Affinage

ANKRD28

Serine/threonine-protein phosphatase 6 regulatory ankyrin repeat subunit A · UniProt O15084

Length
1053 aa
Mass
113.0 kDa
Annotated
2026-04-28
24 papers in source corpus 7 papers cited in narrative 7 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

ANKRD28 is an ankyrin-repeat scaffold protein that functions as a regulatory subunit of the protein phosphatase 6 (PP6) heterotrimer and as a modulator of DOCK180-Rac1 signaling at focal adhesions. Within the PP6 holoenzyme, ANKRD28 associates with SAPS-domain subunits (PP6R1, PP6R3) via the C-terminal region of PP6R1 and determines substrate specificity, including stabilization of IκBε downstream of TNFα; BRCA1 binds ANKRD28 in the cytoplasm to modulate this PP6-dependent IκBε stability (PMID:18186651, PMID:27026398). ANKRD28 protein levels are controlled by Rab40c/Cullin5 E3 ligase-mediated ubiquitylation and lysosomal degradation, and loss of this regulatory axis increases PP6 activity, decreasing FAK and MOB1 (Hippo pathway) phosphorylation at focal adhesions (PMID:35512830). Independently of PP6, ANKRD28 binds the SH3 domain of DOCK180, competing with ELMO to promote p130Cas hyperphosphorylation and regulate focal adhesion distribution and cell migration (PMID:19118547, PMID:19458477).

Mechanistic history

Synthesis pass · year-by-year structured walk · 6 steps
  1. 2007 Medium

    Discovery that ANKRD28 participates in a NUP98 fusion oncoprotein in leukemia established that ANKRD28 is exclusively cytoplasmic and that mislocalization of its fusion product to the nucleolus is associated with oncogenic transformation, raising questions about ANKRD28's normal cellular function.

    Evidence FISH breakpoint mapping, RT-PCR, focus formation assay in NIH/3T3, fluorescence microscopy

    PMID:17988990

    Open questions at the time
    • Single lab study with transient overexpression; stable transformation not demonstrated
    • Mechanism by which NUP98 fusion drives transformation versus wild-type ANKRD28 function not delineated
    • No endogenous ANKRD28 loss-of-function studied in leukemia context
  2. 2008 High

    Identification of ANKRD28 as a PP6-specific regulatory subunit within a >440 kDa heterotrimer with SAPS-domain scaffolds resolved how PP6 achieves substrate selectivity distinct from related phosphatases PP2A and PP4, and linked ANKRD28 to IκBε stability control in TNFα signaling.

    Evidence Co-IP (FLAG-tag and reciprocal), mass spectrometry, size-exclusion chromatography, siRNA knockdown with IκBε degradation readout in HEK293 cells

    PMID:18186651

    Open questions at the time
    • Structural basis of ANKRD28–PP6R1 interaction not resolved
    • Direct PP6 substrates beyond IκBε not identified
    • Functional non-redundancy with ANKRD44 and ANKRD52 not tested
  3. 2008 High

    Parallel identification of ANKRD28 as a DOCK180 SH3-domain binding partner revealed a PP6-independent role in focal adhesion regulation, where ANKRD28 competes with ELMO to specify distinct DOCK180-Rac1 pathway outputs—p130Cas hyperphosphorylation and process formation versus lamellipodia.

    Evidence Nano-LC/MS/MS pulldown, siRNA knockdown migration assays, co-expression studies in HeLa cells

    PMID:19118547 PMID:19458477

    Open questions at the time
    • Whether ANKRD28's PP6 and DOCK180 functions are coordinated or occur in separate pools is unknown
    • Direct binding interface between ANKRD28 ankyrin repeats and DOCK180 SH3 domain not mapped at residue level
    • In vivo relevance of ANKRD28-DOCK180 competition with ELMO not established
  4. 2016 Medium

    Identification of BRCA1 as a cytoplasmic ANKRD28 interactor that stabilizes IκBε upon TNFα stimulation extended the PP6 regulatory network, showing that BRCA1's disordered central region engages ANKRD28 to modulate PP6 signaling output.

