ANKRD13D

Summary: Negative regulation of receptor internalization (IEA). Overexpression of ANKRD13D blocks rapid EGFR internalization, but the source paper argues the endogenous role is positive, as UniProt's own function text says; the sign-neutral parent is the defensible term.

Reason: The inhibition is an overexpression phenotype [PMID:22298428 "In striking contrast, EGFR was mostly retained at the surface in cells overexpressing FLAG-tagged Ankrd 13A, 13B, or 13D (Figure 7, C–E′′)."]. Truncated mutants [PMID:22298428 "which were expected to act as dominant-negative versions"] inhibit as wild type does, which the authors read as [PMID:22298428 "ectopic overexpression of not only truncated but also wild-type Ankrd 13 inhibits the function of endogenous Ankrd 13"]. They therefore pose the sign as open [PMID:22298428 "raising the question of whether Ankrd 13 proteins have a stimulatory or inhibitory role in the process"] and conclude [PMID:22298428 "we propose that Ankrd 13A, 13B, and 13D positively regulate the internalization of ligand-activated EGFR"]; UniProt follows this [file:human/ANKRD13D/ANKRD13D-uniprot.txt "ubiquitin. Positively regulates the internalization of ligand-activated"]. RNAi could not test it because the siRNAs [PMID:22298428 "failed to deplete the three Ankrd 13 mRNAs/proteins simultaneously"]. GO:0002090 regulation of receptor internalization keeps what is shown without asserting the direction. GO:0002092 positive regulation of receptor internalization, which the UniProt text would suggest, is not proposed: it is the authors' interpretation, and no loss-of-function experiment has tested it.

Proposed replacements: regulation of receptor internalization

Summary: Negative regulation of receptor internalization (IMP). Overexpression of ANKRD13D blocks rapid EGFR internalization, but the source paper argues the endogenous role is positive, as UniProt's own function text says; the sign-neutral parent is the defensible term.

Reason: The inhibition is an overexpression phenotype [PMID:22298428 "In striking contrast, EGFR was mostly retained at the surface in cells overexpressing FLAG-tagged Ankrd 13A, 13B, or 13D (Figure 7, C–E′′)."]. Truncated mutants [PMID:22298428 "which were expected to act as dominant-negative versions"] inhibit as wild type does, which the authors read as [PMID:22298428 "ectopic overexpression of not only truncated but also wild-type Ankrd 13 inhibits the function of endogenous Ankrd 13"]. They therefore pose the sign as open [PMID:22298428 "raising the question of whether Ankrd 13 proteins have a stimulatory or inhibitory role in the process"] and conclude [PMID:22298428 "we propose that Ankrd 13A, 13B, and 13D positively regulate the internalization of ligand-activated EGFR"]; UniProt follows this [file:human/ANKRD13D/ANKRD13D-uniprot.txt "ubiquitin. Positively regulates the internalization of ligand-activated"]. RNAi could not test it because the siRNAs [PMID:22298428 "failed to deplete the three Ankrd 13 mRNAs/proteins simultaneously"]. GO:0002090 regulation of receptor internalization keeps what is shown without asserting the direction. GO:0002092 positive regulation of receptor internalization, which the UniProt text would suggest, is not proposed: it is the authors' interpretation, and no loss-of-function experiment has tested it.

Proposed replacements: regulation of receptor internalization

Summary: Protein binding (IPI) with ENTREP1 (FAM189A2), an endosomal ITCH activator. The interaction is reproducible but GO:0005515 carries no functional information and ENTREP1 has no activity term that would name it.

Reason: UniProt records the pair across three experiments [file:human/ANKRD13D/ANKRD13D-uniprot.txt "Q6ZTN6; Q15884: ENTREP1; NbExp=3; IntAct=EBI-947028, EBI-8636612;"], from the BioPlex screens and the ENTREP paper, where [PMID:34927784 "we searched for its binding partners by yeast two‐hybrid screening"]. ENTREP1, in the words of that paper, [PMID:34927784 "FAM189A2, a downregulated gene in breast cancer, encodes a new type of ITCH"] activator. The pairing is biologically coherent with the ANKRD13 proteins' links to ubiquitin ligases [PMID:31985874 "We show that ANKRD13A, ANKRD13B and ANKRD13D form a complex with RNF11 in vivo and that the UIMs are required for complex formation."], but no informative MF term exists for it; it is raised as a question instead.

