The F-box protein Skp2 is a ubiquitylation target of a Cul1-based core ubiquitin ligase complex: evidence for a role of Cul1 in the suppression of Skp2 expression in quiescent fibroblasts - PubMed (original) (raw)

The F-box protein Skp2 is a ubiquitylation target of a Cul1-based core ubiquitin ligase complex: evidence for a role of Cul1 in the suppression of Skp2 expression in quiescent fibroblasts

C Wirbelauer et al. EMBO J. 2000.

Abstract

The ubiquitin protein ligase SCF(Skp2) is composed of Skp1, Cul1, Roc1/Rbx1 and the F-box protein Skp2, the substrate-recognition subunit. Levels of Skp2 decrease as cells exit the cell cycle and increase as cells re-enter the cycle. Ectopic expression of Skp2 in quiescent fibroblasts causes mitogen-independent S-phase entry. Hence, mechanisms must exist for limiting Skp2 protein expression during the G(0)/G(1) phases. Here we show that Skp2 is degraded by the proteasome in G(0)/G(1) and is stabilized when cells re-enter the cell cycle. Rapid degradation of Skp2 in quiescent cells depends on Skp2 sequences that contribute to Cul1 binding and interference with endogenous Cul1 function in serum-deprived cells induces Skp2 expression. Furthermore, recombinant Cul1-Roc1/Rbx1-Skp1 complexes can catalyse Skp2 ubiquitylation in vitro. These results suggest that degradation of Skp2 in G(0)/G(1) is mediated, at least in part, by an autocatalytic mechanism involving a Skp2-bound Cul1-based core ubiquitin ligase and imply a role for this mechanism in the suppression of SCF(Skp2) ubiquitin protein ligase function during the G(0)/G(1) phases of the cell cycle.

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Figures

Fig. 1. Expression of Skp2 mRNA during the G0 to S phase transition. (A) T98G cells were arrested by serum deprivation and then stimulated to re-enter the cell cycle by addition of fresh serum. At the indicated times thereafter, total RNAs were prepared and processed for northern blotting using 32P-labelled cDNAs encoding Skp2 (upper panel) or GAPDH (middle panel). Whole cell extracts were prepared at the same time points, equalized for protein content and processed for western blotting with antibodies against Skp2 (lower panel). The percentages of S-phase cells at various times after serum stimulation were calculated from FACS analysis during the ensuing cell cycle. (B) HDFs were synchronized and processed for FACS (upper panel), northern (middle panel) and western blot (lower panel) analysis as described in (A), except that equal loading of RNA was determined by staining of the 18S RNA by methylene blue following transfer of RNAs to the membrane. (C) Exponentially growing HDFs (lanes 1–3), HDFs arrested by serum deprivation (lanes 4–6) or HDFs induced to re-enter the cell cycle by addition of fresh serum (lanes 7–9) were either left untreated (lanes 1, 4 and 7), treated with DMSO (lanes 2, 5 and 8) or with LLnL (lanes 3, 6 and 9) for 5 h. Whole cell extracts were prepared and processed for immunoblotting using anti-(fl)Skp2 (upper panel) or anti-Cdk2 (lower panel) antibodies.

