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Cited by 122Ubiquitin and proteasome pathwaysCellular transport and secretionHER2/EGFR in Cancer Research

The DOI matches its Crossref record; 52 of the 52 references it cites were found in a registry or an index; 1 of the 77 sources its claims cite were checked for support.

Regulation of Epidermal Growth Factor Receptor Ubiquitination and Trafficking by the USP8·STAM Complex

Open access
Berlin, Ilana, Schwartz, Heather, Nash, Piers D.·2010·Journal of Biological Chemistry
DOI 10.1074/jbc.m109.016287PMC PMC2966105
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Abstract

Reversible ubiquitination of activated receptor complexes signals their sorting between recycling and degradation and thereby dictates receptor fate. The deubiquitinating enzyme ubiquitin-specific protease 8 (USP8/UBPy) has been previously implicated in the regulation of the epidermal growth factor receptor (EGFR); however, the molecular mechanisms governing its recruitment and activity in this context remain unclear. Herein, we investigate the role of USP8 in countering ligand-induced ubiquitination and down-regulation of EGFR and characterize a subset of protein-protein interaction determinants critical for this function. USP8 depletion accelerates receptor turnover, whereas loss of hepatocyte growth factor-regulated substrate (Hrs) rescues this phenotype, indicating that USP8 protects EGFR from degradation via an Hrs-dependent pathway. Catalytic inactivation of USP8 incurs EGFR hyperubiquitination and promotes receptor localization to endosomes marked by high ubiquitin content. These phenotypes require the central region of USP8, containing three extended Arg-X-X-Lys (RXXK) motifs that specify direct low affinity interactions with the SH3 domain(s) of ESCRT-0 proteins, STAM1/2. The USP8·STAM complex critically impinges on receptor ubiquitination status and modulates ubiquitin dynamics on EGFR-positive endosomes. Consequently, USP8-mediated deubiquitination slows progression of EGFR past the early-to-recycling endosome circuit in a manner dependent upon the RXXK motifs. Collectively, these findings demonstrate a role for the USP8·STAM complex as a protective mechanism regulating early endosomal sorting of EGFR between pathways destined for lysosomal degradation and recycling.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

The paper shows that USP8 protects EGFR from ligand-induced degradation via an Hrs-dependent pathway and that its central RXXK motifs mediate low-affinity binding to STAM1/2 SH3 domains. This USP8·STAM interaction regulates EGFR ubiquitination and slows progression through the early-to-recycling endosome circuit.

Key findings

  • USP8 depletion accelerated EGFR turnover, and concomitant Hrs knockdown rescued this phenotype, indicating that USP8 protects EGFR via an Hrs-dependent pathway; USP8 depletion also increased EGFR ubiquitination at 5 min after 2.5 ng/ml EGF.
  • Catalytic inactivation of USP8 (C748A or ΔC) caused EGFR hyperubiquitination and a 2.5-fold increase in EGFR co-localization with HA-ubiquitin; these effects required the central region of USP8, whereas MIT or MIT-Rhod fragments were insufficient.
  • USP8 contains three conserved RXXK motifs that bind STAM1/2 SH3 domains with low affinity; triple RXXK mutation (R3K) or STAM1 SH3 mutation abrogated co-immunoprecipitation/BiFC complex formation. STAM1/2 overexpression increased EGFR ubiquitination by 50%, and SH3-domain mutations abrogated this effect.
  • USP8 overexpression delayed EGFR progression past transferrin receptor-positive endosomes at 45 min after EGF, whereas the R3K mutant abrogated this delay; R3K also compromised USP8-mediated EGFR deubiquitination, while ΔMIT did not.

Methods

  • HeLa cells were used for all cell-culture experiments; transient siRNA knockdown of USP8 or Hrs and overexpression of wild-type or mutant USP8, STAM1/2, Gads, ubiquitin, and other constructs were performed, including site-directed mutants C748A, R3K, and SH3-domain mutants.
  • Degradation assays used 2.5 ng/ml EGF with Western blot against endogenous EGFR; ubiquitination assays used HA-ubiquitin, EGFR immunoprecipitation, and 2.5 or 10 ng/ml EGF for 5–10 min; co-immunoprecipitation, immunofluorescence/confocal microscopy, BiFC, peptide SPOT arrays, and fluorescence polarization were also used.
  • Quantification included Western band intensities, degradation plots, Mander’s/JACoP colocalization coefficients, and equilibrium dissociation constants, with n = 2–3 independent experiments and S.D. reported.

