically conserved α/β-fold core domain of ∼ 150 residues. Four α-helices (α1–α4) compose one face of the protein, and a four-stranded antiparallel β-sheet (β1–β4) sits on the back of the enzyme between helices α1 and α2 (Fig. 4). Every E2 core domain can bind three other proteins: the E1 ubiquitin-activating enzyme, a cognate E3 ligase enzyme, and activated ubiquitin via a labile thioester linkage. E2 enzymes can gain additional functionality via association with additional domains or subunits.3, 4
The complex of c-Cbl and UbcH7 was the first RING E3–E2 structure to be solved,5 but further structural insight into E2–E3 interactions comes from the HECT E3 E6AP complexed to UbcH7,6 the NMR-based model of the RING E3 CNOT4 bound to UbcH5B,7 and the U-box domain E3 CHIP/Ubc13–Uev1a complex.1, 8 All these complex structures show equivalent elements in the E2–E3 interface and similarities in overall structure. The core interface is formed by a hydrophobic groove on the E3 and two main regions on E2: loop L1, located between strands β3 and β4, and loop L2, connecting strand β4 and helix α2. In sharp contrast to the wealth of knowledge on E2 structures, the best structural insight of the E2∼Ub thioester intermediate is limited to models based on NMR chemical shift perturbation data due to the inherent instability of the thioester complex.4, 9
The human genome encodes a few E1s,1, 10 around 30 E2s, several hundreds of E3s, and thousands of substrates for ubiquitin, which makes the ubiquitination pathway very versatile and highly complex. An E3 may have more than one substrate and some substrates can be recognized by multiple E3s.11 The large abundance of E3s relative to E2s implies that E2s can function with multiple E3 ligases, but the inverse situation also exists. For example, the RING E3 complex SCF shows in vitro polyubiquitination activity with both CDC34 and Ubc4,12, 13 and the activity of the E3 APC is observed with both UbcH10 and UbcH5A, C.14, 15
It is generally assumed that the binding ability of E2–E3 is the determinant of functional E2–E3 pairs, because decreasing the interaction between E2 and E3 also diminishes their poly- or monoubiquitination activity in vitro. Interestingly, Brzovic et al. found that the BRCA1/BARD1 E3 heterodimer can interact with UbcH5C and UbcH7 with similar affinity and a similar interface, but only UbcH5C was active in Ub-ligase activity assays.16 Other examples of E3s that can physically interact with some E2s but fail to support ubiquitin transfer to targets include the RING E3 heterodimer Ring1b/Bmr117 and the U-box E3 CHIP.8 These results suggest that binding between E2–E3 pairs does not suffice for function. To address this paradox, we have analyzed and compared the c-Cbl/UbcH5B and c-Cbl/UbcH7 complexes both at a structural and functional level.
The E3 ubiquitin ligases of the Cbl family are key regulators of signaling by many surface receptors. From the N- to the C-terminus, c-Cbl contains a tyrosine kinase binding (TKB) domain, a linker region, and a RING domain, followed by an extensive proline-rich region and a ubiquitin-associated (UBA) domain. The UBA domain of Cbl-b, a homolog of c-Cbl, has recently been shown to bind ubiquitin noncovalently and to promote Cbl-b UBA dimerization and is required for RTK ubiquitination in vivo.18 The RING domain can recruit the E2 and function as an E3 ligase.19 The c-Cbl RING E3 ligase can regulate receptor kinases via their ubiquitin conjugation and catalyze the ubiquitination of c-Cbl itself as well. This autoubiquitination of E3 enzymes is suggested to represent a regulatory mechanism to control the abundance of Ub protein ligases in cells, since E3 autoubiquitination mediates their proteasome-dependent degradation.20, 21 Here, we studied the binding and functional specificity of UbcH5B and UbcH7 to c-Cbl RING using NMR structural analysis, chimeric UbcH5B/UbcH7 proteins, and in vitro activity assays, mostly by studying autoubiquitination of the c-Cbl RING domain. We found that although both UbcH5B and UbcH7 can bind specifically to the c-Cbl RING domain, only UbcH5B can facilitate ubiquitination of c-Cbl and substrate, which suggests that the binding specificity between E2–E3 pairs does not necessarily suffice for their functional specificity.