Summary: Protein binding (IPI) with ENTREP1 (FAM189A2), an endosomal ITCH activator. The interaction is reproducible but GO:0005515 carries no functional information and ENTREP1 has no activity term that would name it.

Reason: UniProt records the pair across three experiments [file:human/ANKRD13D/ANKRD13D-uniprot.txt "Q6ZTN6; Q15884: ENTREP1; NbExp=3; IntAct=EBI-947028, EBI-8636612;"], from the BioPlex screens and the ENTREP paper, where [PMID:34927784 "we searched for its binding partners by yeast two‐hybrid screening"]. ENTREP1, in the words of that paper, [PMID:34927784 "FAM189A2, a downregulated gene in breast cancer, encodes a new type of ITCH"] activator. The pairing is biologically coherent with the ANKRD13 proteins' links to ubiquitin ligases [PMID:31985874 "We show that ANKRD13A, ANKRD13B and ANKRD13D form a complex with RNF11 in vivo and that the UIMs are required for complex formation."], but no informative MF term exists for it; it is raised as a question instead.

Summary: Protein binding (IPI) with ENTREP1 (FAM189A2), an endosomal ITCH activator. The interaction is reproducible but GO:0005515 carries no functional information and ENTREP1 has no activity term that would name it.

Reason: UniProt records the pair across three experiments [file:human/ANKRD13D/ANKRD13D-uniprot.txt "Q6ZTN6; Q15884: ENTREP1; NbExp=3; IntAct=EBI-947028, EBI-8636612;"], from the BioPlex screens and the ENTREP paper, where [PMID:34927784 "we searched for its binding partners by yeast two‐hybrid screening"]. ENTREP1, in the words of that paper, [PMID:34927784 "FAM189A2, a downregulated gene in breast cancer, encodes a new type of ITCH"] activator. The pairing is biologically coherent with the ANKRD13 proteins' links to ubiquitin ligases [PMID:31985874 "We show that ANKRD13A, ANKRD13B and ANKRD13D form a complex with RNF11 in vivo and that the UIMs are required for complex formation."], but no informative MF term exists for it; it is raised as a question instead.

Summary: Cytoplasm (IDA). ANKRD13D has a cytoplasmic reticular distribution in addition to its membrane pool; accepted.

Reason: [PMID:22298428 "FLAG-tagged Ankrd 13A, 13B, and 13D were also distributed intracellularly"] [PMID:22298428 "the cytoplasmic reticular distribution pattern of Ankrd 13A and 13D did not overlap with that of CD3δ, a marker for the endoplasmic reticulum"]

Summary: Late endosome (IBA). Late-endosome colocalization is strong for ANKRD13A and ANKRD13B but only marginal for ANKRD13D; kept as non-core.

Reason: [PMID:22298428 "13A and 13B colocalized considerably with a late endosome marker—cation-independent mannose 6-phosphate receptor (CI-M6PR)—in the perinuclear region"], whereas [PMID:22298428 "Ankrd 13D marginally colocalized with CI-M6PR (Supplemental Figure S1, C–C′′)."] The PAINT node for this term is seeded by ANKRD13A and ANKRD13B only. The marginal signal is consistent with a minor late-endosome pool, and the evidence rests on FLAG-tagged overexpression in HeLa cells.

Summary: Late endosome (IEA, from UniProt subcellular location). UniProt records late endosome for ANKRD13D as an experimental finding from PMID:22298428; the colocalization reported there is marginal, so kept as non-core.

Reason: UniProt asserts [file:human/ANKRD13D/ANKRD13D-uniprot.txt "endosome {ECO:0000269|PubMed:22298428}. Note=Interaction with EGFR may"], citing the paper whose text says [PMID:22298428 "Ankrd 13D marginally colocalized with CI-M6PR (Supplemental Figure S1, C–C′′)."] The localization is real but minor compared with ANKRD13A and ANKRD13B [PMID:22298428 "13A and 13B colocalized considerably with a late endosome marker—cation-independent mannose 6-phosphate receptor (CI-M6PR)—in the perinuclear region"].