Limitations

  • No limitations section is stated in the paper.
  • Evident from the text: all cell-culture experiments used HeLa cells, and no in vivo validation of the proposed USP8·STAM mechanism is presented in this study.
  • Evident from the text: conclusions rely on overexpression, siRNA depletion, and BiFC, which forms an irreversible fluorescent complex; the RXXK–SH3 interactions are low affinity, and the paper notes that USP8’s broad distribution has made ascribing loss of a specific protein-protein interaction difficult.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1074/jbc.m109.016287

Cites

52 of 52 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Verification

Claims the paper makes

1 of 77 cited sources checked · 1 supported · 0 flagged

  • Findingown data

    Loss of Hrs rescues the accelerated EGFR turnover caused by USP8 depletion.

    “USP8 depletion accelerates receptor turnover, whereas loss of hepatocyte growth factor-regulated substrate (Hrs) rescues this phenotype”
    • Also stated in: § USP8 Depletion Accelerates EGFR Turnover through an · boosted
      “Both effects of USP8 depletion on EGFR stability were effectively counteracted by concomitant Hrs knockdown (Fig. 1, D and E).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The effects of USP8 depletion on EGFR turnover and ubiquitination are due to specific loss of USP8 and not off-target effects of siRNA.
  • Findingown data

    The RXXK motifs of USP8 specify direct low-affinity interactions with SH3 domains of STAM1/2.

    “that specify direct low affinity interactions with the SH3 domain(s) of ESCRT-0 proteins, STAM1/2.”
    • Also stated in: § JOURNAL OF BIOLOGICAL CHEMISTRY 34915 · plain
      “Individually, each of the three RXXK motifs of USP8 was found to exhibit lower affinities for the RXXK subclass of SH3 domains”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The in vitro binding affinities and peptide array interactions reflect the interactions of full-length USP8 and STAM in cells.
  • Findingown data

    Catalytic inactivation of USP8 causes EGFR hyperubiquitination.

    “Catalytic inactivation of USP8 incurs EGFR hyperubiquitination and promotes receptor localization to endosomes marked by high ubiquitin content.”
    • Also stated in: § USP8 Depletion Accelerates EGFR Turnover through an · plain
      “inactivation of USP8 through either a point mutation in the catalytic cysteine residue (C748A) or a truncation of the entire catalytic domain (⌬C, Fig. 2A) resulted in acute hyperubiquitination of EGFR (Fig. 2, B and C)”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The USP8 C748A and ΔC mutants are catalytically inactive and act as loss-of-function forms.
  • Findingown data

    The central region of USP8 is required for the enhanced localization of EGFR to ubiquitinated endosomes.

    “As in the case of EGFR hyperubiquitination, enhanced receptor localization to ubiquitinated endosomes was abrogated upon deletion of the central region of USP8 (Fig. 2, D and E).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The RXXK motifs within the central region of USP8 mediate its effect on EGFR ubiquitination.
  • Findingown data

    USP8 acts to constrain EGFR ubiquitination in a manner dependent upon the RXXK motifs of USP8.

    “Mutational analysis reveals that USP8 acts to constrain EGFR ubiquitination in a manner dependent upon the RXXK motifs of USP8”
    • Also stated in: § Abstract · plain
      “USP8-mediated deubiquitination slows progression of EGFR past the earlyto-recycling endosome circuit in a manner dependent upon the RXXK motifs.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The RXXK motifs within the central region of USP8 mediate its effect on EGFR ubiquitination.
  • Probably not a substantial claim. 17
  • It may state two separate claims as one. 32

  • Probably not a substantial claim.

    Premisecitation

    USP8/UBPy has been previously implicated in the regulation of EGFR.

    “The deubiquitinating enzyme ubiquitin-specific protease 8 (USP8/UBPy) has been previously implicated in the regulation of the epidermal growth factor receptor (EGFR); however, the molecular mechanisms governing its recruitment and activity in this context remain unclear.”
    • Also stated in: § USP8 Depletion Accelerates EGFR Turnover through an · boosted
      “The current consensus in the literature indicates that USP8 functions to protect EGFR from ligand-induced down-regulation by virtue of direct deubiquitination of activated receptors (24,27,41).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [24] Niendorf, 2007, Mol. Cell. Biol. source
    • cites [27] Mizuno, 2005, Mol. Biol. Cell source
    • cites [41] Bowers, 2006, J. Biol. Chem. source

  • It may state two separate claims as one.