Fig. 2. Determinants of Skp2 instability in quiescent fibroblasts. (A) Rat1 cells were infected with vector or Skp2(wt) retrovirus and, after selection in neomycin, cell pools of stably infected, neomycin-resistant cells were established. Vector- or Skp2-infected cell pools were growth-arrested by serum deprivation (lanes 1–4) or growth-arrested and then stimulated to re-enter the cell cycle by addition of fresh serum (lanes 5–8). Each of these cultures was treated with DMSO (lanes 1, 3, 5 and 7) or LLnL (lanes 2, 4, 6 and 8) for 5 h. Lysates were prepared from each cell pool, equalized for protein content and processed for western blotting with anti-(fl)Skp2 antibodies. (B) Skp2-infected Rat1 cells were growth-arrested (upper panel) or growth-arrested and stimulated to re-enter the cell cycle by addition of fresh serum (lower panel) and then pulse-labelled with [35S]methionine and chased for the indicated times. Denatured cell lysates were then processed for immunoprecipitation with anti-(fl)Skp2 antibodies. (C) Rat1 cells were infected with Skp2(wt), (ΔF6), (AxA) or (ΔF6/AxA) retrovirus and, after selection in neomycin, cell pools of stably infected, neomycin-resistant cells were established and either growth-arrested (lanes 1, 3, 5 and 7) or growth-arrested and stimulated to re-enter the cell cycle by addition of fresh serum (lanes 2, 4, 6 and 8). Lysates were prepared from each cell pool, equalized for protein content and processed for western blotting with anti-(fl)Skp2 (upper panel) and anti-Cdk2 (lower panels) antibodies. (D) Stably infected Rat1 cell pools as described in (C) were growth-arrested and then either left untreated (lanes 1, 4, 7 and 10) or were treated with DMSO (lanes 2, 5, 8 and 11) or MG132 (lanes 3, 6, 9 and 12) for 5 h and processed for western blotting as described in (C). (E) Stably infected Rat1 cell pools as described in (C) were first growth-arrested and then stimulated to re-enter the cell cycle by addition of fresh serum and were then either left untreated (lanes 1, 4, 7 and 10) or were treated with DMSO (lanes 2, 5, 8 and 11) or MG132 (lanes 3, 6, 9 and 12) for 5 h. Normalized lysates were processed for western blotting as described in (D). (F) HDFs were growth-arrested by serum deprivation for 64 h and were then either left untreated (lane 1) or were treated with transfection agent GS2888 alone (lane 2), antisense oligonucleotides (100 nmol) (lane 3) or control oligonucleotides (100 nmol) (lane 4) for 24 h. Whole cell extracts were prepared, equalized for protein content and processed for immunoblotting using anti-Cul1 (upper panel), anti-Cdk2 (middle panel) and anti-(fl)Skp2 (lower panel). A representative result from three independent experiments using different concentrations of oligonucleotides (100 or 200 nmol) is displayed.

Fig. 3. The F-box protein Skp2 is a ubiquitylation target of the bound Cul1-based core ubiquitin ligase complex. (A) Sf9 insect cells were infected with baculovirus vectors that express GST (lane 1) or GST–Skp1 (lane 2) together with HisSkp2, HisCul1HA and PyRoc1/Rbx1. Lysates were prepared and then aliquots immunoblotted with anti-GST (upper panel), anti-(fl)Skp2 (second panel), anti-HA (panel 3) or anti-Py (lower panel) antibodies. (B) Other aliquots prepared as in (A) were incubated with GS beads and bound proteins were processed for western blotting with indicated antibodies. Note that HisSkp2, HisCul1HA and PyRoc1/Rbx1 bound to GST–Skp1 (lanes 2, 4, 6, 8) but not to GST alone (lanes 1, 3, 5 and 7). (C) Recombinant SCFSkp2 complexes were produced in insect Sf9 cells and bound to GS beads as described in (B) and processed for the in vitro ubiquitylation reaction as described in Materials and methods. Lane 1, all components; lane 2, no ubiquitin; lane 3, no E1; lane 4, no Cdc34; lane 5, mutant K48R ubiquitin. Samples were processed for western blotting with anti-(fl)Skp2 antibodies. The bracket on the left marks ubiquitylated Skp2. (D) Sf9 insect cells were infected with various combinations of recombinant baculoviruses as indicated by the plus and minus signs at the top of the figure. Lysates were prepared and aliquots were incubated with GS beads. The corresponding Sepharose beads were either processed for in vitro ubiquitylation in the absence (lanes 2, 4, 6, 8, 10, 12, 14 and 16) or presence (lanes 1, 3, 5, 7, 9, 11, 13 and 15) of ubiquitin and western blotting with the antibodies described in (A). Asterisks in the upper panel (lanes 5 and 6) indicate a Skp2 signal that derives from spill-over from lane 4. (E) Other aliquots of cell lysates prepared as in (D) were processed directly for western blotting with the antibodies described in (A).