Summary: Plasma membrane (IBA). Endogenous ANKRD13D is bound to cell-surface molecules, more so after EGF, and colocalizes with EGFR at the cell periphery; accepted.

Reason: [PMID:22298428 "Similarly, endogenous Ankrd 13A and 13D, but not 13C, were coprecipitated with biotinylated molecules from untransfected cells, and the amounts of plasma membrane-bound endogenous 13A and 13D were elevated after 5 min of EGF stimulation (Figure 6B)."] [PMID:22298428 "On double staining with anti-FLAG and anti-EGFR antibodies at 5 min after EGF treatment, colocalization of Ankrd 13A, 13B, and 13D with EGFR was observed at the cell periphery in a proportion of the cells (Figure 5, arrowheads)."]

Summary: Plasma membrane (IDA). Endogenous ANKRD13D is bound to cell-surface molecules, more so after EGF, and colocalizes with EGFR at the cell periphery; accepted.

Reason: [PMID:22298428 "Similarly, endogenous Ankrd 13A and 13D, but not 13C, were coprecipitated with biotinylated molecules from untransfected cells, and the amounts of plasma membrane-bound endogenous 13A and 13D were elevated after 5 min of EGF stimulation (Figure 6B)."] [PMID:22298428 "On double staining with anti-FLAG and anti-EGFR antibodies at 5 min after EGF treatment, colocalization of Ankrd 13A, 13B, and 13D with EGFR was observed at the cell periphery in a proportion of the cells (Figure 5, arrowheads)."]

Summary: Plasma membrane (IEA). Endogenous ANKRD13D is bound to cell-surface molecules, more so after EGF, and colocalizes with EGFR at the cell periphery; accepted.

Reason: [PMID:22298428 "Similarly, endogenous Ankrd 13A and 13D, but not 13C, were coprecipitated with biotinylated molecules from untransfected cells, and the amounts of plasma membrane-bound endogenous 13A and 13D were elevated after 5 min of EGF stimulation (Figure 6B)."] [PMID:22298428 "On double staining with anti-FLAG and anti-EGFR antibodies at 5 min after EGF treatment, colocalization of Ankrd 13A, 13B, and 13D with EGFR was observed at the cell periphery in a proportion of the cells (Figure 5, arrowheads)."]

Summary: Regulation of receptor-mediated endocytosis (IBA). ANKRD13 proteins regulate the rate of rapid EGFR internalization; this sign-neutral term matches the evidence; accepted.

Reason: [PMID:22298428 "On the basis of these results, we propose that Ankrd 13A, 13B, and 13D regulate the rate of rapid internalization but are not essential for internalization."] Because truncated mutants [PMID:22298428 "which were expected to act as dominant-negative versions"] also inhibit, the authors leave the sign open [PMID:22298428 "raising the question of whether Ankrd 13 proteins have a stimulatory or inhibitory role in the process"] and favour a positive role [PMID:22298428 "we propose that Ankrd 13A, 13B, and 13D positively regulate the internalization of ligand-activated EGFR"].

Summary: Perinuclear region of cytoplasm (IDA). The text of PMID:22298428 places ANKRD13A and ANKRD13B, not ANKRD13D, in the perinuclear region; the curator may have read this from a figure not in the cached text.

Reason: The perinuclear statement names 13A and 13B [PMID:22298428 "13A and 13B colocalized considerably with a late endosome marker—cation-independent mannose 6-phosphate receptor (CI-M6PR)—in the perinuclear region"], and for ANKRD13D the text says only [PMID:22298428 "Ankrd 13D marginally colocalized with CI-M6PR (Supplemental Figure S1, C–C′′)."] and describes [PMID:22298428 "the cytoplasmic reticular distribution pattern of Ankrd 13A and 13D did not overlap with that of CD3δ, a marker for the endoplasmic reticulum"]. The supplementary figure is not available here, so the curator's reading cannot be checked.