    Findingown data

    The USP8·STAM complex impinges on receptor ubiquitination status and modulates ubiquitin dynamics on EGFR-positive endosomes.

    “The USP8⅐STAM complex critically impinges on receptor ubiquitination status and modulates ubiquitin dynamics on EGFR-positive endosomes.”
    • Also stated in: § Abstract · plain
      “The USP8⅐STAM complex impinges upon the ESCRT-0 ubiquitination status and modulates ubiquitin dynamics on EGFR-positive endosomes.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    The three RXXK motifs of USP8 are highly conserved between mouse and human.

    “An alignment of the three RXXK motifs of USP8 from the mouse and human proteins indicates a high level of conservation (Fig. 3A).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    All three GST-RXXK fragments of USP8 precipitate full-length Gads but not Gads-WA.

    “All three GST-RXXK fragments of USP8 were able to precipitate full-length Gads but not Gads-WA”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingcitation

    The second RXXK motif (R2) lacks consensus residues N-terminal to Asp at -1 relative to the conserved arginine but maintains the core DRXXKP region.

    “The second RXXK motif in USP8 (R2) lacks consensus residues N-terminal to Asp located at Ϫ1 relative to the conserved arginine in both species but maintains the core region corresponding to DRXXKP”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [43] Liu, 2003, Mol. Cell source

  • It may state two separate claims as one.

    Findingown data

    EGFR internalized in response to EGF stimulation encounters the USP8·STAM complex and, in the presence of a catalytically inactive USP8, overlaps with subcellular regions of high ubiquitin content.

    “As visualized with BiFC, EGFR internalized in response to EGF stimulation encounters the USP8⅐STAM complex and, in the presence of a catalytically inactive USP8, overlaps with subcellular regions of high ubiquitin content (Fig. 5).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    The USP8·STAM2 VFP extensively colocalized with endogenous ESCRT-0 components STAM1 and Hrs but did not exhibit appreciable colocalization with the late MVB marker CD63.

    “the USP8⅐STAM2 VFP extensively colocalized with endogenous ESCRT-0 components, STAM1 and Hrs (Fig. 5, A, B, and D) but did not exhibit appreciable colocalization with a late MVB marker, CD63 (Fig. 5, C and D).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Ubiquitination of Grb2 was not affected by USP8, revealing specificity of USP8 toward the SH3 domains of STAM proteins.

    “Conversely, ubiquitination of Grb2 was not affected by USP8, revealing specificity of USP8 toward the SH3 domains of STAM proteins.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    USP8·STAM2 BiFC produced robust VFP-positive puncta that were not observed with the triple RXXK mutant of USP8, confirming that the complex forms in a manner requiring the RXXK/SH3 interaction.

    “As in the case of STAM1, USP8⅐STAM2 BiFC produced robust VFP-positive puncta that were not observed with the triple RXXK mutant of USP8 (Fig. 4C, panels 4 and 5), confirming that the complex forms in a manner requiring the RXXK/ SH3 interaction.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    USP8 overexpression attenuates progression of EGFR away from transferrin receptor-positive endosomes, whereas disruption of the USP8·STAM complex through mutations in the RXXK motifs is sufficient to abrogate the trafficking delay.

    “This model is supported by the observations demonstrating that USP8 overexpression attenuates progression of EGFR away from transferrin receptor-positive endosomes, whereas disruption of the USP8⅐STAM complex through mutations in the RXXK motifs is sufficient to abrogate the trafficking delay (Fig. 6).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    The effects of USP8 inactivation are observed nearly immediately after EGF stimulation (5-10 min), a time when a substantial portion of the receptor travels on STAM-positive endosomes.

    “The above effects of USP8 inactivation are observed nearly immediately after stimulation with EGF (5-10 min), a time when a substantial portion of the receptor travels on STAMpositive endosomes (supplemental Fig. S2).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Ectopic expression of a catalytically inactive USP8 leads to enhanced EGFR ubiquitination, which occurs in a manner dependent upon the central region of USP8 harboring STAM binding determinants.

