Expression of Skp1 and Skp2 in normal and transformed cells. (A) Cell extracts were prepared from growing normal human fibroblasts (cell line AG1523) (lane 1), AG1523 cells at 24 h after treatment with UV irradiation (lane 2), and HUT12 (lane 3), HepG2 (lane 4), 293 (lane 5), H4 (lane 6), HBL100 (lane 7), MG63 (lane 8), K562 (lane 9), HeLa (lane 10), and HaCaT (lane 11) cells (see Materials and Methods for descriptions of cell lines). The extracts were dissolved in SDS sample buffer, and 10 μg each was subjected to SDS-PAGE on a 17.5% gel. The proteins were transferred onto a membrane and immunoblotted with antibodies against Skp2 (top), Skp1 (middle), and tubulin (bottom). (B) Cell extracts prepared from the indicated cell lines were dissolved in SDS sample buffer, and 10 μg each was subjected to SDS-PAGE on a 17.5% gel. The proteins were transferred onto a membrane and immunoblotted with a monoclonal antibody against cyclin A.

Interactions between cyclin A-Cdk2, Skp2, and p21^Cip1/WAF1. (A) HtTA1 cells were transfected with either pUHD-P1 vector alone (lanes 1, 3, and 5) or FLAG-p21 in pUHD-P1 (lanes 2, 4, and 6). Cell extracts were prepared, and 60 μg was immunoprecipitated (IP) with either NRS (lanes 3 and 4) or anti-Cdk2 serum (lanes 5 and 6). The immunoprecipitates and total cell extracts (10 μg) (lanes 1 and 2) were subjected to SDS-PAGE on a 17.5% gel, transferred onto a membrane, and immunoblotted with antibodies against Skp2, cyclin A, Cdk2, or FLAG tag to detect FLAG-p21, as indicated at the left. Positions of Skp2, cyclin A, Cdk2, FLAG-p21 (F-p21), and IgG chains are indicated on the right. The asterisk indicates the position of an extra cyclin A band (which can also be immunoprecipitated with Cdk2) seen after transfection with the p21-expressing construct. The positions of molecular size standards (in kilodaltons) are shown on the left. (B) HeLa cell extracts (120 μg) were immunoprecipitated with antiserum against Skp2 (lanes 2 to 4). Buffer (lane 2) or purified recombinant p21-H6 protein expressed in bacteria (1 μg [lane 3] or 5 μg [lane 4]) was incubated with the Skp2 immunoprecipitates at 30°C for 30 min. After washing, the immunoprecipitates were dissolved in SDS sample buffer and subjected to SDS-PAGE on a 17.5% gel. Total cell lysate (10 μg) was loaded in lane 1. The proteins were transferred onto a membrane and immunoblotted with a monoclonal antibody against PSTAIRE to detect Cdk2 (anti-PSTAIRE monoclonal antibody was used instead of anti-Cdk2 polyclonal antibody because it gave a cleaner background of the IgG bands) (top), anti-cyclin A monoclonal antibody E72 (middle), and anti-Skp2 antiserum (bottom).

Binding of Skp2 to cyclin A-Cdk2 and Skp1. (A) Skp2 wild type (WT) and mutants. The Skp2 truncation and site-directed point mutants were constructed as described in Materials and Methods. The position of the F box (∼112 to 152) is indicated. Abilities to bind GST-Skp1 and GST-Cdk2-cyclin A, and inhibition of cyclin A-Cdk2 kinase activity, are indicated (see text). ND, not determined. CΔ96 does not contain an H6 tag and hence cannot be purified from bacteria for cyclin A-Cdk2 inhibition assay. (B) Binding of Skp1 and cyclin A-Cdk2 to Skp2 truncation mutants. Wild-type Skp2 (WT) or truncated mutants of Skp2 were translated in a coupled transcription-translation rabbit reticulocyte lysate system in the presence of [³⁵S]methionine. The amount of ³⁵S label in the translated proteins was quantitated by SDS-PAGE (17.5% gel) and phosphorimagery (lanes 1 to 8). The proteins were adjusted to the same amount of labeling (the strongest band was about 10% of the input for the binding experiments) and incubated with bacterially expressed GST-Skp1 (lanes 9 to 16) or GST-Cdk2 and PA-cyclin A (lanes 17 to 24). The GST fusion proteins and associated proteins were then precipitated with GSH-agarose and analyzed by SDS-PAGE (17.5% gel) and phosphorimagery. No significant binding to these proteins was detected when GST was used (data not shown). The positions of molecular size standards (in kilodaltons) are shown on the left.