Summary: Perinuclear region of cytoplasm (IEA, ARBA). Rule-based transfer from the ANKRD13 family; the ANKRD13D-specific text does not place it in the perinuclear region, and the IDA row for the same term cannot be checked either.

Reason: The family's perinuclear late-endosome signal comes from ANKRD13A and ANKRD13B [PMID:22298428 "13A and 13B colocalized considerably with a late endosome marker—cation-independent mannose 6-phosphate receptor (CI-M6PR)—in the perinuclear region"], while [PMID:22298428 "Ankrd 13D marginally colocalized with CI-M6PR (Supplemental Figure S1, C–C′′)."]

Summary: Ubiquitin-modified protein reader activity (IBA). ANKRD13D's UIMs bind Lys63-linked polyubiquitin and ubiquitinated, ligand-activated EGFR; accepted.

Reason: [PMID:22298428 "As shown in Figure 4A, Ankrd 13A, 13B, and 13D, but not 13C, pulled down Lys-63–linked, but not Lys-48–linked, Ub chains."] [PMID:22298428 "Blotting with anti-FLAG antibody showed that Ankrd 13A, 13B, and 13D, but not 13C, bind to EGFR upon EGF treatment (Figure 1B)."]

Summary: Ubiquitin-modified protein reader activity (IDA). ANKRD13D's UIMs bind Lys63-linked polyubiquitin and ubiquitinated, ligand-activated EGFR; accepted.

Reason: [PMID:22298428 "As shown in Figure 4A, Ankrd 13A, 13B, and 13D, but not 13C, pulled down Lys-63–linked, but not Lys-48–linked, Ub chains."] [PMID:22298428 "Blotting with anti-FLAG antibody showed that Ankrd 13A, 13B, and 13D, but not 13C, bind to EGFR upon EGF treatment (Figure 1B)."]

Summary: Ubiquitin-modified protein reader activity (IEA). ANKRD13D's UIMs bind Lys63-linked polyubiquitin and ubiquitinated, ligand-activated EGFR; accepted.

Reason: [PMID:22298428 "As shown in Figure 4A, Ankrd 13A, 13B, and 13D, but not 13C, pulled down Lys-63–linked, but not Lys-48–linked, Ub chains."] [PMID:22298428 "Blotting with anti-FLAG antibody showed that Ankrd 13A, 13B, and 13D, but not 13C, bind to EGFR upon EGF treatment (Figure 1B)."]

Summary: Ubiquitin-modified protein reader activity (IPI). ANKRD13D's UIMs bind Lys63-linked polyubiquitin and ubiquitinated, ligand-activated EGFR; accepted.

Reason: [PMID:22298428 "As shown in Figure 4A, Ankrd 13A, 13B, and 13D, but not 13C, pulled down Lys-63–linked, but not Lys-48–linked, Ub chains."] [PMID:22298428 "Blotting with anti-FLAG antibody showed that Ankrd 13A, 13B, and 13D, but not 13C, bind to EGFR upon EGF treatment (Figure 1B)."] [PMID:22298428 "Blotting of the precipitates with the anti–Ankrd 13 antibodies showed that endogenous Ankrd 13A and 13D, but not 13C, bind to EGFR upon EGF treatment with the same time course as transfected Ankrd 13 proteins (Figure 2B)."]

Summary: K63-linked polyubiquitin modification-dependent protein binding (NEW, IDA). ANKRD13D pulls down Lys63-linked but not Lys48-linked ubiquitin chains; this linkage selectivity is not captured by GO:0140036.

Reason: [PMID:22298428 "As shown in Figure 4A, Ankrd 13A, 13B, and 13D, but not 13C, pulled down Lys-63–linked, but not Lys-48–linked, Ub chains."] UniProt records the same selectivity [file:human/ANKRD13D/ANKRD13D-uniprot.txt "binds 'Lys-63'-linked ubiquitin. Does not bind 'Lys-48'-linked"]. GO:0070530 is not an ancestor or descendant of GO:0140036 (both sit under modification-dependent protein binding), so it adds the linkage specificity without redundancy.