    “Consistent with these findings, ectopic expression of a catalytically inactive USP8 leads to enhanced EGFR ubiquitination, which occurs in a manner dependent upon the central region of USP8, harboring STAM binding determinants (Fig. 2).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    The fluorescent BiFC complex was readily reconstituted in an RXXK- and SH3 domain-dependent manner between USP8 and either STAM1 or STAM2, whereas BiFC between USP8 and Grb2 was not observed.

    “Using split VFP fluorophore protein fusions, we found the fluorescent BiFC complex to be readily reconstituted in an RXXK-and SH3 domain-dependent manner between USP8 and either STAM1 or STAM2, whereas BiFC between USP8 and Grb2 was not observed (Fig. 4).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    BiFC of the USP8·STAM1 complex depended on the SH3 domain of STAM1, as the mutant VC-STAM1-WA lacked bright VFP puncta.

    “BiFC of the USP8⅐STAM1 complex exhibited dependence on the SH3 domain of STAM1, as illustrated by the lack of bright VFP puncta in cells expressing the mutant VC-STAM1-WA (Fig. 4C, panel 2).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    The RXXK mutations (R3K-CFP) abrogated the USP8 overexpression phenotype, with EGFR exhibiting a localization profile similar to that of the control.

    “whereas the RXXK mutations (R3K-CFP) abrogated this phenotype, with EGFR exhibiting a localization profile similar to that of the control (Fig. 6, D and E).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Peptides corresponding to each of the three RXXK sites bound with measurable affinity, suggesting each may contribute to binding in the context of the USP8 protein.

    “although peptides corresponding to each of the three sites bound with measurable affinity, suggesting that each one may contribute to binding in the context of the USP8 protein.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The in vitro binding affinities and peptide array interactions reflect the interactions of full-length USP8 and STAM in cells.

  • It may state two separate claims as one.

    Findingown data

    We identified a novel partial consensus RXXK site in USP8 and determined that all three USP8 motifs constitute low affinity SH3 binding partners, with equilibrium dissociation constants in the M to 10s of M range.

    “In the course of these studies, we identified a novel partial consensus RXXK site in USP8 and determined that all three USP8 motifs constitute low affinity SH3 binding partners, with equilibrium dissociation constants in the M to 10s of M range (Fig. 3).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingreasoning

    Overexpression of STAM proteins results in elevated levels of EGFR ubiquitination, presumably due to titration of cognate DUB activity away from the receptor substrate.

    “Not surprisingly, overexpression of STAM proteins under these conditions results in elevated levels of EGFR ubiquitination, presumably due to titration of cognate DUB activity away from the receptor substrate (supplemental Fig. S2).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Mutation of all three RXXK motifs in USP8 or a single mutation in the SH3 domain of STAM1 abrogates binding between the full-length proteins.

    “The mutation of all three RXXK motifs in USP8 or a single mutation in the SH3 domain of STAM1 abrogated binding between the full-length proteins (Fig. 3D)”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Our loss-of-function studies indicate a requirement for USP8 in EGFR stabilization and demonstrate that accelerated receptor degradation resulting from USP8 depletion proceeds through a pathway controlled by Hrs.

    “In agreement with published evidence, our loss-of-function studies indicate a requirement for USP8 in EGFR stabilization (24,27,41) and demonstrate that accelerated receptor degradation, resulting from USP8 depletion, proceeds through a pathway controlled by Hrs (Fig. 1).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [24] Niendorf, 2007, Mol. Cell. Biol. source
    • cites [27] Mizuno, 2005, Mol. Biol. Cell source
    • cites [41] Bowers, 2006, J. Biol. Chem. source

  • It may state two separate claims as one.

    Findingown data

    Expression of USP8 did not significantly alter the extent of EGFR localization to TrfR-positive endosomes at the time point after receptor stimulation, implying that the delay in receptor trafficking did not occur before its encounter with the sorting endosome.

    “Expression of USP8 was not found to significantly alter the extent of EGFR localization to TrfR-positive endosomes at the time point after receptor stimulation, implying that the delay in receptor trafficking did not occur before its encounter with the sorting endosome (data not shown).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingcitation

    Hyperubiquitination of EGFR in cells compromised for USP8 activity occurs quickly (5-10 min) after stimulation, on a timescale adequate for receptor internalization and possibly progression to the sorting endosome but not sufficient for late trafficking events.

