ZWINT

ZWINT-1 is an outer kinetochore protein essential for spindle assembly checkpoint (SAC) signaling and accurate chromosome segregation during mitosis and meiosis. It is recruited to kinetochores downstream of HEC1/NDC80 as part of the Mis12 complex network and is required for the subsequent kinetochore localization of ZW10, the RZZ complex, dynein/dynactin, CENP-F, and Mad2 (PMID:15502821, PMID:15485811, PMID:16732327). Aurora B kinase phosphorylates ZWINT-1 at three sites to promote RZZ and dynein assembly at kinetochores, and phospho-mimetic mutations sustain SAC activity while blocking poleward streaming needed for checkpoint silencing (PMID:21775627). ZWINT-1 protein levels are controlled by APC/C-Cdc20–mediated D-box-dependent ubiquitination during mitotic exit and by TRIM17 E3 ligase–directed proteasomal degradation (PMID:31945194, PMID:22023800).

1. 2004 High

   Establishing ZWINT-1 as an outer kinetochore component within the Mis12 complex network resolved where the protein sits in the centromere-to-kinetochore hierarchy and linked it to the ZW10 recruitment pathway.

   Evidence Co-IP/MS of hMis12-associated polypeptides in HeLa cells; siRNA depletion of ZWINT-1 abolished ZW10 kinetochore localization, caused premature segregation and SAC failure

   PMID:15485811 PMID:15502821

   Open questions at the time

   • Direct binding interface between ZWINT-1 and ZW10 not structurally resolved
   • How ZWINT-1 couples to dynein/dynactin recruitment was unclear
2. 2006 High

   Defining an HEC1→ZWINT-1→ZW10 sequential recruitment hierarchy during M phase established the epistatic order through which the outer kinetochore assembles checkpoint-competent signaling platforms.

   Evidence M-phase-specific reciprocal Co-IP; epistatic siRNA knockdown showing HEC1 depletion removes ZWINT-1 and ZW10, but ZWINT-1 depletion does not affect HEC1

   PMID:16732327

   Open questions at the time

   • Whether ZWINT-1–ZW10 interaction is direct or bridged by Zwilch/ROD was unresolved
   • Regulation of the HEC1–ZWINT-1 interaction was unknown
3. 2011 High

   Identifying Aurora B phosphorylation of ZWINT-1 at three sites as the switch for RZZ/dynein kinetochore assembly answered how the checkpoint recruitment cascade is activated and how its silencing requires dephosphorylation-dependent poleward streaming.

   Evidence In vitro kinase assay with MS site mapping; phospho-dead (3A) mutant blocked RZZ assembly, phospho-mimetic (3E) mutant sustained SAC and blocked dynein streaming

   PMID:21775627

   Open questions at the time

   • Identity of the phosphatase(s) that reverse Aurora B phosphorylation on ZWINT-1 is unknown
   • Structural basis of phosphorylation-dependent RZZ binding not determined
4. 2011 Medium

   Discovery that TRIM17 E3 ligase promotes ZWINT-1 proteasomal degradation via its coiled-coil domain established a first post-translational mechanism controlling ZWINT-1 protein abundance.

   Evidence Yeast two-hybrid, Co-IP, and overexpression/knockdown in cell proliferation assays

   PMID:22023800

   Open questions at the time

   • Ubiquitination sites on ZWINT-1 targeted by TRIM17 not mapped
   • Cell-cycle timing of TRIM17-mediated degradation not established
   • Not independently confirmed by a second group
5. 2015 Medium

   Demonstrating ZWINT-1's requirement for SAC function and Aurora C–mediated error correction in oocyte meiosis I extended its role beyond mitosis to female germ cell chromosome fidelity.

   Evidence siRNA in mouse oocytes; ZWINT-1 depletion abolished Mad2 kinetochore recruitment and caused aneuploidy; epistasis with Aurora C inhibition

   PMID:26486467

   Open questions at the time

   • Whether Aurora C directly phosphorylates ZWINT-1 (analogous to Aurora B) was not tested
   • Relevance to human oocyte meiosis not confirmed
6. 2020 Medium

   Identification of APC/C-Cdc20 as the E3 ligase that ubiquitinates ZWINT-1 through a D-box motif during mitotic exit provided the molecular timer for ZWINT-1 destruction and kinetochore disassembly.

   Evidence In vivo ubiquitination assay, D-box deletion mutant escapes Cdc20-mediated degradation; MG132 and cycloheximide chase experiments

   PMID:31945194

   Open questions at the time

   • Specific lysine residues ubiquitinated by APC/C-Cdc20 not identified
   • Functional consequences of stabilized ZWINT-1 (D-box mutant) on subsequent cell cycles not characterized
7. 2021 Medium

   Finding that ZWINT-1 promotes p53 ubiquitination and degradation under hypoxia revealed a non-kinetochore function linking ZWINT-1 overexpression to oncogenic proliferation via suppression of p53/p21 signaling.

   Evidence Co-IP, ubiquitination assay, ChIP showing HIF1α drives ZWINT transcription, functional proliferation assays in pancreatic cancer cells

   PMID:34900978

   Open questions at the time

   • Whether ZWINT-1 acts as an E3 ligase adaptor or scaffold for p53 ubiquitination is unknown
   • Not independently replicated; mechanism may be indirect through kinetochore-independent pool
   • Physiological relevance beyond cancer cell lines not established

• A structural model of the ZWINT-1–RZZ interface, the identity of phosphatases counteracting Aurora B on ZWINT-1, and the in vivo significance of ZWINT-1's reported p53-regulatory function remain unresolved.
• No crystal or cryo-EM structure of ZWINT-1 in complex with RZZ or HEC1
• No genetic mouse model assessing ZWINT-1 loss in vivo
• Potential non-kinetochore functions (Rab3c/SNAP25 interaction in neurons) not mechanistically developed