SCCRO (DCUN1D1) promotes nuclear translocation and assembly of the neddylation E3 complex

Abstract

SCCRO/DCUN1D1/DCN1 (squamous cell carcinoma-related oncogene/defective in cullin neddylation 1 domain containing 1/defective in cullin neddylation) serves as an accessory E3 in neddylation by binding to cullin and Ubc12 to allow efficient transfer of Nedd8. In this work we show that SCCRO has broader, pleiotropic effects that are essential for cullin neddylation in vivo. Reduced primary nuclear localization of Cul1 accompanying decreased neddylation and proliferation in SCCRO(-/-) mouse embryonic fibroblasts led us to investigate whether compartmentalization plays a regulatory role. Decreased nuclear localization, neddylation, and defective proliferation in SCCRO(-/-) mouse embryonic fibroblasts were rescued by transgenic expression of SCCRO. Expression of reciprocal SCCRO and Cul1-binding mutants confirmed the requirement for SCCRO in nuclear translocation and neddylation of cullins in vivo. Nuclear translocation of Cul1 by tagging with a nuclear localization sequence allowed neddylation independent of SCCRO, but at a lower level. We found that in the nucleus, SCCRO enhances recruitment of Ubc12 to Cul1 to promote neddylation. These findings suggest that SCCRO has an essential role in neddylation in vivo involving nuclear localization of neddylation components and recruitment and proper positioning of Ubc12.

FIGURE 1.

SCCRO enhances cell proliferation by promoting cullin neddylation. A, representative SCCRO^+/+ and SCCRO^−/− mice at embryo (top), birth (middle), and weaning (bottom) stages. B, cell size of SCCRO^+/+, SCCRO^+/−, and SCCRO^−/− MEFs determined by forward scatter analysis (FSC), showing no differences. C, cell proliferation assay showing reduced proliferation in SCCRO^−/− relative to SCCRO^+/+ and SCCRO^+/− MEFs. Retroviral delivery of SCCRO but not SCCRO-D241N rescued the proliferation defect in SCCRO^−/− MEFs.

FIGURE 2.

Neddylation, nuclear localization of Cul1, and levels of ubiquitinated proteins are reduced in SCCRO^−/− MEFs. A, immunoblotting with antibodies as indicated on lysates from MEFs before and after UV irradiation showing activation of cullin neddylation in SCCRO^+/+ but not SCCRO^−/− MEFs. B, immunoblots with antibodies against Cul1 and Ub on lysates from 2A after activation of neddylation by addition of E1, E2, Nedd8, and ATP. Levels of neddylated Cul1 and ubiquitinated proteins was higher is SCCRO^+/+ MEFs. C, immunoblot of cell lysates from MEFs subjected to activated neddylation assay. Levels of neddylated Cul1 were higher in SCCRO^+/+ MEFs. Defective neddylation in SCCRO^−/− MEFs was rescued by retroviral delivery of SCCRO but not SCCRO-D241A. D, immunofluorescence using anti-Cul1 antibodies on MEFs showing higher proportion of SCCRO^+/+ with primary nuclear localization (compare lanes 1 and 2). Retroviral delivery of SCCRO (lane 3) but not SCCRO-D241A (lane 4) promoted nuclear translocation of Cul1 in SCCRO^−/− MEFs. Exposure of MEFs to UV irradiation resulted in increased nuclear translocation of Cul1 in SCCRO^+/+ MEFs (lane 5) but had no effect on SCCRO^−/− MEFs (lane 6).

FIGURE 3.

SCCRO-mediated nuclear translocation of Cul1 is required for its neddylation in vivo. A, immunoblots on lysates from U2OS cells transfected with the indicated constructs and probed with antibodies against Cul1, Cul3, HA, and tubulin. SCCRO is required for cullin neddylation in vivo (top and third panels, lane 2) and augments the in vitro reaction when co-expressed with Ubc12 (second and fourth panels, lane 6). B, immunoblot on lysates from U2OS cells transfected with the indicated constructs showing no increase in Cul1 neddylation in HA-NES-SCCRO-transfected cells.