    “Our work demonstrates that hyperubiquitination of EGFR in cells compromised for USP8 activity occurs quickly (5-10 min) after stimulation on a timescale adequate for receptor internalization and possibly progression to the sorting endosome but not sufficient to allow late trafficking events to take place (Ref.49, Fig. 6, and data not shown).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Ref.49

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • It may state two separate claims as one.

    Premisecitation

    Reversible ubiquitination of activated receptor complexes signals sorting between recycling and degradation and dictates receptor fate.

    “Reversible ubiquitination of activated receptor complexes signals their sorting between recycling and degradation and thereby dictates receptor fate.”
    • Also stated in: § Abstract · boosted
      “Reversible ubiquitination, orchestrated by the opposition of ubiquitin ligases and deubiquitinating enzymes (DUBs) 3 (2), specifies sorting of endocytosed cargo (3,4), with mono-ubiquitination and lysine 63-linked polyubiquitination extensively documented to promote trafficking of EGFR to the preproteolytic multivesicular body (MVB) compartment (5)(6)(7).”
    Quote in the paperSubstantial claim0 of 6 sources checked
    • cites [2] Pickart, 2001, Annu. Rev. Biochem. source
    • cites [3] Haglund, 2003, Nat. Cell Biol. source
    • cites [4] Hicke, 1996, Cell source
    • cites [5] Duan, 2003, J. Biol. Chem. source
    • cites [6] Huang, 2006, Mol. Cell source
    • cites [7] Pennock, 2008, Mol. Cell. Biol. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Two consensus RXXK motifs had previously been identified in USP8 and shown to interact with the STAM2 SH3 domain.

    “Two consensus RXXK motifs had previously been identified in USP8 and shown to interact with the STAM2 SH3 domain (19 -21).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [19] Berry, 2002, Curr. Biol. source
    • cites [20] Kaneko, 2003, J. Biol. Chem. source
    • cites [21] Kato, 2000, J. Biol. Chem. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    USP8 activity has previously been implicated at the late endosome stage.

    “USP8 activity has previously been implicated at the late endosome stage (35).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [35] Hislop, 2009, J. Biol. Chem. source

  • Probably not a substantial claim.

    Premisecitation

    A subfamily of SH3 domains binds atypical peptide ligands centered around a consensus RXXK peptide motif.

    “We and others have previously reported a subfamily of SH3 domains that bind to atypical peptide ligands centered around a consensus RXXK peptide motif (19 -21, 43-46).”
    Quote in the paperSubstantial claim0 of 7 sources checked
    • cites [19] Berry, 2002, Curr. Biol. source
    • cites [20] Kaneko, 2003, J. Biol. Chem. source
    • cites [21] Kato, 2000, J. Biol. Chem. source
    • cites [43] Liu, 2003, Mol. Cell source
    • cites [44] Lewitzky, 2004, J. Biol. Chem. source
    • cites [45] Lewitzky, 2001, Oncogene source
    • cites [46] Harkiolaki, 2003, EMBO J. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    A truncation of USP8 containing both MIT and Rhod interacts with activated EGFR complexes.

    “A truncation of USP8 containing both MIT and Rhod has been shown to interact with activated EGFR complexes (26)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] Alwan, 2007, J. Biol. Chem. source

  • It may state two separate claims as one.

    Premisecitation

    Hrs and STAM deficiencies impair EGFR trafficking and degradation and lead to endocytic swelling.

    “Similarly, deficiencies in Hrs and STAM proteins impair EGFR trafficking and degradation and lead to gross endocytic swelling, characteristic of defects in cargo progression (11).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] Kanazawa, 2003, Biochem. Biophys. Res. Commun. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Cellular distribution of USP8 straddles the endosomal continuum between early and late extremes.

    “cellular distribution of USP8 has been reported to straddle the endosomal continuum between the early and late extremes (27,34,35)”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [27] Mizuno, 2005, Mol. Biol. Cell source
    • cites [34] Hasdemir, 2009, J. Biol. Chem. source
    • cites [35] Hislop, 2009, J. Biol. Chem. source

  • Probably not a substantial claim.

