PPP6R3

PPP6R3 (PP6R3/SAPS3) is a SAPS-domain regulatory subunit of the protein phosphatase 6 (PP6) holoenzyme that recruits the PP6 catalytic subunit and dictates its substrate selectivity across diverse cellular processes (PMID:16769727, PMID:19835610). Its conserved SAPS domain folds into helical repeats whose negatively charged interhelical-loop residues mediate specific binding to the PP6 catalytic subunit, distinguishing PP6 from PP2A and PP4; charge-reversal mutations in these residues disrupt the interaction (PMID:16769727, PMID:19835610). Within cells PPP6R3 assembles into a >440 kDa heterotrimer comprising the PP6 catalytic subunit and an ankyrin-repeat subunit such as Ankrd28 (PMID:18186651). Through this complex PPP6R3 specifies dephosphorylation of defined substrates: it removes TRF2-Ser365 phosphorylation specifically during S phase, opening a window for RTEL1 to unwind telomeric t-loops and enabling telomere replication while restraining inappropriate ATM activation (PMID:31723267), and it dephosphorylates the translation initiation factors EIF3C-Ser39 and EIF4G1-Ser1217 in KIT+ spermatogonia, stabilizing these factors and sustaining mRNA translation required for spermatogonial differentiation (PMID:40721635). Germline deletion of PPP6R3 causes male infertility with translation inhibition, a defect rescued by phospho-dead EIF3C(S39A)/EIF4G1(S1217A) mutants (PMID:40721635). PPP6R3-containing PP6 complexes also act redundantly with PP6R1/PP6R2 to promote RIPK1-dependent PANoptosis by sustaining pro-death RIPK1-Ser166 autophosphorylation and reducing pro-survival Ser321 phosphorylation (PMID:38807188), and PP6 holoenzymes containing PPP6R3 engage DNA-PKcs in the DNA damage response and the influenza A virus RdRP during viral replication (PMID:20065038, PMID:25187537).

1. 2006 Medium

   Establishing that the SAPS domain confers phosphatase specificity answered whether PP6R3 is a dedicated PP6 adaptor rather than a promiscuous regulatory subunit.

   Evidence FLAG co-immunoprecipitation in HEK293 cells showing PP6 but not PP2A/PP4 binding, plus siRNA knockdown with an IkappaBepsilon degradation readout

   PMID:16769727

   Open questions at the time

   • Did not resolve the structural basis of specificity
   • PP6R3 found dispensable for the IkappaBepsilon substrate, leaving its true substrates undefined
2. 2008 Medium

   Defining the holoenzyme composition established that PP6R3 functions as one arm of a heterotrimer with the catalytic and ankyrin-repeat subunits.

   Evidence FLAG co-IP, mass spectrometry, and size-exclusion chromatography in a single lab demonstrating co-elution of PP6R3, PP6c, and Ankrd28 at >440 kDa

   PMID:18186651

   Open questions at the time

   • Stoichiometry within the assembled trimer not quantified
   • Functional consequence of the ankyrin subunit not separated from PP6R3
3. 2009 Medium

   Mapping the SAPS domain fold and the charged residues that contact PP6c provided the structural mechanism of subunit recognition.

   Evidence Charge-reversal mutagenesis, co-precipitation, circular dichroism, protease protection, and homology modeling to golgin p115

   PMID:19835610

   Open questions at the time

   • No experimentally determined high-resolution structure
   • Model rests on homology rather than direct structure determination
4. 2009 Medium

   Cross-species complementation tested whether human PP6R3 is a functionally conserved phosphatase adaptor by rescuing yeast SAP-deficient phenotypes.

   Evidence Yeast quadruple sap mutant rescue, rapamycin sensitivity and cell-cycle assays, and Sit4 co-immunoprecipitation

   PMID:19621075

   Open questions at the time

   • Heterologous yeast context does not establish native mammalian substrates
   • Sit4 is not PP6c, so conservation is functional rather than identical
5. 2010 Medium

   Linking PP6 subunits to DNA-PKcs and gamma-H2AX placed the holoenzyme in the DNA double-strand break response, though PP6R3's individual contribution was not isolated.