Regulation of cyclin A-Cdk2 kinase activity by Skp1 and Skp2. (A) Bacterially expressed and CAK-activated PA-cyclin A-GST-Cdk2 complexes (250 nM) were incubated with 250 nM bacterially expressed Skp2-H6 (lanes 2, 4, and 6), Skp1-H6 (lanes 3 and 4), or boiled Skp1-H6 (B) (lanes 5 and 6) at 23°C for 30 min. The kinase activity against histone H1 was then assayed, and phosphorylation was detected by SDS-PAGE followed by phosphorimagery. Quantitation from the phosphorimagery is shown in the lower panel. (B) PA-cyclin A-GST-Cdk2 complexes (250 nM) (lanes 1, 2, and 5 to 11) or buffer (lanes 3 and 4) were incubated with bacterially expressed Skp2-H6 (250 nM [lanes 1, 3, 10, and 11], 25 nM [lane 9], or 2.5 nM [lane 8]) and Skp1-H6 (6.5 μM [lanes 2, 4, 7, and 11] or 650 nM [lane 6]) at 23°C for 30 min. In lanes 1 and 2, Skp2-H6 and Skp1-H6, respectively, were boiled (B) prior to incubation. The kinase activity against histone H1 was then assayed, and phosphorylation was detected by SDS-PAGE (17.5 gel) followed by phosphorimagery. The positions of histone H1 and Skp2-H6 are indicated. Quantitation of histone H1 phosphorylation from the phosphorimagery is shown in the lower panel. The levels of the proteins added were confirmed by immunoblotting with antibodies against Cdk2, Skp2, or Skp2; PA-cyclin A was detected by virtue of the PA tag binding to IgG in the immunoblotting reaction. (C) Skp2 has no effect on p25-Cdk5 kinase activity. GST-p25 (250 nM) and GST-Cdk5 (250 nM) were incubated with Skp1-H6 (650 nM [lane 2] or 6.5 μM [lane 3]) or Skp2-H6 (25 nM [lane 4] or 250 nM [lane 5]) at 23°C for 30 min. The kinase activity against histone H1 was then assayed, and phosphorylation was detected by SDS-PAGE followed by phosphorimagery.

Inhibition of endogenous cyclin-CDK kinase activity by Skp2. (A) HeLa cell extracts (250 μg) were immunoprecipitated with antiserum against cyclin A or Cdk2, NRS, or serum against Cdc2, Skp2, or cyclin E, as indicated above the lanes. The immunoprecipitates were incubated with buffer (odd-numbered lanes) or 1 μg of bacterially expressed Skp2-H6 (even-numbered lanes) at 30°C for 30 min. The kinase activity against histone H1 was then assayed, and phosphorylation was analyzed by SDS-PAGE (17.5% gel) followed by phosphorimagery. Quantitation of histone H1 phosphorylation from the phosphorimagery is shown in the lower panel; the kinase activity associated with NRS immunoprecipitates in lanes 5 and 6 were very low, and the quantitation is not shown. (B) Binding of cyclin A and cyclin E to Skp2. HeLa cells were transiently transfected with a control vector (lanes 1 and 3) or plasmid expressing FLAG-Skp2 (lanes 2 and 4). Extracts were prepared; 200 μg was immunoprecipitated (IP) with anti-FLAG antiserum and dissolved in 30 μl of SDS sample buffer; 10 μl was loaded onto an SDS–17.5% polyacrylamide gel, transferred onto a membrane, and immunoblotted with the anti-cyclin A monoclonal antibody E72 and anti-cyclin E monoclonal antibody HE12.