    Premisecitation

    The current consensus in the literature indicates that USP8 functions to protect EGFR from ligand-induced down-regulation by direct deubiquitination of activated receptors.

    “The current consensus in the literature indicates that USP8 functions to protect EGFR from ligand-induced down-regulation by virtue of direct deubiquitination of activated receptors (24,27,41).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [24] Niendorf, 2007, Mol. Cell. Biol. source
    • cites [27] Mizuno, 2005, Mol. Biol. Cell source
    • cites [41] Bowers, 2006, J. Biol. Chem. source

  • Probably not a substantial claim.

    Premisecitation

    The ESCRT-0 complex partitions cargo arriving at the sorting endosome between recycling and progression to the MVB.

    “The ESCRT-0 complex, consisting of adaptor proteins hepatocyte growth factor-regulated substrate (Hrs) and signal transducing adaptor molecule (STAM) (9), partitions cargo arriving at the sorting endosome between recycling and progression to the MVB (10 -13)”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [10] Hanyaloglu, 2005, EMBO J. source
    • cites [11] Kanazawa, 2003, Biochem. Biophys. Res. Commun. source
    • cites [12] Raiborg, 2002, Nat. Cell Biol. source
    • cites [13] Urbé, 2003, J. Cell Sci. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    The Rhodanese-like domain of USP8 binds E3 ligase Nrdp1 and indirectly influences degradation of Erb-B3.

    “the rhodanese-like domain (Rhod) of USP8 is known to bind the E3 ligase, Nrdp1, and in so doing indirectly influences the degradation of Erb-B3 (33,36,37).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [33] Avvakumov, 2006, J. Biol. Chem. source
    • cites [36] Cao, 2007, Mol. Cell. Biol. source
    • cites [37] Wu, 2004, Mol. Cell. Biol. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    The N-terminal DUF1873 of USP8 constitutes a structurally atypical MIT domain.

    “The N-terminal domain of unknown function (DUF) 1873 of USP8 reportedly constitutes a structurally atypical low homology microtubule interacting and transport (MIT) domain (32,33)”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [32] Row, 2007, J. Biol. Chem. source
    • cites [33] Avvakumov, 2006, J. Biol. Chem. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    The MIT domain of USP8 interacts in vitro with CHMP-1A/B components of ESCRT-III.

    “which has been shown to interact in vitro with the CHMP-1A/B protein components of the late endosomal sorting complex, ESCRT-III.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [32] Row, 2007, J. Biol. Chem. source
    • cites [33] Avvakumov, 2006, J. Biol. Chem. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    The yeast ESCRT-0 complex interacts with ligase Rsp5 and DUBs Ubp2 and Ubp7 to allow cargo sorting into the MVB.

    “the yeast ESCRT-0 complex, composed of the Hrs and STAM orthologs Vps27 and Hse1, respectively, interacts with the ligase Rsp5 and the DUBs Ubp2 and Ubp7 to allow cargo sorting into the MVB (17).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [17] Ren, 2007, Mol. Biol. Cell source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Classical SH3 domains bind to proline-rich ligands that assume a polyproline type II helix, whereas this subfamily of SH3 domains interacts with R-XXK ligands, which take on a characteristic 3_10 helical conformation.

    “Although the classical SH3 domains bind to proline-rich ligands that assume a polyproline type II helix (52), this subfamily of SH3 domains interacts with R-XXK ligands, which take on a characteristic 3 10 helical conformation (43).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [52] Feng, 1994, Science source
    • cites [43] Liu, 2003, Mol. Cell source

  • Probably not a substantial claim.

    Premisecitation

    Ubiquitin-dependent trafficking of endocytosed EGFR proceeds in at least two phases, each characterized by a discrete ubiquitination event.

    “Ubiquitin-dependent trafficking of endocytosed EGFR proceeds in at least two phases, with each characterized by a discrete ubiquitination event (7).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [7] Pennock, 2008, Mol. Cell. Biol. source

  • It may state two separate claims as one.

    Interpretationreasoning

    Mutations in the SH3 domains of STAM1/2 abrogate the overexpression effect, implying a role for the SH3 domains in recruitment of a critical DUB to the ubiquitinated EGFR.

