Analysis of cyclin D3-cdk4 complexes in fibroblasts expressing and lacking p27(kip1) and p21(cip1)

Abstract

Our studies examined the effects of p27(kip1) and p21(cip1) on the assembly and activity of cyclin D3-cdk4 complexes and determined the composition of the cyclin D3 pool in cells containing and lacking these cyclin-dependent kinase inhibitors. We found that catalytically active cyclin D3-cdk4 complexes were present in fibroblasts derived from p27(kip1)-p21(cip1)-null mice and that immunodepletion of extracts of wild-type cells with antibody to p27(kip1) and/or p21(cip1) removed cyclin D3 protein but not cyclin D3-associated activity. Similar results were observed in experiments assaying cyclin D1-cdk4 activity. Data obtained using mixed cell extracts demonstrated that p27(kip1) interacted with cyclin D3-cdk4 complexes in vitro and that this interaction was paralleled by a loss of cyclin D3-cdk4 activity. In p27(kip1)-p21(cip1)-deficient cells, the cyclin D3 pool consisted primarily of cyclin D3 monomers, whereas in wild-type cells, the majority of cyclin D3 molecules were complexed to cdk4 and either p27(kip1) or p21(cip1) or were monomeric. We conclude that neither p27(kip1) nor p21(cip1) is required for the formation of cyclin D3-cdk4 complexes and that cyclin D3-cdk4 complexes containing p27(kip1) or p21(cip1) are inactive. We suggest that only a minor portion of the total cyclin D3 pool accounts for all of the cyclin D3-cdk4 activity in the cell regardless of whether the cell contains p27(kip1) and p21(cip1).

FIG. 1

Lack of effect of p27^kip1 immunodepletion on cyclin D3-associated kinase activity in BALB/c 3T3 cells. Density-arrested BALB/c 3T3 cells were mitogenically stimulated with 10 ng of platelet-derived growth factor per ml and 10% serum and harvested at the indicated times. (A) Cell lysates (40 μg) were immunoblotted with antibody to cyclin D3. (B and C) Cell lysates (80 μg) were incubated with antibody to p27^kip1. Immune complexes were pelleted with protein A-agarose beads and immunoblotted with antibody to cyclin D3 (C). The p27^kip1-depleted supernatant was immunoprecipitated and immunoblotted with cyclin D3 antibody (B). (D) Cell lysates (40 μg) were immunoblotted with antibody to p27^kip1. (E and F) Cell lysates were immunoprecipitated with preimmune serum (E) or antibody to p27^kip1 (F). Immune complexes were removed by centrifugation with protein A-agarose beads, and supernatants were immunoprecipitated with cyclin D3 antibody. Immunoprecipitated material was assayed for cyclin D3-associated kinase activity using GST-Rb as substrate.

FIG. 2

Lack of effect of p27^kip1 immunodepletion on cyclin D3-associated kinase activity in p21^−/− MEFs. Confluent p21^−/− MEFs were incubated in medium containing 0.1% serum for 30 h and subsequently stimulated with 10 ng of PDGF per ml and 10% serum for 18 h. Cell lysates were immunoprecipitated with preimmune serum (mock depletion, left panel) or antibody to p27^kip1 (right panel). Immune complexes were removed by centrifugation with protein A-agarose beads and immunoblotted with monoclonal antibody to cyclin D3 (C). Supernatants were immunoprecipitated with polyclonal antibody to cyclin D3, and immunoprecipitated material was assayed for cyclin D3-associated kinase activity using GST-Rb as substrate (A) or immunoblotted with monoclonal antibody to cyclin D3 (B).

FIG. 3

Cyclin D3-cdk4 association and activity in MEFs lacking both p27^kip1 and p21^cip1. Asynchronously cycling wild-type MEFs, p27N/p21^−/− MEFs (derived from mice 816 and 780), and p27/p21^−/− MEFs (derived from mouse Rob6) were harvested and assayed as follows. (A and E) Cell lysates were immunoprecipitated with antibody to cyclin D3, and immune complexes were assayed for kinase activity using GST-Rb as substrate. (B and F) Cell lysates (80 μg) were immunoblotted with antibody to cyclin D3. (C and G) Cell lysates (80 μg) were immunoblotted with antibody to cdk4. (D and H) Cell lysates (200 μg) were immunoprecipitated with antibody to cdk4 and immunoblotted with antibody to cyclin D3. As different amounts of lysate were used for the assays in panels B and F versus those in panels D and H, the results obtained cannot be compared on a quantitative basis.

