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1.  ULK3 regulates cytokinetic abscission by phosphorylating ESCRT-III proteins 
eLife  null;4:e06547.
The endosomal sorting complexes required for transport (ESCRT) machinery mediates the physical separation between daughter cells during cytokinetic abscission. This process is regulated by the abscission checkpoint, a genome protection mechanism that relies on Aurora B and the ESCRT-III subunit CHMP4C to delay abscission in response to chromosome missegregation. In this study, we show that Unc-51-like kinase 3 (ULK3) phosphorylates and binds ESCRT-III subunits via tandem MIT domains, and thereby, delays abscission in response to lagging chromosomes, nuclear pore defects, and tension forces at the midbody. Our structural and biochemical studies reveal an unusually tight interaction between ULK3 and IST1, an ESCRT-III subunit required for abscission. We also demonstrate that IST1 phosphorylation by ULK3 is an essential signal required to sustain the abscission checkpoint and that ULK3 and CHMP4C are functionally linked components of the timer that controls abscission in multiple physiological situations.
eLife digest
Our cells multiply by dividing into two. Many proteins are involved in this process, including a group called the ESCRT-III complex. This group is required to complete the final stage of cell division when the single membrane that surrounds the two new daughter cells separates. Before the cell divides, its DNA—which is packaged in structures called chromosomes—is copied, and the two sets of chromosomes are pulled to opposite ends of the cell. This ensures that each daughter cell will have a complete set of DNA.
If the cell divides before the chromosomes have finished moving to opposite ends of the cell, the daughter cells may end up with the wrong number of chromosomes. This can lead to cancer or other diseases. To prevent this, cells have evolved a quality control system called the ‘abscission checkpoint’, which delays cell division until the chromosomes have properly separated. Previous studies have shown that when the checkpoint is active, an ESCRT-III complex protein called CHMP4C is inactivated by an enzyme, which prevents the cell from dividing.
Other signals that indicate that the new daughter cells are not yet ready to separate can also delay cell division, but it is not clear how those defects are detected by the checkpoint. Here, Caballe, Wenzel et al. found that a protein called ULK3 can bind to several proteins in the ESCRT-III complex, including one called IST1. In doing so, ULK3 is able to delay cell division if the chromosomes have not finished separating, if there are defects in the nucleus of the cell, or if the cell is experiencing high levels of mechanical tension at the site where the membrane will separate.
The experiments also show that ULK3 needs to bind to and regulate the activity of IST1 to sustain the abscission checkpoint, and that CHMP4C is required for this process. Caballe, Wenzel et al.'s findings reveal that ULK3 plays an essential role in controlling when a cell divides and imply that there may be additional proteins involved that release cells from the checkpoint delay imposed by ULK3. The next challenges will be to identify these proteins and to understand how all checkpoint proteins work together to regulate cell division.
PMCID: PMC4475061  PMID: 26011858
cytokinesis; abscission checkpoint; ESCRT; ULK3; phosphorylation; human
2.  Intrinsic disorder drives N-terminal ubiquitination by Ube2w 
Nature chemical biology  2014;11(1):83-89.
Ubiquitination of the αN-terminus of protein substrates has been reported sporadically over the past twenty years. However the identity of an enzyme responsible for this unique ubiquitin (Ub) modification has only recently been elucidated. We show the ubiquitin-conjugating enzyme (E2) Ube2w employs a novel mechanism to facilitate the specific ubiquitination of the α-amino group of its substrates that involves recognition of backbone atoms of intrinsically disordered N-termini. We present the NMR-based solution ensemble of full-length Ube2w that reveals a structural architecture unlike any other E2, in which its C-terminus is partly disordered and flexible to accommodate variable substrate N-termini. Flexibility of the substrate is critical for recognition by Ube2w and point mutations in, or removal of, the flexible C-terminus of Ube2w inhibits substrate binding and modification. Mechanistic insights reported here provide guiding principles for future efforts to define the N-terminal-Ubiquitome in cells.
PMCID: PMC4270946  PMID: 25436519
3.  Biochemical and Structural Characterization of the Ubiquitin-Conjugating Enzyme UBE2W Reveals the Formation of a Noncovalent Homodimer 
Cell biochemistry and biophysics  2013;67(1):103-110.