    “As mutations in the SH3 domains of STAM1/2 abrogate the overexpression effect, these results imply a role for the SH3 domains in recruitment of a critical DUB to the ubiquitinated EGFR.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    USP8 and the ESCRT-0 machinery constitute a key regulatory mechanism for the determination of EGFR fate after endocytosis and link reversible ubiquitination to the spatiotemporal regulation of receptor trafficking.

    “Taken together, these findings demonstrate that USP8 and the ESCRT-0 machinery constitute a key regulatory mechanism for the determination of EGFR fate after endocytosis and in doing so link reversible ubiquitination to the spatiotemporal regulation of receptor trafficking.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationcitation

    The MIT domain likely constitutes a late endosomal localization determinant irrelevant to the early phase of EGFR trafficking directly controlled by USP8.

    “As the MIT domain reportedly interacts with the ESCRT-III proteins (32), it likely constitutes a late endosomal localization determinant irrelevant to the early phase of EGFR trafficking directly controlled by USP8.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Row, 2007, J. Biol. Chem. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    AMSH does not directly regulate EGFR but functions to generally promote cargo trafficking through the sorting endosome.

    “Unlike AMSH, which does not directly regulate EGFR (27) but functions to generally promote cargo trafficking through the sorting endosome (48)”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [27] Mizuno, 2005, Mol. Biol. Cell source
    • cites [48] Sierra, 2010, J. Biol. Chem. source

  • It may state two separate claims as one.

    Interpretationown data

    The biochemical characterization of these interactions is consistent with transient association between USP8 and relevant proteins in vivo, well suited to support recruitment of USP8 to a dynamic cellular compartment, such as the endocytic pathway.

    “The biochemical characterization of these interactions is consistent with transient association between USP8 and relevant proteins in vivo, well suited to support recruitment of USP8 to a dynamic cellular compartment, such as the endocytic pathway.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    USP8 employs RXXK-mediated interaction(s), but not those afforded by the MIT domain, to influence ligand-induced ubiquitination of EGFR.

    “These results demonstrate that USP8 employs RXXK-mediated interaction(s), but not those afforded by the MIT domain, to influence ligandinduced ubiquitination of EGFR.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    We and others have previously reported two motifs in USP8 that interact with atypical SH3 domains of proteins belonging to the Grb2 and STAM adaptor families.

    “We and others have previously reported two motifs in USP8 that interact with atypical SH3 domains of proteins belonging to the Grb2 and STAM adaptor families (19,45,51).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [19] Berry, 2002, Curr. Biol. source
    • cites [45] Lewitzky, 2001, Oncogene source
    • cites [51] Lock, 2000, J. Biol. Chem. source

  • It may state two separate claims as one.

    Premisecitation

    Subsequently, Cbl-b ubiquitinates EGFR in the second phase, which is required for the irrevocable commitment to degradation.

    “Subsequently, Cbl-b ubiquitinates EGFR in the second phase, required for the irrevocable commitment to degradation.”
    Quote in the paperNot a substantial claim
45 of 45

  • The paper relies on this without support.

    The effects of USP8 depletion on EGFR turnover and ubiquitination are due to specific loss of USP8 and not off-target effects of siRNA.

    “Ambion oligonucleotide 105117 targeted against USP8 (siUSP8) and Silencer Negative Control #1 were obtained from Applied Biosystems.”
    “A previously reported sequence (27) coding an anti-USP8 siRNA was custom-synthesized by Integrated DNA Technologies and cloned into the pSilencer vector according to the manufacturer's instructions.”

  • The paper relies on this without support.

    The USP8 C748A and ΔC mutants are catalytically inactive and act as loss-of-function forms.

    “inactivation of USP8 through either a point mutation in the catalytic cysteine residue (C748A) or a truncation of the entire catalytic domain (⌬C, Fig. 2A) resulted in acute hyperubiquitination of EGFR”

  • The paper relies on this without support.

    The RXXK motifs within the central region of USP8 mediate its effect on EGFR ubiquitination.

    “These phenotypes require the central region of USP8, containing three extended Arg-X-X-Lys (RXXK) motifs that specify direct low affinity interactions with the SH3 domain(s) of ESCRT-0 proteins, STAM1/2.”

  • The paper relies on this without support.

    The in vitro binding affinities and peptide array interactions reflect the interactions of full-length USP8 and STAM in cells.

