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. 2012;7(10):e47736.
doi: 10.1371/journal.pone.0047736. Epub 2012 Oct 17.

The Stomatin-Like Protein SLP-1 and Cdk2 Interact With the F-Box Protein Fbw7-γ

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Free PMC article

The Stomatin-Like Protein SLP-1 and Cdk2 Interact With the F-Box Protein Fbw7-γ

Wei Zhang et al. PLoS One. .
Free PMC article

Abstract

Control of cellular proliferation is critical to cell viability. The F-box protein Fbw7 (hAgo/hCdc4/FBXW7) functions as a specificity factor for the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase complex and targets several proteins required for cellular proliferation for ubiquitin-mediated destruction. Fbw7 exists as three splice variants but the mechanistic role of each is not entirely clear. We examined the regulation of the Fbw7-γ isoform, which has been implicated in the degradation of c-Myc. We show here that Fbw7-γ is an unstable protein and that its turnover is proteasome-dependent in transformed cells. Using a two-hybrid screen, we identified a novel interaction partner, SLP-1, which binds the N-terminal domain of Fbw7-γ. Overexpression of SLP-1 inhibits the degradation of Fbw7-γ, suggesting that this interaction can happen in vivo. When Fbw7-γ is stabilized by overexpression of SLP-1, c-Myc protein abundance decreases, suggesting that the SCF(Fbw7-γ) complex maintains activity. We demonstrate that Cdk2 also binds the N-terminal domain of Fbw7-γ as well as SLP-1. Interestingly, co-expression of Cdk2 and SLP-1 does not inhibit Fbw7-γ degradation, suggesting that Cdk2 and SLP-1 may have opposing functions.

Conflict of interest statement

Competing Interests: D.M. Koepp is an Academic Editor for PLOS ONE. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Fbw7-γ is an unstable protein and its proteolysis is dependent on the proteasome.
A) Diagram of Fbw7 isoforms. The F-box motif and WD40 repeats are marked as shown. B) Fbw7-β and γ are unstable proteins, and their degradation is proteasome-dependent. Flag-tagged Fbw7 isoforms were expressed in HEK293T cells and protein stability assays were performed as described in Materials and Methods. Quantitation of a representative experiment is shown in the graph. C) The N-terminal unique region is critical for Fbw7-γ degradation. Protein stability assays were performed as described in (B) with cells expressing the indicated Flag-tagged proteins. Quantitation of three independent experiments is shown on the graph. Error bars indicate standard deviations. D) The lysine residues within the unique region of Fbw7-γ are critical for degradation. Protein stability assays were performed as described in (B) with cells expressing the indicated Flag-tagged proteins. Quantitation of a representative experiment is shown in the graph.
Figure 2
Figure 2. SLP1 is an Fbw7-γ-specific interacting protein.
A) Fbw7-γ co-immunoprecipitates (co-IP) with SLP-1. Flag tagged SLP-1 was co-expressed with Myc-tagged Fbw7-γ (lane1) or vector (lane2) in HEK293T cells. Immunoprecipitation was performed as described in Materials and Methods. Reciprocal co-IP is shown in the right panel (lanes 3, 4). B) SLP-1 and Fbw7-γ co-fractionate. Standard fractionation assays from cells co-expressing Flag-tagged SLP-1 and Flag-tagged Fbw7-γ were performed as described in Materials and Methods. α-tubulin, a cytosolic protein, was used as a control. Asterisks indicate modified forms of SLP-1, C = cytosolic, N = nuclear. C) SLP-1 and Fbw7-γ co-immunoprecipitate in both nuclear and cytosolic fractions. Co-IPs were performed as in (A), except that cellular fractions from (B) were used. Asterisks indicate non-specific cross-reacting band. D) The Fbw7-γ and SLP-1 interaction is specific. Flag-tagged Fbw7 isoforms were co-expressed with myc-tagged SLP1. Immunoprecipitations were performed as in (A). IgG refers to heavy chain of the anti-Myc antibody, * indicates a non-specific band.
Figure 3
Figure 3. A) SLP-1 overexpression inhibits Fbw7-γ turnover.
Flag-tagged Fbw7-γ was co-transfected with Flag-tagged SLP-1 (lane 1–4) or empty vector (lane 5–8) into HEK293T cells and protein stability assays were performed as described in Materials and Methods. Quantitation of three independent experiments is shown on the graph. Error bars indicate standard deviations. B) Abundance of co-expressed c-Myc is decreased in cells expressing Flag-tagged SLP-1 and Flag-tagged Fbw7-γ. HEK293T cells were transfected with the indicated expression constructs and cell extracts probed with anti-Flag, anti-Myc (9E10) or anti-GAPDH antibodies. Quantitation of three independent experiments is shown on the graph. Error bars indicate standard deviations.
Figure 4
Figure 4. SLP-1 and Fbw7-γ interact with Cdk2. A) SLP-1 interacts with Cdk2.
HA-tagged Cdk2 was transfected with Flag-tagged SLP-1 or empty vector into HEK293T cells. Co-immunoprecipitations were performed as described in Materials and Methods. B) Cdk2 interacts with Fbw7-γ. Co-IPs were performed as in (A) in cells expressing the indicated proteins. C) Co-expression of Cdk2 and SLP-1 promotes turnover of Flag-tagged Fbw7-γ. Protein stability assays were performed as described in Materials and Methods in cells expressing the indicated proteins. Quantitation of a representative experiment is shown in the graph.

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