FIG. 4

Verification of cyclin D3-associated cdk4 activity as the GST-Rb-phosphorylating activity in p27^kip1-p21^cip1-deficient MEFs. (A) Lysates of exponentially proliferating p27/p21^−/− MEFs were immunoprecipitated with the indicated antibodies (lanes 1 to 7) or preimmune serum (lane 8) or were mock precipitated (lane 9). Immune complexes were assayed for kinase activity using GST-Rb as substrate (top panel) or were immunoblotted with antibody to cyclin D3 (bottom panel). The buffer control (lane 10) contained kinase buffer in place of resuspended immune complex. Cyclin D3 antibodies 1 and 2 were purchased from Santa Cruz and prepared by us, respectively. Cell extracts were also immunoprecipitated with antibody to cyclin D3 (Santa Cruz) that had been preincubated with either buffer alone (lane 11) or the peptide against which the antibody was generated (lane 12). (B) Cell extracts prepared from logarithmically growing p27/p21^−/− MEFs (top panels) and BALB/c 3T3 cells (bottom panels) were immunoprecipitated with antibody to cyclin D3. Immunoprecipitated material was resuspended in kinase buffer containing the indicated concentrations of flavopiridol and incubated for 30 min at 30°C. Immune complexes were assayed for cyclin D3-associated activity using GST-Rb as substrate or were immunoblotted with antibody to cyclin D3. (C) Cell extracts prepared from asynchronously cycling p27/p21^−/− cells were immunoprecipitated with antibody to cyclin D3 (top panels) or cyclin A (bottom panels). Immune complexes were resuspended in kinase buffer containing the indicated concentrations of roscovitine and incubated for 30 min at 30°C. Immunoprecipitated material was assayed for kinase activity using GST-Rb as substrate or was immunoblotted with antibody to cyclin D3 (cyclin D3 immunoprecipitates) or cyclin A (cyclin A immunoprecipitates). Ab, antibody; IP, immunoprecipitation.

FIG. 5

Cyclin D1-cdk4 activity in p27/p21^−/− MEFs. (A) Cell lysates of growing wild-type and p27/p21^−/− MEFs were immunoprecipitated with antibody to cdk4 and immunoblotted with antibody to cyclin D1 (top panel) or were only immunoblotted with antibody to cyclin D1 (bottom panel). (B) Extracts of wild-type and cyclin D1-deficient MEFs were immunoblotted with antibody to cyclin D1. (C) Extracts of wild-type and p27/p21^−/− MEFs were immunoprecipitated with antibody to cyclin D1 (lanes 1 and 4), preimmune serum (PI) (lanes 3 and 6), or cyclin D1 antibody that had been preincubated with the peptide against which the antibody was generated (lanes 2 and 5). Immune complexes were assayed for kinase activity using GST-Rb as substrate. Ab, antibody.

FIG. 6

Inhibition of cyclin D3-cdk4 activity by p27^kip1 in mixed cell extracts. Extracts of density-arrested BALB/c 3T3 cells were boiled for 5 min and clarified by centrifugation (designated “G₀ extracts”). G₀ extracts were mixed at the indicated ratios with extracts (200 μg) of proliferating p27/p21^−/− cells and incubated at 30°C for 30 min. Mixed cell extracts were immunoprecipitated with antibody to cyclin D3 (A and B) or cyclin A (C and D). Immune complexes were assayed for cyclin D3-associated activity (A) or cyclin A-associated activity (C) or were immunoblotted with antibody to p27^kip1 (B and D). Based on comparisons with known amounts of recombinant p27^kip1, 100 μg of G₀ extract contains approximately 7 ng of p27^kip1. Ab, antibody. IP, immunoprecipitation.

FIG. 7

High levels of uncomplexed cyclin D3 in p27/p21^−/− MEFs. (A) Boiled and clarified extracts of G₀-arrested BALB/c 3T3 cells were mixed with extracts of exponentially growing p27/p21^−/− cells at the indicated ratios and incubated for 30 min at 30°C. (Top panel) Mixed cell extracts were immunoprecipitated and immunoblotted with antibody to cyclin D3. (Middle panel) Mixed cell extracts were immunoprecipitated with antibody to p27^kip1 and immunoblotted with antibody to cyclin D3. (Bottom panel) Mixed cell extracts were immunodepleted of p27^kip1, and depleted extracts were immunoprecipitated and immunoblotted with antibody to cyclin D3. (B) Mixed cell extracts were prepared and incubated as for panel A and were immunodepleted of p27^kip1 (+) or mock depleted (−). Depleted extracts were immunoprecipitated (IP) with preimmune serum (PI) or antibody to cdk4 and immunoblotted with antibody to cyclin D3.

FIG. 8

Low levels of binary cyclin D3-cdk4 complexes in wild-type MEFs. (A) Extracts of growing wild-type MEFs were immunodepleted of p27^kip1, p21^cip1, or cdk4 or were mock depleted. Depleted extracts were immunoprecipitated with antibody to cdk4 (lanes 1 to 4) or cyclin D3 (lanes 5 to 9) and immunoblotted with antibody to cyclin D3. Depleted extracts were also immunoblotted with antibody to actin (lanes 10 to 13). A longer exposure of lanes 1 to 4 is also shown (lanes denoted by asterisks). (B) Extracts were immunodepleted of p27^kip1, p21^cip1, or both or were mock depleted. Depleted extracts were immunoprecipitated with antibody to cyclin D3 and assayed for kinase activity using GST-Rb as substrate. (C) Extracts of wild-type MEFs were immunodepleted using preimmune serum (−) or antibodies to p27^kip1 and p21^cip1 (+). Depleted extracts were immunoprecipitated with antibody to cdk4 or preimmune serum (PI), and immune complexes were assayed for kinase activity (top panel) or immunoblotted with cdk4 antibody (bottom panel). (D) Extracts of growing p21^−/− MEFs were depleted of p27^kip1 or mock depleted. Depleted extracts were immunoprecipitated with antibody to cyclin D3 for determination of kinase activity (top panel) or were immunoprecipitated with antibody to cdk4 and immunoblotted with antibody to cyclin D3 (bottom panel). Ab, antibody. IP, immunoprecipitation.