Phosphorylation of Skp2 by cyclin A-Cdk2. (A) Inhibition of Cdk2 kinase activity by Skp2 wild type (WT) and mutants. Assay of the inhibition of cyclin A-Cdk2 kinase activity against histone H1 by Skp2 truncation and site-directed point mutants was as described for Fig. 4. Quantitation from the phosphorimagery is shown in the lower panel. (B) Phosphorylation of site-directed Skp2 mutants by cyclin A-Cdk2. Buffer (lane 1), Skp2-H6 (lane 2), and mutants S76A (lane 3), S191A (lane 4), and S76A S191A (lane 5) were incubated with reconstituted PA-cyclin A-GST-Cdk2 in the presence of [γ-³²P]ATP. The reactions were terminated by addition of SDS sample buffer, and phosphorylations were analyzed by SDS-PAGE (17.5% gel) followed by phosphorimagery. (C) Phosphoamino acid analysis of Skp2. Skp2-H6 (left) and the S76T mutant (right) were phosphorylated by reconstituted PA-cyclin A-GST-Cdk2 in vitro. The proteins were separated by SDS-PAGE (17.5% gel) and transferred to an Immobilon membrane. The phosphorylated Skp2 bands were cut out and subjected to phosphoamino acid analysis using thin-layer electrophoresis (TLE) in the first dimension and thin-layer chromatography (TLC) in the second dimension, followed by analysis with phosphorimagery. The positions of phosphoamino acids standards are indicated.

Direct interaction between Skp1 and cyclin A-Cdk2. (A) Purified bacterially expressed GST (lanes 1 and 6) or GST-Cdk2 (other lanes) was incubated with Skp1-H6 in the presence of Skp2-H6 (lanes 1, 4, 5, 6, 9, and 10) and PA-cyclin A (lanes 1, 3, 5, 6, 8, and 10). Input samples (10% of that used for binding) were loaded in lanes 1 to 5. GST-Cdk2 and associated proteins were precipitated with GSH-agarose (lanes 6 to 10), and the bound proteins were detected by immunoblotting with antibodies against Skp1, Skp2, and GST; PA-cyclin A was detected by virtue of the PA tag binding to IgG in the immunoblotting reaction. (B) Purified bacterially expressed GST-Skp1 was incubated with PA-cyclin A (lanes 2 and 3) and Cdk2 (lanes 1 and 3). GST-Skp1 and associated proteins were precipitated with GSH-agarose, and the bound proteins were detected by immunoblotting with antibodies against Skp1 and PSTAIRE (detected Cdk2), as indicated on the left; in the top panel, PA-cyclin A was detected by virtue of the PA tag binding to IgG in the immunoblotting reaction.

Inhibition of Cdk2 Thr160 and Tyr15 phosphorylation by Skp2. GST-Cdk2(K33R)-PA-cyclin A (lanes 1 to 4) was mixed with bacterially expressed Skp2-H6 (lanes 2, 3, and 5) and Skp1-H6 (lanes 3 to 5). The reactions were incubated with an immunoprecipitate of CAK (A) or bacterially expressed GST-Wee1 (B) in the presence of [γ-³²P]ATP. Phosphorylation was detected by SDS-PAGE (17.5% gel) followed by phosphorimagery analysis. The positions of GST-Cdk2(K33R) are indicated on the right; positions of molecular size standards (in kilodaltons) are shown on the left.

Cell cycle arrest after overexpression of Skp2 in mammalian cells. (A) HtTA1 cells were transiently transfected with the same amount of vector pcDNA3.1(−) (lane 1) or Skp2 in pcDNA3.1(−) (lane 2). Cell extracts were prepared and subjected to SDS-PAGE (17.5% gel). Proteins were transferred to a membrane and immunoblotted with antibodies against Skp2 and Skp1. Positions of the endogenous Skp1 and transfected Skp2 are shown on the right; positions of molecular size standards (in kilodaltons) are shown on the left. (B) HtTA1 cells were transiently transfected with the same amount of vector pUHD-P1 (lane 1) or FLAG-Skp1 in pUHD-P1 (F-Skp1; lane 2). Cell extracts were prepared and subjected to SDS-PAGE (17.5% gel). Proteins were transferred to a membrane and immunoblotted with antibodies against Skp2 (top) and FLAG-tag (bottom). Positions of the endogenous Skp2 and transfected FLAG-tagged Skp1 are shown on the right. (C) HtTA1 cells were cotransfected with Skp2 in pcDNA3.1(−) construct and a plasmid expressing CD20. Immediately after harvest, the cells were incubated with a fluorescein isothiocyanate-conjugated anti-CD20 monoclonal antibody, fixed, and stained with propidium iodide. DNA content of the transfected cells (upper portion of the cell population diagram) and nontransfected cells (lower portion of the cell population diagram) was analyzed by FACS. (D) HtTA1 cells were cotransfected with FLAG-tagged Skp1 in pUHD-P1 and a CD20-expressing plasmid as for panel C. FACS analyses of the transfected and nontransfected cells are shown.