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

In the paper

“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).”

What the sources say

• Partly supported

  The Discussion (page unavailable) supports a protective role for USP8 and cites evidence of direct EGFR deubiquitination. But the paper presents its proposed mechanism as a suggestion, not an established consensus; its introduction (page unavailable) explicitly describes conflicting reports on EGFR degradation after USP8 knockdown. (LLM, conf=0.87)

  cites [24] Niendorf, 2007, Mol. Cell. Biol. sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [27] Mizuno, 2005, Mol. Biol. Cell source
• Disputed

  In “EGF Degradation Does Not” (page unavailable), the authors report that UBPY depletion significantly inhibits EGF degradation, the opposite of the predicted effect if UBPY protected the receptor from down-regulation. They also speculate that UBPY acts on MVB sorting machinery rather than directly on EGFR; they identify AMSH, not UBPY, as thought to act directly on EGFR. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  cites [41] Bowers, 2006, J. Biol. Chem. sourceExperimental — accuracy not yet measured

In the paper

“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).”

What the sources say

• Supported

  The Abstract reports that loss of UBPy (USP8) strongly reduces EGFR levels, directly supporting its role in EGFR regulation. The passage describing earlier RNA-interference studies also says they found altered EGFR degradation, though they disagreed on its direction. (LLM, conf=0.94)

  cites [24] Niendorf, 2007, Mol. Cell. Biol. source
• ?Not checked — no full text of the sourcecites [27] Mizuno, 2005, Mol. Biol. Cell source
• Supported

  In “EGF Degradation Does Not Require Mammalian ESCRTII” (Fig. 6b; page not provided), the paper reports that UBPY depletion significantly inhibits degradation of EGF, which it describes as internalized with EGFR. This supports the broad claim that UBPY had been implicated in EGFR regulation, without establishing a direct action on the receptor. (LLM, conf=0.88)

  cites [41] Bowers, 2006, J. Biol. Chem. source

In the paper

“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).”

What the sources say

• ?Not checked — no full text of the sourcecites [19] Berry, 2002, Curr. Biol. source
• ?Not checked — no full text of the sourcecites [20] Kaneko, 2003, J. Biol. Chem. source
• ?Not checked — no full text of the sourcecites [21] Kato, 2000, J. Biol. Chem. source
• ?Not checked — no full text of the sourcecites [43] Liu, 2003, Mol. Cell source
• Supported

  In the comparison with other SH3-domain complexes (page not available), the paper reports that Mona/Gads SH3C binds RXXK-containing peptides and describes an RXXK-containing peptide bound to STAM2 SH3. It also identifies Mona/Gads SH3C and STAM2 SH3 as members of a homologous SH3-domain subfamily. The paper notes that lysine can sometimes substitute for the motif’s arginine, but this does not undermine the cited generalization. (LLM, conf=0.88)

  cites [44] Lewitzky, 2004, J. Biol. Chem. source
• ?Not checked — no full text of the sourcecites [45] Lewitzky, 2001, Oncogene source
• Supported

  The Introduction describes atypical SH3-binding motifs that lack the canonical PxxP sequence. The Abstract identifies RxxK as the central motif of the SLP-76 peptide bound by Mona/Gads SH3C, and the Figure 1 caption identifies multiple SH3 domains reported to bind peptides with a longer consensus containing RxxK. (LLM, conf=0.90)

  cites [46] Harkiolaki, 2003, EMBO J. source

In the paper

“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.”

In the paper

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

In the paper

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

In the paper

“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”

What the sources say

• ?Not checked — no full text of the sourcecites [43] Liu, 2003, Mol. Cell source

In the paper

“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).”

In the paper

“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).”

In the paper

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

In the paper

“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.”

In the paper

“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).”

In the paper

“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).”

In the paper

“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).”

In the paper

“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).”

In the paper

“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).”

In the paper

“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).”

In the paper

“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.”

In the paper

“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).”

In the paper

“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).”

In the paper

“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)”

In the paper

“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).”

What the sources say

• Not checked yetcites [24] Niendorf, 2007, Mol. Cell. Biol. source
• ?Not checked — no full text of the sourcecites [27] Mizuno, 2005, Mol. Biol. Cell source
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [41] Bowers, 2006, J. Biol. Chem. source

In the paper

“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).”

In the paper

“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).”

What the sources say

• ?Not matched

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

  cites Ref.49

In the paper

“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).”