    Evidence Yeast two-hybrid, reciprocal co-IP (overexpressed and endogenous), proximity ligation assay, IκBε stability assay

    PMID:27026398

    Open questions at the time
    • Whether BRCA1 directly affects PP6 catalytic activity or substrate access via ANKRD28 is not determined
    • Single lab finding; independent confirmation lacking
    • Physiological contexts where BRCA1-ANKRD28 interaction is functionally relevant beyond TNFα not explored
  5. 2022 High

    Demonstration that Rab40c/Cullin5 ubiquitylates ANKRD28 for lysosomal degradation established a post-translational mechanism controlling PP6 activity, linking ANKRD28 turnover to FAK and Hippo (MOB1) pathway phosphorylation at focal adhesions.

    Evidence Rab40c knockout MDA-MB-231 cells, ubiquitylation assays, phosphoproteomics (FAK/MOB1), lysosomal inhibitor rescue

    PMID:35512830

    Open questions at the time
    • Ubiquitylation sites on ANKRD28 not mapped
    • Whether ANKRD44 and ANKRD52 are similarly regulated by Rab40c/CRL5 not tested
    • How ANKRD28 degradation is spatially coordinated at focal adhesions versus bulk cytoplasm is unclear
  6. 2024 Medium

    Confirmation of ANKRD28 as one of three interchangeable PP6 scaffold subunits in the context of colorectal cancer stem cell regulation reinforced its role within the PP6 holoenzyme but did not resolve subunit-specific contributions.

    Evidence siRNA knockdown, colony formation, xenograft, RNA-seq in CRC cell lines

    PMID:39014521

    Open questions at the time
    • Specific contribution of ANKRD28 versus ANKRD44/ANKRD52 to cancer phenotypes not dissected
    • PP6 substrates in cancer stemness context not identified
    • No ANKRD28-specific perturbation performed in this study

Open questions

Synthesis pass · forward-looking unresolved questions
  • The structural basis of ANKRD28 assembly into the PP6 heterotrimer, the functional non-redundancy among ANKRD28/ANKRD44/ANKRD52 scaffold subunits, and how ANKRD28's PP6 and DOCK180 functions are spatially and temporally coordinated remain unresolved.
  • No structural model of the ANKRD28-containing PP6 heterotrimer exists
  • Subunit-specific substrate selectivity among the three ankyrin-repeat scaffolds is not defined
  • In vivo physiological roles of ANKRD28 (e.g., animal models) have not been reported

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0098772 molecular function regulator activity 3 GO:0060090 molecular adaptor activity 2
Localization
GO:0005829 cytosol 2
Pathway
R-HSA-162582 Signal Transduction 3 R-HSA-168256 Immune System 2
Partners
BRCA1CUL5DOCK1PPP6CPPP6R1PPP6R3RAB40C
Complex memberships
PP6 holoenzyme

