Oncofusion

start / graph / hyp-kmt2a-14-3-3-association-is-bridged-by-aff-family

Hypothesisspeculative confidence 1 of 5: Our own inference or analogy. Not shown to be true.Q22-phospho-contacts

KMT2A is a 14-3-3 client by proxy: the contact is on an AFF-family scaffold, not on KMT2A

also called bridged 14-3-3 hypothesis · AFF1 S588 bridge

KMT2A keeps showing up near 14-3-3 proteins, but it has no good docking site of its own. Its partner AF4 does have one, at a spot that is already known and already phosphorylated - so KMT2A may simply be travelling with a partner that 14-3-3 holds.

The same thing, for a scientist

Three facts sit awkwardly together. KMT2A is recovered as a 14-3-3 client by six paralogues in one interactome and by YWHAG in a second study. A canonical-motif scan of KMT2A finds three matches at chance rate, **none of whose phospho-acceptors is observed in a 130-site phosphoproteomic re-analysis**. And the one demonstrated 14-3-3 contact in this system is **AFF1 Ser588**, non-canonical, phosphorylated, and bound by **14-3-3 theta (YWHAQ) - one of the same paralogues that recovers KMT2A**. The hypothesis: **the KMT2A-14-3-3 association is indirect, bridged by an AFF-family scaffold (AFF1 or AFF4), and there is no 14-3-3 motif on KMT2A to find.** This explains the proximity data, the motif-scan negative and the phosphosite negative at once, and it predicts a specific, cheap experiment.

Go deeper
What it predicts
  1. Phosphatase-sensitive, AFF-dependent recovery. 14-3-3 immunoprecipitation from a KMT2A-expressing cell should recover KMT2A, and that recovery should be abolished by AFF1/AFF4 double knockdown while 14-3-3's recovery of AFF1 is unaffected.
  2. S588A should break the bridge. An AFF1 S588A knock-in should reduce KMT2A recovery by 14-3-3 theta without reducing AFF1 protein levels.
  3. Paralogue concordance. The paralogues that recover KMT2A should be a subset of those that recover AFF1/AFF4. YWHAQ appearing in both observations is the first, weak, already-available piece of that.
  4. No KMT2A site will be found. Targeted phospho-IP of KMT2A should fail to find a 14-3-3-competent site, which is the prediction that is already partly confirmed by the S821 verdict.
  5. In t(4;11) the bridge is on the fusion itself, because AFF1 S588 is retained in KMT2A::AFF1 - so a fusion protein carries both halves of the bridge in one chain, and the 14-3-3 contact becomes intramolecular in a way it is not in normal cells.
What would kill it
  • 14-3-3 recovery of KMT2A that survives AFF1/AFF4 depletion.
  • A direct KMT2A peptide-14-3-3 binding measurement at physiological affinity.
  • A KMT2A phosphosite in a 14-3-3-competent context found by targeted phospho-IP, which the pooled databases would have missed.
  • Structural or crosslinking evidence of a direct KMT2A-14-3-3 contact.

And a caution against over-reading a confirmation: if the bridge hypothesis is right, 14-3-3 is not a handle on the retained KMT2A fragment at all, and the cycle-28 menin-only picture survives this particular challenge. The hypothesis is as likely to defend the menin-interface position as to threaten it.

Facts and figures
statementKMT2A's reproduced association with 14-3-3 proteins is indirect, mediated by an AFF-family scaffold (AFF1 or AFF4) carrying a non-canonical phospho-dependent 14-3-3 site such as AFF1 Ser588, rather than by a 14-3-3 binding motif on KMT2A itself.
origindata-pattern
prior probability0.35
predictions
[
  "14-3-3 recovery of KMT2A is abolished by AFF1/AFF4 depletion",
  "AFF1 S588A reduces 14-3-3 theta recovery of KMT2A without reducing AFF1 levels",
  "targeted phospho-IP of KMT2A finds no 14-3-3-competent site",
  "in KMT2A::AFF1 the bridge is intramolecular because S588 is retained"
]
experiments
[
  "exp-14-3-3-bridge-pulldown-t411"
]
analyses
[
  "ana-14-3-3-kmt2a-direct-binding-bounded-absence",
  "ana-kmt2a-s821-phosphosite-verdict"
]
falsifiers
[
  "14-3-3 recovery of KMT2A survives AFF1/AFF4 double knockdown",
  "a direct KMT2A peptide binds a 14-3-3 paralogue at physiological affinity in vitro",
  "a 14-3-3-competent KMT2A phosphosite is found by targeted phospho-IP",
  "structural or crosslinking evidence of a direct KMT2A-14-3-3 contact"
]
How this could bite us back
  • If this hypothesis is right it removes a candidate second handle rather than adding one, and the menin-only picture of the retained fragment survives intact on this axis.
  • Targeting a bridged interaction means targeting the bridge, and AFF1/AFF4 are core elongation scaffolds whose loss is not tolerable in normal haematopoiesis.
  • A confirmed bridge would make the 14-3-3 proximity signal uninformative about KMT2A regulation, retiring what is currently the graph's best-reproduced KMT2A proximity relation.
Still open
  1. Does the published interactome's raw data allow a bridged-versus-direct test without new experiments? Recovery of KMT2A in an AFF1-depleted condition, if that condition exists in the deposition, would settle it for free.
  2. If the bridge exists, is it intramolecular in the fusion? That would be a structural feature unique to KMT2A::AFF1 among the fusions and worth modelling.
  3. Does AFF4, which carries 53 observed phosphosites, have its own 14-3-3 site?
Every source behind this entry (2)
  1. E1moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper

    14-3-3 theta binds AF4 at Ser588 and forms a complex with MLL-AF4, giving the AFF-family scaffold a demonstrated, phosphorylation-gated 14-3-3 contact that KMT2A itself lacks.

    PMID 31493143DOI
    What this evidence cannot show

    Abstract-level; the binding assay format and whether S588A abolishes binding are not established from what was readable. One laboratory, two t(4;11) cell lines, no independent replication found.

    Evidence that argues against this (1)
    • The interactome that makes KMT2A a client reports it as prey of six paralogues while the AF4 result concerns only theta. If the bridge were a single AFF1 site bound by a single paralogue, it would be an odd explanation for a six-paralogue recovery pattern; a shared, degenerate, multi-paralogue contact somewhere else is at least as consistent with the data. Segal D et al. 2023
  2. E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.database

    No phospho-acceptor of any canonical 14-3-3 motif match in KMT2A is observed as a phosphosite in a 130-site PRIDE/PTMeXchange re-analysis, and the same scan finds zero canonical motifs in AFF1 despite AFF1 carrying a demonstrated 14-3-3 contact, so the absence of a canonical KMT2A site is compatible either with no site or with a non-canonical one.

    What this evidence cannot show

    The motif scan covers modes 1 and 2 only. A non-canonical KMT2A site would be invisible to it, which is precisely why the hypothesis is framed as bridged rather than as absent-therefore-nonexistent. The phosphosite resource has no KMT2A-rearranged leukaemia data in it.

    Evidence that argues against this (1)
    • A substantial share of validated 14-3-3 clients bind through divergent motifs, and the study that makes KMT2A a client reports no motif for it. Its own headline mechanism is chaperone-like engagement of clients, which in that model does not require a canonical site - so 'no canonical site' is an unremarkable property of a real client rather than evidence for a bridge. Segal D et al. 2023
Connected to