The biochemical and structural characterization of ubiquitin-conjugating enzymes (E2s) over the past 30 years has fostered important insights into ubiquitin transfer mechanisms. Although many of these enzymes share high sequence and structural conservation, their functional roles in the cell are decidedly diverse. Here, we report that the mono-ubiquitinating E2 UBE2W forms a homodimer using two distinct protein surfaces. Dimerization is primarily driven by residues in the ß-sheet region and Loops 4 and 7 of the catalytic domain. Mutation of two residues in the catalytic domain of UBE2W is capable of disrupting UBE2W homodimer formation, however, we find that dimerization of this E2 is not required for its ubiquitin transfer activity. In addition, residues in the C-terminal region, although not compulsory for the dimerization of UBE2W, play an ancillary role in the dimer interface. In all current E2 structures, the C-terminal helix of the UBC domain is at least 15Å away from the primary dimerization surface shown here for UBE2W. This leads to the proposal that the C-terminal region of UBE2W adopts a noncanonical position that places it closer to the UBC ß-sheet, providing the first indication that at least some E2s adopt C-terminal conformations different from the canonical structures observed to date.
PMCID: PMC3758794  PMID: 23709311
Ubiquitin; Ubiquitin-conjugating enzyme; UBE2W; Dimerization; NMR
4.  UbcH7 reactivity profile reveals Parkin and HHARI to be RING/HECT hybrids 
Nature  2011;474(7349):105-108.
Although the functional interaction between ubiquitin conjugating enzymes (E2s) and ubiquitin ligases (E3s) is essential in ubiquitin (Ub) signaling, the criteria that define an active E2–E3 pair are not well-established. The human E2 UbcH7 (Ube2L3) shows broad specificity for HECT-type E3s1, but often fails to function with RING E3s in vitro despite forming specific complexes2–4. Structural comparisons of inactive UbcH7/RING complexes with active UbcH5/RING complexes reveal no defining differences3,4, highlighting a gap in our understanding of Ub transfer. We show that, unlike many E2s that transfer Ub with RINGs, UbcH7 lacks intrinsic, E3-independent reactivity with lysine, explaining its preference for HECTs. Despite lacking lysine reactivity, UbcH7 exhibits activity with the RING-In Between-RING (RBR) family of E3s that includes Parkin and human homologue of ariadne (HHARI)5,6. Found in all eukaryotes7, RBRs regulate processes such as translation8 and immune signaling9. RBRs contain a canonical C3HC4-type RING, followed by two conserved Cys/His-rich Zn2+-binding domains, In-Between-RING (IBR) and RING2 domains, which together define this E3 family7. Here we show that RBRs function like RING/HECT hybrids: they bind E2s via a RING domain, but transfer Ub through an obligate thioester-linked Ub (denoted ‘~Ub’), requiring a conserved cysteine residue in RING2. Our results define the functional cadre of E3s for UbcH7, an E2 involved in cell proliferation10 and immune function11, and suggest a novel mechanism for an entire class of E3s.
PMCID: PMC3444301  PMID: 21532592
5.  Following Ariadne's thread: a new perspective on RBR ubiquitin ligases 
BMC Biology  2012;10:24.
Ubiquitin signaling pathways rely on E3 ligases for effecting the final transfer of ubiquitin from E2 ubiquitin conjugating enzymes to a protein target. Here we re-evaluate the hybrid RING/HECT mechanism used by the E3 family RING-between-RINGs (RBRs) to transfer ubiquitin to substrates. We place RBRs into the context of current knowledge of HECT and RING E3s. Although not as abundant as the other types of E3s (there are only slightly more than a dozen RBR E3s in the human genome), RBRs are conserved in all eukaryotes and play important roles in biology. Re-evaluation of RBR ligases as RING/HECT E3s provokes new questions and challenges the field.
PMCID: PMC3305615  PMID: 22420831
6.  E2s: Structurally Economical and Functionally Replete 
The Biochemical journal  2010;433(1):31-42.
Ubiquitination is a post-translational modification pathway involved in myriad cellular regulation and disease pathways. The ubiquitin (Ub) transfer cascade requires three enzyme activities: a Ub-activating (E1) enzyme, a Ub-conjugating (E2) enzyme, and a Ub ligase (E3). Because the E2 is responsible both for E3 selection and substrate modification, E2s function at the heart of the Ub transfer pathway and are responsible for much of the diversity of Ub cellular signaling. There are currently over ninety three-dimensional structures of E2s, both alone and in complex with protein binding partners, providing a wealth of information regarding how E2s are recognized by a wide variety of proteins. In this review, we describe the prototypical E2/E3 interface and discuss limitations of current methods to identify cognate E2/E3 partners. We present non-canonical E2-protein interactions and highlight the economy of E2s in their ability to facilitate many protein-protein interactions at nearly every surface on their relatively small, compact catalytic domain. Lastly, we compare the structures of conjugated E2~Ub species, their unique protein interactions, and the mechanistic insights provided by species that are poised to transfer Ub.
PMCID: PMC3118098  PMID: 21158740
ubiquitin; ubiquitin-conjugating enzyme; ubiquitin ligase enzyme; ubiquitination; ubiquitylation

Results 1-6 (6)