Editors' ChoiceCell Biology

Pseudophosphatase as E3 ubiquitin ligase inhibitor

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Sci. Signal.  14 Feb 2017:
Vol. 10, Issue 466,
DOI: 10.1126/scisignal.aam9607

The pseudophosphatase STYX prevents the substrate recognition subunit FBXW7 from binding the catalytic E3 ubiquitin ligase complex.

Enzymes with mutations in residues essential for catalytic activity are referred to as pseudoenzymes. To investigate the function of the pseudophosphatase STYX, Reiterer et al. performed mass spectrometry analysis of proteins that coimmunopreciptated with a tagged form of STYX overexpressed in transfected cells. The putative interacting partners were enriched in F-box proteins (FBPs), which function as the substrate recognition subunit, most commonly for proteins tagged with phosphodegrons, of the three subunit E3 ubiquitin ligases of the SKP1/CUL1/F-box (SCF) family, including the tumor suppressor FBXW7. STYX coimmunoprecipitated and colocalized with FBXW7—specifically FBXW7α and FBXW7γ and not FBXW7β—and recombinant forms of STYX and FBXW7 directly interacted in vitro. Overexpression or knockdown of FBXW7 did not alter the abundance of STYX, indicating that STYX was not a substrate of FBXW7 and was not targeted for degradation by this SCF complex. However, overexpression of STYX or knockdown of STYX increased or decreased the abundance of FBXW7 substrates, including c-myc, cyclin E, and MCL1, consistent with STYX inhibiting FBXW7 function. Analysis of the ability of FBXW7 mutants to interact with various partners indicated that STYX competed with SKP1 of the E3 ubiquitin ligase complex for interacting with FBXW7 but not with substrates, indicating that STYX inhibited FBXW7 by preventing its binding to the catalytic portion of the complex. Knockdown of STYX in a breast cancer cell line reduced the abundance of MCL1, increased susceptibility to apoptosis in response to the DNA-damaging agent doxorubicin, and decreased anchorage-free colony formation. Transcript abundance for STYX and FBXW7 were inversely correlated, with STYX transcripts more abundant in breast cancer relative to normal tissue and FBXW7 transcripts less abundant in cancer tissue relative to normal tissue. Furthermore, relapse-free survival was significantly lower in patients with a high STYX and low FBXW7 phenotype than in those with the inverse phenotype. These data provide clues into the function of this pseudophosphatase, show how a nontargeted analysis of interacting partners can reveal function, and suggest that the STYX-FBXW7 interaction may be a target for cancer therapy.

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