What the sources say

• ?Not checked — no full text of the sourcecites [2] Pickart, 2001, Annu. Rev. Biochem. source
• ?Not checked — no full text of the sourcecites [3] Haglund, 2003, Nat. Cell Biol. source
• ?Not checked — no full text of the sourcecites [4] Hicke, 1996, Cell source
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [5] Duan, 2003, J. Biol. Chem. source
• ?Not checked — no full text of the sourcecites [6] Huang, 2006, Mol. Cell source
• ?Not checked — no full text of the sourcecites [7] Pennock, 2008, Mol. Cell. Biol. source

In the paper

“Two consensus RXXK motifs had previously been identified in USP8 and shown to interact with the STAM2 SH3 domain (19 -21).”

What the sources say

• ?Not checked — no full text of the sourcecites [19] Berry, 2002, Curr. Biol. source
• ?Not checked — no full text of the sourcecites [20] Kaneko, 2003, J. Biol. Chem. source
• ?Not checked — no full text of the sourcecites [21] Kato, 2000, J. Biol. Chem. source

In the paper

“USP8 activity has previously been implicated at the late endosome stage (35).”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [35] Hislop, 2009, J. Biol. Chem. source

In the paper

“A truncation of USP8 containing both MIT and Rhod has been shown to interact with activated EGFR complexes (26)”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [26] Alwan, 2007, J. Biol. Chem. source

In the paper

“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).”

What the sources say

• ?Not checked — no full text of the sourcecites [11] Kanazawa, 2003, Biochem. Biophys. Res. Commun. source

In the paper

“cellular distribution of USP8 has been reported to straddle the endosomal continuum between the early and late extremes (27,34,35)”

What the sources say

• ?Not checked — no full text of the sourcecites [27] Mizuno, 2005, Mol. Biol. Cell source
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [34] Hasdemir, 2009, J. Biol. Chem. source
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [35] Hislop, 2009, J. Biol. Chem. source

In the paper

“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)”

What the sources say

• ?Not checked — no full text of the sourcecites [10] Hanyaloglu, 2005, EMBO J. source
• ?Not checked — no full text of the sourcecites [11] Kanazawa, 2003, Biochem. Biophys. Res. Commun. source
• ?Not checked — no full text of the sourcecites [12] Raiborg, 2002, Nat. Cell Biol. source
• ?Not checked — no full text of the sourcecites [13] Urbé, 2003, J. Cell Sci. source

In the paper

“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).”

What the sources say

• ?Not checked — no full text of the sourcecites [33] Avvakumov, 2006, J. Biol. Chem. source
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [36] Cao, 2007, Mol. Cell. Biol. source
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [37] Wu, 2004, Mol. Cell. Biol. source

In the paper

“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)”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [32] Row, 2007, J. Biol. Chem. source
• ?Not checked — no full text of the sourcecites [33] Avvakumov, 2006, J. Biol. Chem. source

In the paper

“which has been shown to interact in vitro with the CHMP-1A/B protein components of the late endosomal sorting complex, ESCRT-III.”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [32] Row, 2007, J. Biol. Chem. source
• ?Not checked — no full text of the sourcecites [33] Avvakumov, 2006, J. Biol. Chem. source

In the paper

“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).”

What the sources say

• ?Not checked — no full text of the sourcecites [17] Ren, 2007, Mol. Biol. Cell source

In the paper

“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).”

What the sources say

• ?Not checked — no full text of the sourcecites [52] Feng, 1994, Science source
• ?Not checked — no full text of the sourcecites [43] Liu, 2003, Mol. Cell source

In the paper

“Ubiquitin-dependent trafficking of endocytosed EGFR proceeds in at least two phases, with each characterized by a discrete ubiquitination event (7).”

What the sources say

• ?Not checked — no full text of the sourcecites [7] Pennock, 2008, Mol. Cell. Biol. source

In the paper

“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.”

In the paper

“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.”

In the paper

“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.”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [32] Row, 2007, J. Biol. Chem. source

In the paper

“Unlike AMSH, which does not directly regulate EGFR (27) but functions to generally promote cargo trafficking through the sorting endosome (48)”

What the sources say

• ?Not checked — no full text of the sourcecites [27] Mizuno, 2005, Mol. Biol. Cell source
• ?Not checked — no full text of the sourcecites [48] Sierra, 2010, J. Biol. Chem. source

In the paper

“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.”

In the paper

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

In the paper

“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).”

What the sources say

• ?Not checked — no full text of the sourcecites [19] Berry, 2002, Curr. Biol. source
• ?Not checked — no full text of the sourcecites [45] Lewitzky, 2001, Oncogene source
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [51] Lock, 2000, J. Biol. Chem. source

In the paper

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