    “Individually, each of the three RXXK motifs of USP8 was found to exhibit lower affinities for the RXXK subclass of SH3 domains than any of the previously measured interactions with single RXXK motifs in Slp76, Blnk, Gab1, or Gab2 (19).”

  • The paper relies on this without support.

    HeLa cells are a valid model for EGFR trafficking in vivo.

    “HeLa cells were used for all experiments requiring cell cultures in this study.”

  • The paper relies on this without support.

    No experimental evidence had been produced that the USP8 RXXK–STAM SH3 complex promotes USP8 function on endosomes.

    “Although the complex between the RXXK motifs of USP8 and the SH3 domains of STAM has previously been speculated to promote USP8 function on endosomes, no experimental evidence to that end has been produced.”

  • The paper relies on this without support.

    The ability of USP8 to influence receptor ubiquitination and turnover through its interaction with the ESCRT machinery had not been described.

    “Although the association of USP8 with the ESCRT machinery has been previously speculated to target the DUB to its EGFR substrate on endosomes, the ability of USP8 to influence receptor ubiquitination and turnover by virtue of such interaction(s) has not been described.”

  • The paper relies on this without support.

    Previous studies had not addressed the temporal aspects of USP8 function in EGFR deubiquitination.

    “Although it has been appreciated for some time that USP8 promotes EGFR deubiquitination, previous studies have not addressed the temporal aspects of USP8 function in this context.”

  • The paper relies on this without support.

    The molecular determinants responsible for targeting USP8 activity to EGFR and STAM in their cellular context had not been elucidated.

    “Although both EGFR and STAM have formerly been identified as USP8 substrates, the molecular determinants responsible for targeting USP8 activity to these proteins in their cellular context have not been elucidated.”

  • The paper relies on this without support.

    No phenotypic outcomes for disrupting the USP8·STAM interaction in endocytosis had been identified.

    “Similarly, no phenotypic outcomes for disrupting the USP8⅐STAM interaction in endocytosis have been identified.”

  • The paper relies on this without support.

    No direct experimental evidence in support of the STAM-mediated recruitment of USP8 to the sorting endosome model had been produced.

    “Although it has previously been speculated that STAMmediated recruitment of USP8 to the sorting endosome may constitute an important regulatory mechanism in EGFR trafficking (21,29), no direct experimental evidence in support of this model has been produced (27).”

  • The paper relies on this without support.

    EGFR ubiquitination status dictates partitioning of endocytosed receptors between recycling and degradation.

    “Because EGFR ubiquitination status dictates partitioning of endocytosed receptors between recycling and degradation”
12 of 12

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Citations

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Research works printed but not linked: 1

  1. Hu, 2005, Curr. Protoc. Protein Sci., 19.10.1

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Metadata

Authors
Berlin, I., Schwartz, H., & Nash, P. D.
Affiliations
University of Chicago (US)
Corresponding author
Nash, Piers D.
ORCID
Berlin, Ilana
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2010-11
Volume
285
Issue
45
Pages
34909-34921
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m109.016287
PMC
PMC2966105
OpenAlex
W1583797852
Cited by
121
References
52
Citations by year
2026: 7 · 2025: 8 · 2024: 15 · 2023: 10 · 2022: 6 · 2021: 5 · 2020: 5 · 2019: 3 · 2018: 4 · 2017: 7 · 2016: 5 · 2015: 5 · 2014: 9 · 2013: 12 · 2012: 10
FWCI
3.29
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • U.S. Department of Defense
  • American Cancer Society
  • V Foundation for Cancer Research
  • Concern Foundation
  • Purdue University
  • Congressionally Directed Medical Research Programs
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Health Sciences › Medicine › Oncology › HER2/EGFR in Cancer Research
Keywords
epidermal growth factor receptor, USP8, STAM, deubiquitination, receptor trafficking, receptor degradation, Hrs, ESCRT-0, SH3 domain, early endosomes, endosomal sorting, receptor recycling
MeSH terms
Animals, HeLa Cells, Humans, Lysosomes, Endopeptidases, Phosphoproteins, Endosomes, Protein Structure, Tertiary, Amino Acid Motifs, Protein Transport, Ubiquitin Thiolesterase, Multiprotein Complexes, Adaptor Proteins, Signal Transducing, Mice, Ubiquitination, Endosomal Sorting Complexes Required for Transport, ErbB Receptors

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

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