Evidence

Reading pass · 7 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2008 ANKRD28 is a regulatory subunit of protein phosphatase 6 (PP6), forming a heterotrimer with the PP6 catalytic subunit and SAPS-domain scaffold subunits (PP6R1, PP6R3). Tagged ANKRD28 co-precipitated with PP6 but not PP2A or PP4, and the C-terminal region of PP6R1 was sufficient to co-precipitate ANKRD28 but not PP6, demonstrating PP6R1 acts as a scaffold with separate binding regions. Endogenous PP6 holoenzymes with PP6R1, PP6R3, and ANKRD28 were resolved at >440 kDa by size-exclusion chromatography. Knockdown of PP6R1 or ANKRD28 produced equivalent enhancement of IκBε degradation in response to TNFα, placing ANKRD28 as a functional determinant of PP6 specificity. Co-immunoprecipitation (FLAG-tag and reciprocal), mass spectrometry, DEAE chromatography, size-exclusion chromatography, siRNA knockdown with IκBε degradation readout Biochemistry High 18186651
2008 ANKRD28 binds to the SH3 domain of DOCK180 (a Rac1 guanine nucleotide exchange factor) and competes with ELMO for this interaction. Knockdown of ANKRD28 in HeLa cells reduced migration velocity and altered focal adhesion protein distribution (Crk, paxillin, p130Cas). Co-expression of ANKRD28 with p130Cas, Crk, and DOCK180 induced hyper-phosphorylation of p130Cas and multiple long cellular processes, indicating ANKRD28 specifies localization and activity of the DOCK180-Rac1 pathway. Nano-LC/MS/MS pulldown, RNA interference knockdown, migration assays, immunofluorescence of focal adhesion proteins, co-expression overexpression studies Experimental cell research High 19118547
2007 ANKRD28 is fused to NUP98 in a cryptic translocation t(3;5;11)(p25;q35;p15) generating an in-frame ANKRD28-NUP98 fusion transcript. Transient overexpression of ANKRD28-NUP98 in NIH/3T3 cells caused significantly increased focus formation (oncogenic transformation), whereas a C-terminal deletion mutant (ΔC-ANKRD28-NUP98) did not. ANKRD28-NUP98 localized to the nucleolus and cytoplasm, whereas wild-type ANKRD28 and ΔC-ANKRD28 were exclusively cytoplasmic, indicating altered subcellular localization contributes to leukemogenesis. FISH breakpoint mapping, RT-PCR for fusion transcripts, transient overexpression focus formation assay in NIH/3T3 cells, fluorescence microscopy for subcellular localization International journal of hematology Medium 17988990
2016 BRCA1 interacts with ANKRD28 as identified by yeast two-hybrid screen and confirmed by reciprocal co-immunoprecipitation of overexpressed proteins and endogenous co-immunoprecipitation. The interaction is located in the cytoplasm (confirmed by proximity ligation assay) and maps to the intrinsically disordered scaffold central region of BRCA1. BRCA1 overexpression stabilizes IκBε upon TNFα stimulation, an effect lost with a truncated BRCA1 that cannot bind ANKRD28, suggesting BRCA1 modulates PP6 signaling through ANKRD28. Yeast two-hybrid screen, reciprocal co-immunoprecipitation (overexpressed and endogenous), proximity ligation assay, siRNA knockdown, IκBε stability assay upon TNFα stimulation The Biochemical journal Medium 27026398
2022 Rab40c forms a ubiquitin E3 ligase complex with Cullin5 (Rab40c/CRL5) that ubiquitylates ANKRD28, leading to its lysosomal degradation. Loss of Rab40c in MDA-MB-231 cells alters focal adhesion number, size, and distribution, decreases phosphorylation of FAK and MOB1, and increases PP6 activity. This places ANKRD28 ubiquitylation by Rab40c/CRL5 as a regulatory node controlling PP6 activity, FAK signaling, and Hippo pathway signaling at focal adhesions. Rab40c knockout cells, co-immunoprecipitation, ubiquitylation assays, focal adhesion imaging, phosphoproteomics (FAK, MOB1 phosphorylation), lysosomal inhibitor rescue experiments Life science alliance High 35512830
2009 ANKRD28 interacts with the SH3 domain of DOCK180 to regulate focal adhesion dynamics and cell migration through dual modes: ANKRD28 promotes p130Cas hyperphosphorylation and focal process formation while ELMO promotes lamellipodia, demonstrating ANKRD28 and ELMO as competing regulators that specify distinct outputs of the DOCK180-Rac1 pathway. Knockdown (siRNA), co-expression overexpression, immunofluorescence, migration assays Cell adhesion & migration Medium 19458477
2024 The PP6 catalytic subunit forms a complex with regulatory subunits PP6R1/R2/R3 and scaffold subunits ANKRD28/ANKRD44/ANKRD52 as a heterotrimer. The PP6c-PP6R3 complex specifically regulates cancer stem cell markers in colorectal cancer, and PP6c knockdown decreases colony-forming ability and in vivo proliferation of CRC cell lines. siRNA knockdown, colony formation assay, in vivo xenograft, transcriptome analysis (RNA-seq), sphere formation assay Cancer science Medium 39014521