Section snippets
Ub-charged UbcH7 cannot mediate the autoubiquitination of c-Cbl
Early studies indicated that c-Cbl can ubiquitinate epidermal growth factor receptor and Src in cooperation with UbcH7,22, 23 while more recent work showed that c-Cbl promoted epidermal growth factor receptor and Src ubiquitination in a UbcH5B-dependent fashion.24, 25 We tested if c-Cbl can form functional complexes with UbcH7 and/or UbcH5B using an in vitro autoubiquitination assay, which is a characteristic commonly used to identify functional E2–E3 pairs.26 To enable accurate quantitation of
Discussion
In our experiments, we found that UbcH5B can support the in vitro autoubiquitination of c-Cbl and the c-Cbl-facilitated substrate ubiquitination but UbcH7 cannot. NMR titrations of c-Cbl to UbcH5B and UbcH7 did not show structural distinctions that could explain the functional differences. More likely, the different reactivity of the thioesters UbcH5B∼Ub and UbcH7∼Ub might explain why c-Cbl/UbcH7 is almost “nonproductive” in vitro. The E2∼Ub thioester intermediate is a key intermediate in the
Construction of plasmids
The construct of wild-type human UbcH5B was amplified from a human cDNA library and cloned into the plasmid pLICHIS, a pET15B-derived expression vector, by enzyme-free cloning.34 The chimera 7H1, 7L1, and 7L2 (Table 2) plasmids were amplified from the plasmid pLICHIS-UbcH5B, and further details are provided as Supplementary material. The fragment encoding the c-Cbl linker and RING domain (358–437) was cloned from a human cDNA library into pLISHISGST vector.34 Correct constructions of these
Acknowledgements
We would like to thank Dr. E. AB for help with structure calculations, Dr. Tammo Diercks and Dr. Rainer Wechselberger for expert NMR assistance, and Dr. Gert E. Folkers, M. Hilbers, and J. van der Zwan for laboratory and technical support. This work was supported by grants from the Netherlands Organization for Scientific Research (NWO-CW TOP 700-52-303 and 700-53-103) and by European Commission funding through the SPINE2-COMPLEXES project LSHG-CT-2006-031220. G.S.W. and H.Th.M.T. were supported
References (43)
- et al.
A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination
Mol. Cell
(2006) - et al.
Structure of a c-Cbl–UbcH7 complex: RING domain function in ubiquitin-protein ligases
Cell
(2000) - et al.
Structural model of the UbcH5B/CNOT4 complex revealed by combining NMR, mutagenesis, and docking approaches
Structure
(2004) - et al.
Chaperoned ubiquitylation—crystal structures of the CHIP U box E3 ubiquitin ligase and a CHIP–Ubc13–Uev1a complex
Mol. Cell
(2005) - et al.
Structure of a conjugating enzyme–ubiquitin thiolester intermediate reveals a novel role for the ubiquitin tail
Structure
(2001) - et al.
Mechanism of lysine 48-linked ubiquitin-chain synthesis by the cullin-RING ubiquitin–ligase complex SCF-Cdc34
Cell
(2005) - et al.
Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex
Cell
(2008) - et al.
Structural basis for ubiquitin-mediated dimerization and activation of the ubiquitin protein ligase Cbl-b
Mol. Cell
(2007) - et al.
Regulation of Smurf2 ubiquitin ligase activity by anchoring the E2 to the HECT domain
Mol. Cell
(2005) - et al.
Ligand-induced ubiquitination of the epidermal growth factor receptor involves the interaction of the c-Cbl RING finger and UbcH7
J. Biol. Chem.
(1999)
Src-catalyzed phosphorylation of c-Cbl leads to the interdependent ubiquitination of both proteins
J. Biol. Chem.
Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1
Mol. Cell
Lysine 63 polyubiquitination of the nerve growth factor receptor TrkA directs internalization and signaling
Mol. Cell
An altered-specificity ubiquitin-conjugating enzyme/ubiquitin-protein ligase pair
J. Mol. Biol.
Sequence determinants of E2-E6AP binding affinity and specificity
J. Mol. Biol.
Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients
Prog. Nucl. Magn. Reson. Spectrosc.
Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA
J. Mol. Biol.
Solution structure of the ubiquitin-conjugating enzyme UbcH5B
J. Mol. Biol.
Mechanisms underlying ubiquitination
Annu. Rev. Biochem.
Modification of proteins by ubiquitin and ubiquitin-like proteins
Annu. Rev. Cell Dev. Biol.
Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A
J. Cell Biol.
Cited by (37)
In Vitro Ubiquitination Platform Identifies Methyl Ellipticiniums as Ubiquitin Ligase Inhibitors
2021, SLAS DiscoveryCitation Excerpt :In our autoubiquitination assay, these compounds do not inhibit CBLB E3 activity (Suppl. Fig. S7). UbcH7 has been crystallized in complex with CBL but does not function with CBL proteins in E3 assays, possibly explaining their lack of activity in our hands.13,54–57 In addition, a pharmaceutical company has described a CBLB inhibitor in a conference abstract in which no chemical or screening assay information was provided.45
When Worlds Collide—Mechanisms at the Interface between Phosphorylation and Ubiquitination
2017, Journal of Molecular BiologySelective recruitment of an E2∼ubiquitin complex by an E3 ubiquitin ligase
2012, Journal of Biological ChemistryRINGs hold the key to ubiquitin transfer
2012, Trends in Biochemical SciencesCitation Excerpt :In addition, the complexes formed between the RING domain of CBL and either Ube2D2 or Ube2L3 (two E2 enzymes) are comparable, although only Ube2D2 allows ubiquitin transfer. This suggests that no major structural change in the E2 protein is associated with activity [19,23], and the interaction of an E2 enzyme and an E3 ligase, although necessary, is insufficient to predict activity. To elicit ubiquitin transfer, the E3 protein must interact with an E2∼ubiquitin conjugate, yet most studies have focused on the interaction between RING or U-box domains and E2 enzymes in the absence of ubiquitin.
- 1
Present address: R. N. de Jong, Genmab B.V., Yalelaan 60, 3584 CM Utrecht, The Netherlands.
- 2
Present address: G. S. Winkler, School of Pharmacy, University of Nottingham, University Park, NG7 2RD Nottingham, UK.