   Evidence Co-immunoprecipitation of endogenous proteins, siRNA knockdown, gamma-H2AX immunofluorescence, and G2/M checkpoint assays

   PMID:20065038

   Open questions at the time

   • The gamma-H2AX phenotype was attributed to PP6R1, not PP6R3 specifically
   • PP6R3-specific role in DSB repair untested
6. 2014 Medium

   Affinity purification of the influenza RdRP interactome implicated PP6 holoenzyme membership in viral replication.

   Evidence Strep-tag affinity purification from infected cells with label-free quantitative MS and siRNA knockdown of PPP6C with viral RNA and plaque readouts

   PMID:25187537

   Open questions at the time

   • Functional viral phenotype tested for PPP6C, not PPP6R3
   • Direct PP6R3-RdRP contact not demonstrated
7. 2019 High

   Identifying TRF2-Ser365 as a cell-cycle-staged substrate established a precise phospho-switch mechanism by which PP6R3 gates telomere replication.

   Evidence Phospho-specific antibodies, co-IP, knockdown, CDK-site mutagenesis, and cell-cycle-staged telomere replication and ATM activation assays

   PMID:31723267

   Open questions at the time

   • Recruitment mechanism of the complex to telomeres not fully defined
   • Whether other SAPS subunits can substitute at TRF2 not tested
8. 2024 Medium

   Defining PP6 control of RIPK1 phosphorylation placed PPP6R3 in cell-death signaling, with redundancy among the three SAPS subunits.

   Evidence CRISPR death screen, individual and combined PP6 subunit knockouts, and phospho-specific immunoblotting of RIPK1 S166 and S321

   PMID:38807188

   Open questions at the time

   • Redundancy means PP6R3-unique contribution cannot be isolated
   • Direct RIPK1 dephosphorylation by the PP6R3 complex not reconstituted
9. 2025 High

   In vivo conditional knockout identified EIF3C and EIF4G1 as direct substrates, linking PP6R3-mediated dephosphorylation to translational control of spermatogonial differentiation.

   Evidence Germline conditional knockout in mice, PPP6R3-EIF3C/EIF4G1 co-IP, phosphoproteomics, RNA-IP, and rescue with phospho-dead EIF3C(S39A)/EIF4G1(S1217A) mutants

   PMID:40721635

   Open questions at the time

   • Whether translation control extends beyond germ cells not addressed
   • Mechanism coupling phosphorylation to factor degradation not detailed
10. 2025 Medium

    Zebrafish knockout extended the germline requirement across species, showing Ppp6r3 is needed for gonadal differentiation and the spermatocyte-to-sperm transition.

    Evidence CRISPR/Cas9 knockout in zebrafish with testis histology and fertility assays

    PMID:40962474

    Open questions at the time

    • No biochemical substrate identified in this system
    • Mechanism of all-male offspring skewing unresolved
11. 2025 Low

    Microexon deletion in zebrafish hinted at a neural function for an alternatively spliced PPP6R3 isoform.

    Evidence CRISPR/Cas9 microexon deletion in zebrafish with larval brain activity imaging

    PMID:41252186

    Open questions at the time

    • No molecular mechanism for the microexon
    • Neural phenotype mild and not connected to phosphatase activity
12. 2025 Low

    A Sec16 dephosphorylation role at ER exit sites positioned PP6R3 in secretory pathway regulation.

    Evidence Phosphatase complex identification with ERES formation and secretion assays under genetic and pharmacological perturbation (preprint)

    Open questions at the time

    • Preprint without reconstitution or mutagenesis detail
    • Direct PP6R3-Sec16 contact and phospho-site not established in available evidence

• How a single SAPS subunit partitions PP6 among telomeric, translational, cell-death, secretory, and viral substrates within the cell remains unresolved.
• No mechanism for spatial/temporal substrate switching
• No structure of a substrate-bound holoenzyme
• Relative contribution of distinct ankyrin partners to substrate choice unknown