c-Myc regulates cyclin D-Cdk4 and -Cdk6 activity but affects cell cycle progression at multiple independent points

Abstract

c-myc is a cellular proto-oncogene associated with a variety of human cancers and is strongly implicated in the control of cellular proliferation, programmed cell death, and differentiation. We have previously reported the first isolation of a c-myc-null cell line. Loss of c-Myc causes a profound growth defect manifested by the lengthening of both the G1 and G2 phases of the cell cycle. To gain a clearer understanding of the role of c-Myc in cellular proliferation, we have performed a comprehensive analysis of the components that regulate cell cycle progression. The largest defect observed in c-myc-/- cells is a 12-fold reduction in the activity of cyclin D1-Cdk4 and -Cdk6 complexes during the G0-to-S transition. Downstream events, such as activation of cyclin E-Cdk2 and cyclin A-Cdk2 complexes, are delayed and reduced in magnitude. However, it is clear that c-Myc affects the cell cycle at multiple independent points, because restoration of the Cdk4 and -6 defect does not significantly increase growth rate. In exponentially cycling cells the absence of c-Myc reduces coordinately the activities of all cyclin-cyclin-dependent kinase complexes. An analysis of cyclin-dependent kinase complex regulators revealed increased expression of p27(KIP1) and decreased expression of Cdk7 in c-myc-/- cells. We propose that c-Myc functions as a crucial link in the coordinate adjustment of growth rate to environmental conditions.

FIG. 1

Cdk activities and expression of cell cycle effectors in exponentially growing cells. (A) Cdk activities. Complexes were immunoprecipitated from extracts with the antibodies indicated at the bottom. The substrate used in the kinase assays (histone H1 or Rb) is indicated in parentheses. Cell lines: TGR-1, c-myc^+/+; HO15.19, c-myc^−/−; HOmyc3, HO15.19 with reconstituted c-Myc expression. All data are normalized to the activity measured in TGR-1 immunoprecipitates. CycA, cyclin A. Error bars indicate standard deviations of a minimum of two independent experiments. (B) Immunoblots of cyclin, Cdk, CKI, and Mad proteins. myc rec., HOmyc3.

FIG. 2

Immediate-early serum-induced events. (A) Phosphorylation of Erk-1 and Erk-2 proteins. (B) Induction of c-Fos expression. Both events were analyzed by immunoblotting; in panel A a phospho-specific MAPK antibody was used. In panel B the higher-molecular-weight bands represent phosphorylated forms of the c-Fos protein. Cell lines: TGR-1, c-myc^+/+; HO15.19, c-myc^−/−. Time points are identified above each lane. Extract from an equal number of cells was loaded in each lane.

FIG. 3

Cyclin and Cdk expression during the G₀-to-S transition. (A) RNase protection analysis of cyclins D1 (CycD1) and D3. (B) Immunoblot analysis of cyclins D1 and D3, Cdk4, and Cdk6. (C) RNase protection analysis of cyclins E and A. (D) Immunoblot analysis of cyclins E and A and Cdk2. Cell lines: TGR-1, c-myc^+/+; HO15.19, c-myc^−/−. All data are presented on an equal-cell-number basis. (A and C) The fold induction is expressed relative to the TGR-1 zero time point. (B and D) Time points are identified above each lane, and antibodies are indicated on the left.

FIG. 4

Cdk activities during the G₀-to-S transition. (A) Cyclin D1 immunoprecipitates (IP). (B) Cyclin D3 immunoprecipitates. (C) Cyclin E immunoprecipitates. (D) Cdk2 immunoprecipitates. Complexes were immunoprecipitated from extracts containing equal amounts of total protein, and kinase assays were performed with either a GST-Rb substrate (A and B) or a histone H1 substrate (C and D) as described in Materials and Methods. Cell lines: TGR-1, c-myc^+/+; HO15.19, c-myc^−/−. The fold induction is expressed relative to the TGR-1 zero time point. The kinase assays shown are representative of three independent experiments.

FIG. 5

Phosphorylation of Rb family members during the G₀-to-S transition. Samples were taken at the time points indicated above each lane and analyzed by immunoblotting. Cell lines: TGR-1, c-myc^+/+; HO15.19, c-myc^−/−. Protein from an equal number of cells was loaded in each lane.

FIG. 6

E2F DNA-binding activity and protein expression during the G₀-to-S transition. (A) E2F DNA-binding activity measured by EMSA. Cell lines: TGR-1, c-myc^+/+; HO15.19, c-myc^−/−. The times at which samples were collected are identified above the lanes. +DOC, addition of 0.8% deoxycholate to the reaction mixture. The identities of E2F complexes are marked by arrows between the panels and were determined by supershifts with the indicated antibodies. (B) Immunoblot analysis of E2F family members. The antibodies used are indicated on the left.

FIG. 7

Activation of Cdk2 complexes with CAK. Cdk2 complexes were immunoprecipitated from exponentially growing cells with Cdk2 antibodies, incubated with active Cak1p, and assayed for histone H1 phosphorylation activity, as described in Materials and Methods. Cell lines: TGR-1, c-myc^+/+; HO15.19, c-myc^−/−; HOmyc3, HO15.19 with reconstituted c-Myc expression (myc reconstr.). All data are represented as percentages of the activity measured in TGR-1 immunoprecipitates without CAK addition.

FIG. 8

Expression of p27 and assembly into Cdk4 and -6 complexes. (A) RNase protection analysis during the G₀-to-S transition. (B) Immunoblot analysis during the G₀-to-S transition. (C) RNase protection analysis in exponentially growing cells. (D) Presence of p27 in cyclin D1-Cdk4 and -Cdk6 complexes during the G₀-to-S transition. Complexes were immunoprecipitated (IP) with cyclin D1 antibody from extracts containing equal amounts of total protein and subsequently analyzed by immunoblotting with p27 antibody. Equal loading of lanes was demonstrated by subsequent reblotting with Cdk6 antibody. Cell lines: TGR-1, c-myc^+/+; HO15.19, c-myc^−/−.

FIG. 9

Overexpression of cyclins D1, E, and A in c-myc^−/− cells. (A) Analysis of protein expression. HO15.19 (c-myc^−/−) cells were infected singly with retrovirus vectors, and clonal cell lines were established. Immunoblotting was performed with exponentially growing cells. Cell lines are designated above each lane. HO15LX1 and HO15LX4 are two clones infected with empty LXSH vector. The exponential-phase doubling times for each clone are indicated below the lanes. The antibodies are indicated to the left of each panel. The cyclin D1 and cyclin A antibodies recognize both the human and rodent proteins; the cyclin E antibody is human specific. (B) Phosphorylation of Rb in cyclin D1-overexpressing cells. Cells were rendered quiescent by serum starvation and subsequently stimulated to reenter the cell cycle by serum addition. The zero time point is the time of serum addition. Samples were taken at the time points indicated above each lane and analyzed by immunoblotting. Cell lines and antibodies are indicated on the left. Equal amounts of protein were loaded in each lane.