FIGURE 4.

Binding to SCCRO is required for neddylation and nuclear localization of Cul1. A, schematic representation of the C terminus of Cul1 showing cullin mutants used in these experiments. B, immunoblot on lysates from U2OS cells transfected with Myc-SCCRO and HA-Cul1 or selected mutants probed with the indicated antibodies following HA immunoprecipitation. SCCRO binds to HA-Cul1 and HA-Cul1-Δ610–615 (lanes 1 and 2) but not C-terminal Cul1 mutants (lane 3–7). C, immunoblots for Cul1 on lysates from U2OS cells co-transfected with Cul1 or its mutants with or without SCCRO showing mutants that lose SCCRO binding cannot be neddylated. D, immunofluorescence using Cy3-conjugated anti-Myc antibody and FITC-conjugated anti-HA antibody on U2OS cells transfected with HA-Cul1 or indicated HA-tagged Cul1 mutants with or without Myc-SCCRO. HA-Cul1 (first lane) was primarily nuclear (∼74%) whereas Cul1 mutants (third, fifth, and seventh lanes) were primarily cytoplasmic in the absence of SCCRO expression. Co-expression of SCCRO increased the proportion of HA-Cul1 and rescued the localization defect of Cul1-Δ610–615 with primary nuclear localization. E, U2OS cells were transfected with Cul1 or its mutants with or without SCCRO. The results of Cul1 localization monitored by immunofluorescence by counting 200 cells for each transfection is tabulated. Nuclear localization by SCCRO requires the Cul1 C-terminal sequence.

FIGURE 5.

SCCRO rescues nuclear localization and neddylation of Cul1-Δ610–615. A, immunoblot on lysates from U2OS cells transfected with the indicated constructs and probed with anti-Myc or anti-HA antibodies following HA immunoprecipitation showing increased interaction between Roc1 and HA-Cul1-Δ610–615 with SCCRO co-expression (compare lanes 4 with 2 and 6). B, immunoblot on lysates from U2OS cells transfected with the indicated constructs probed with anti-HA antibodies showing rescue of HA-Cul1-Δ610–615 neddylation defect by SCCRO (top panel, lanes 2 and 6). C, immunofluorescence on U2OS cells co-transfected with FLAG-Cul1-Δ610–615 and HA-NES-SCCRO using Cy3-conjugated anti-FLAG antibody and FITC-conjugated HA antibody showing nuclear exclusion of SCCRO and loss of nuclear translocation of Cul1-Δ610–615.

FIGURE 6.

NLS-Cul1-Δ610–615 cannot be neddylated in vivo. A, immunoblot on lysates from U2OS cells transfected with the indicated constructs showing that neither HA-Ubc12 nor HA-SCCRO expression promotes neddylation of NLS-Cul1-Δ610–615. B and C, immunoblot on cytoplasmic (C) and nuclear (N) fractions of lysates from U2OS cells transfected with the indicated constructs probed with anti-HA antibody showing enrichment of Roc1 in the nuclear fraction of cells co-expressing SCCRO with HA-Cul1-Δ610–615 (B, top panel, lane 4) but not with HA-NLS-Cul1-Δ610–615 (C, top panel, lane 4). P300/CBP-associated factor (PCAF) and tubulin are nuclear and cytoplasmic loading controls, respectively.

FIGURE 7.

SCCRO promotes recruitment of Ubc12 onto Cul1-Roc1 complex after nuclear translocation. A, immunoblot on lysates from U2OS cells transfected with the indicated constructs showing an increase in neddylation of HA-NLS-Cul1 with co-expression of HA-Ubc12 or HA-SCCRO (lanes 2 and 3). A synergistic effect was seen with co-expression of both HA-Ubc12 and HA-SCCRO (lane 4). B, immunoblot on lysates from U2OS cells transfected with the indicated constructs following anti-HA immunoprecipitation showing an increase in interaction between Ubc12 and Cul1 with SCCRO co-expression (lane 2).