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Identification of the von Hippel-lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex - PubMed

  • ️Fri Jan 01 1999

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Identification of the von Hippel-lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex

K Iwai et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Mutations of von Hippel-Lindau disease (VHL) tumor-suppressor gene product (pVHL) are found in patients with dominant inherited VHL syndrome and in the vast majority of sporadic clear cell renal carcinomas. The function of the pVHL protein has not been clarified. pVHL has been shown to form a complex with elongin B and elongin C (VBC) and with cullin (CUL)-2. In light of the structural analogy of VBC-CUL-2 to SKP1-CUL-1-F-box ubiquitin ligases, the ubiquitin ligase activity of VBC-CUL-2 was examined in this study. We show that VBC-CUL-2 exhibits ubiquitin ligase activity, and we identified UbcH5a, b, and c, but not CDC34, as the ubiquitin-conjugating enzymes of the VBC-CUL-2 ubiquitin ligase. The protein Rbx1/ROC1 enhances ligase activity of VBC-CUL-2 as it does in the SKP1-CUL-1-F-box protein ligase complex. We also found that pVHL associates with two proteins, p100 and p220, which migrate at a similar molecular weight as two major bands in the ubiquitination assay. Furthermore, naturally occurring pVHL missense mutations, including mutants capable of forming a complex with elongin B-elongin C-CUL-2, fail to associate with p100 and p220 and cannot exhibit the E3 ligase activity. These results suggest that pVHL might be the substrate recognition subunit of the VBC-CUL-2 E3 ligase. This is also, to our knowledge, the first example of a human tumor-suppressor protein being directly involved in the ubiquitin conjugation system which leads to the targeted degradation of substrate proteins.

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Figures

Figure 1
Figure 1

VBC-CUL-2 exhibits the E3 activity together with E2s of UbcH5 family. (A) The VBC-CUL-2 complex immunoprecipitated from HA-tagged WT-pVHL expressing 786–0 cells was incubated with recombinant E1, 32P-labeled ubiquitin, and ATP regeneration system at 37°C for 30 min in the presence of UBC3 (lane 2), UbcH5a (lane 3), UbcH5b (lane 4), UbcH5c (lane 5), UbcH7 (lane 6), E2–25K (lane 7), or E2–20K (lane 8), or in the absence of any E2 (lane 1). (B) In vitro ubiquitination reactions were performed as described in Materials and Methods, except for the followings: lane 1 lacks VBC-CUL-2, lane 3 lacks ATP regeneration system, lane 4 lacks UbcH5b, and lane 5 lacks E1. (C) Immunoprecipitates with anti-HA agarose beads from HA-tagged WT-pVHL expressing (lane 3), HA-tagged truncated pVHL (amino acids 1–115) expressing (lane 4), or parent 786–0 cells (lane 2) were subjected to the in vitro ubiquitination assay together with anti-HA beads alone (lane 1) in the presence of E1, UbcH5b, 32P-labeled ubiquitin, and ATP regeneration system at 37°C for 30 min. Reactions were stopped by adding 4× sample buffer and electrophoresed in 10% SDS/PAGE, followed by autoradiography.

Figure 2
Figure 2

Identification of P220 and P100 as WT-pVHL specific associated molecules. Immunoprecipitates of HeLa cells expressing Flag-tagged WT, Y98N, R167Q, or vector-transfected HeLa cells with Flag M2 beads were electrophoresed in 5% SDS/PAGE, followed by transfer to nitrocellulose membrane. After proteins were renatured on the membrane, the membrane was incubated with 32P-labeled VBC complex, followed by autoradiography.

Figure 3
Figure 3

Rbx1/ROC1 enhances the E3 activity of VBC-CUL-2. Hi Five cells infected with recombinant baculoviruses encoding CUL-2, elongin B (Elo B), elongin C (Elo C) together with either pVHL (lane 2), or Rbx1 (lane 4) or both (Lane1). Lysates of infected cells or noninfected cells (lane 3) were immunoprecipitated with anti-Flag M2 beads. (A) Immunoprecipitates were subjected to in vitro ubiquitination assay as described in Fig. 1C. Samples were electrophoresed in 10% SDS/PAGE, followed by autoradiography. (B) Immunoprecipitates were electrophoresed in 4–20% gradient SDS/PAGE. After being transferred, the membrane was probed with anti-HA, anti-Flag M2, anti-HSV, or anti-T7 antibodies, to detect CUL-2, pVHL, elongin C (Elo C), or Rbx1, respectively.

Figure 4
Figure 4

VHL mutants found in patients failed to ubiquitinate p220 and p100. Hi Five cells infected with recombinant baculovirus encoding CUL-2, elongin B, elongin C, and Rbx1 (lane 2) together with WT-pVHL (lane 3), P86H (lane 4), Y98N (lane 5), or L158P (lane 6) encoding recombinant baculoviruses. Lysates of infected cells or noninfected cells (lane 1) were immunoprecipitated with anti-Flag M2 beads. (A) Immunoprecipitates were subjected to in vitro ubiquitination assay as described in Fig. 1C. Samples were electrophoresed in 9% SDS/PAGE, followed by autoradiography. (B) Immunoprecipitates were electrophoresed in 4–20% gradient SDS/PAGE. After being transferred, the membrane was probed with anti-HA, anti-Flag M2, anti-HSV, or anti-T7 antibodies, to detect CUL-2, pVHL, elongin C (Elo C), or Rbx1, respectively.

Comment on

  • VHL: a very hip ligase.

    Tyers M, Rottapel R. Tyers M, et al. Proc Natl Acad Sci U S A. 1999 Oct 26;96(22):12230-2. doi: 10.1073/pnas.96.22.12230. Proc Natl Acad Sci U S A. 1999. PMID: 10535903 Free PMC article. No abstract available.

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