Fig. 4. Skp2(wt), (ΔF6) and (AxA) are ubiquitylated in vitro. (A) Sf9 insect cells were infected with baculovirus vectors that express GST–Skp1, HisCul1HA, PyRoc1/Rbx1 together with either HisSkp2(wt) (lanes 1 and 5), HisSkp2(ΔF6) (lanes 2 and 6), HisSkp2(AxA) (lanes 3 and 7) or HisSkp2(ΔF6/AxA) (lanes 4 and 8). Lysates were prepared and aliquots processed for immunoprecipitation using anti-(fl)Skp2 antibody followed by western blotting with anti-Cul1 or anti-(fl)Skp2 antibody (left panels). Note that similar amounts of Skp2 are present in each of the immunoprecipitates. Other aliquots were processed directly for western blotting with anti-GST, anti-(fl)Skp2 and anti-HA antibodies (right panels). (B) Sf9 insect cells were infected with baculovirus vectors expressing GST–Skp1 together with either HisSkp2(wt) (lanes 1 and 5), HisSkp2(ΔF6) (lanes 2 and 6), HisSkp2(AxA) (lanes 3 and 7) or HisSkp2(ΔF6/AxA) (lanes 4 and 8). Lysates were prepared and aliquots were incubated with GS beads and bound proteins were processed for western blotting with anti-(fl)Skp2 or anti-GST antibodies (left panels). Other aliquots were processed directly for western blotting with anti-GST and anti-(fl)Skp2 antibodies (right panels). (C) Recombinant SCFSkp2 complexes were produced in insect Sf9 cells with either HisSkp2(wt) (lanes 1, 2 and 7), HisSkp2(AxA) (lanes 3, 4 and 8) or HisSkp2(ΔF6) (lanes 5, 6 and 9). Lysates were prepared and aliquots incubated with GS beads. Beads were processed for in vitro ubiquitylation reaction in the presence (lanes 1, 3 and 5) or absence (lanes 2, 4 and 6) of ubiquitin and immunoblotted with anti-(fl)Skp2 antibodies. Other aliquots of cell lysates (lanes 7–9) were processed directly for western blotting with anti-GST (upper panel), anti-HA (second panel), anti-Py (third panel) or anti-(fl)Skp2 (lower panel) antibodies.

Fig. 5. The availability of A- and B-box sequences of Cul1 primary structure are independently required for Cul1 to support Skp2 ubiquitylation. (A) Recombinant SCFSkp2 complexes were produced in insect Sf9 cells with either Cul1(wt) or indicated mutant derivatives of it. Lysates were prepared and aliquots were incubated with GS beads. The corresponding Sepharose beads were either processed for in vitro ubiquitylation in the absence (lanes 2, 4, 6, 8, 10, 12 and 14) or presence (lanes 1, 3, 5, 7, 9, 11 and 13) of ubiquitin and western blotting with the antibodies described in the legend to Figure 3A, except that anti-His antibodies were used to detect Cul1. (B) Other aliquots of cell lysates prepared in (A) were processed directly for western blotting with the antibodies described in the legend to Figure 3A, except that anti-His antibodies were used to detect Cul1. (C) Schematic representation of the Cul1 deletion mutants tested and their corresponding Roc1 binding and Skp2 ubiquitylation activity (plus versus minus). (D) Sequence alignment of the CH region of cullin members and Apc2 from different species. The alignment was made with the ClustalX method and the Boxshade program. Black bars on top of the alignment indicate the A- and the B-box. Black lines on the bottom of the alignment represent in-frame deletion mutants of Cul1. Identical residues are indicated by white letters on red boxes and functionally similar residues are in white letters on blue boxes.

Fig. 6. A-box mutants of Cul1 are defective in Cdc34 E2 enzyme binding. (A) Sf9 insect cells were infected with baculovirus vectors encoding Cdc34 (lane 1), HisCul1HA (lane 2) or Cdc34 and HisCul1HA (lane 3). Lysates were prepared and aliquots processed for immuno precipitation using anti-HA antibody followed by western blotting with anti-Cdc34 (upper panel) or anti-HA antibody (second panel). Other aliquots were processed directly for western blotting with anti-HA (third panel) or anti-Cdc34 (lower panel) antibodies. (B) Sf9 insect cells were infected with baculovirus vectors encoding Cdc34 (lane 1) or Cdc34 and HisCul1HA-wt (lane 2), ΔC63 (lane 3), Δ6 (lane 4) or ΔN63 (lane 5). Lysates were prepared and aliquots processed for immunoprecipitation using anti-His antibody followed by western blotting with anti-Cdc34 (upper panel) or anti-HA antibody (second panel). Other aliquots were processed directly for western blotting with anti-HA (third panel) or anti-Cdc34 (lower panel) antibodies.

Fig. 7. A- and B-box mutants of Cul1 fail to restore growth of cdc53-2 yeast cells. (A) Individual transformants of the cdc53-2 strain (YMP740) containing the plasmids indicated were plated on selective media and grown either at the permissive (25°C, right plate) or restrictive (34°C, left plate) temperature. Photographs were taken after 3 days. (B) The protein levels of wild-type or mutant Cul1 were analysed by immunoblotting of cdc53-2 cells grown at the restrictive temperature (25°C).

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The F-box protein Skp2 is a ubiquitylation target of a Cul1-based core ubiquitin ligase complex: evidence for a role of Cul1 in the suppression of Skp2 expression in quiescent fibroblasts - PubMed (original) (raw)