Source papers

Stage 0 corpus · 24 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2008 Protein phosphatase 6 regulatory subunits composed of ankyrin repeat domains. Biochemistry 95 18186651
2012 Genetic and epigenetic analysis of non-small cell lung cancer with NotI-microarrays. Epigenetics 62 22491060
2014 Primary urethral clear-cell adenocarcinoma: comprehensive analysis by surgical pathology, cytopathology, and next-generation sequencing. The American journal of pathology 49 24389164
2020 Blockade of interleukin-2-inducible T-cell kinase signaling attenuates acute lung injury in mice through adjustment of pulmonary Th17/Treg immune responses and reduction of oxidative stress. International immunopharmacology 46 32163900
2020 Inhibition of interleukin-2-inducible T-cell kinase causes reduction in imiquimod-induced psoriasiform inflammation through reduction of Th17 cells and enhancement of Treg cells in mice. Biochimie 41 33007409
2008 Ankyrin repeat domain 28 (ANKRD28), a novel binding partner of DOCK180, promotes cell migration by regulating focal adhesion formation. Experimental cell research 27 19118547
2021 Role of ITK signaling in acute kidney injury in mice: Amelioration of acute kidney injury associated clinical parameters and attenuation of inflammatory transcription factor signaling in CD4+ T cells by ITK inhibition. International immunopharmacology 19 34365077
2021 Targeting of the Tec Kinase ITK Drives Resolution of T Cell-Mediated Colitis and Emerges as Potential Therapeutic Option in Ulcerative Colitis. Gastroenterology 18 34224738
2007 A novel gene, ANKRD28 on 3p25, is fused with NUP98 on 11p15 in a cryptic 3-way translocation of t(3;5;11)(p25;q35;p15) in an adult patient with myelodysplastic syndrome/acute myelogenous leukemia. International journal of hematology 17 17988990
2006 PITK, a PP1 targeting subunit that modulates the phosphorylation of the transcriptional regulator hnRNP K. Cellular signalling 15 16564677
2016 BRCA1 affects protein phosphatase 6 signalling through its interaction with ANKRD28. The Biochemical journal 14 27026398
2015 Polygenic inheritance of cryptorchidism susceptibility in the LE/orl rat. Molecular human reproduction 13 26502805
2012 Detection of significant pathways in osteoporosis based on graph clustering. Molecular medicine reports 12 22992777
2022 Rab40c regulates focal adhesions and PP6 activity by controlling ANKRD28 ubiquitylation. Life science alliance 11 35512830
2016 Interleukin-2-inducible T-cell kinase expression and relation to disease severity in systemic lupus erythematosus. Clinica chimica acta; international journal of clinical chemistry 11 27729219
2009 Regulation of focal adhesion and cell migration by ANKRD28-DOCK180 interaction. Cell adhesion & migration 10 19458477
2006 Dual kinase-mediated regulation of PITK by CaMKII and GSK3. Cellular signalling 9 17023142
2025 Enhancer Extrachromosomal Circular DNA ANKRD28 Elicits Drug Resistance via POU2F2-Mediated Transcriptional Network in Multiple Myeloma. Advanced science (Weinheim, Baden-Wurttemberg, Germany) 8 40167268
2023 Effects of Co-Culture EBV-miR-BART1-3p on Proliferation and Invasion of Gastric Cancer Cells Based on Exosomes. Cancers 6 37345178
2015 Analysis of gene profiles in glioma cells identifies potential genes, miRNAs, and target sites of migratory cells. Tumori 6 25953448
2024 Unveiling diabetic nephropathy: a novel diagnostic model through single-cell sequencing and co-expression analysis. Aging 4 38968594
2024 Protein phosphatase 6 promotes stemness of colorectal cancer cells. Cancer science 4 39014521
2023 Integrative genomic profiling reveals characteristics of lymph node metastasis in small cell lung cancer. Translational lung cancer research 4 36895932
2015 Characterization of PI (breast cancer cell special peptide) in MDA-MB-231 breast cancer cells and its potential therapeutic applications. International journal of oncology 4 26693549