Inhibition of cyclin-dependent kinase 2 by p21 is necessary for retinoblastoma protein-mediated G1 arrest after gamma-irradiation

Abstract

In mammalian cells, activation of certain checkpoint pathways as a result of exposure to genotoxic agents results in cell cycle arrest. The integrity of these arrest pathways is critical to the ability of the cell to repair mutations that otherwise might compromise viability or contribute to deregulation of cellular growth and proliferation. Here we examine the mechanism through which DNA damaging agents result in a G1 arrest that depends on the tumor suppressor p53 and its transcriptional target p21. By using primary cell lines lacking specific cell cycle regulators, we demonstrate that this pathway functions through the growth suppressive properties of the retinoblastoma protein (pRB) tumor suppressor. Specifically, gamma-irradiation inhibits the phosphorylation of pRB at cyclin-dependent kinase 2-specific, but not cyclin-dependent kinase 4-specific, sites in a p21-dependent manner. Most importantly, we show that pRB is a critical component of this DNA damage checkpoint. These data indicate that the p53 --> p21 checkpoint pathway uses the normal cell cycle regulatory machinery to induce the accumulation of the growth suppressive form of pRB and suggest that loss of pRB during the course of tumorigenesis disrupts the function of an important DNA damage checkpoint.

Figure 1

Analysis of the genetic requirements of the DNA damage-induced G₁ arrest response. Asynchronous cultures of wild-type, p53−/−, p21−/−, Rb−/− and p21−/−;Rb−/− fibroblasts were irradiated with a dose of 5.5 Gy and labeled with BrdUrd for 4 hr beginning 14 hr after irradiation. Histogram shows the S-phase fraction of irradiated versus untreated samples with the mean and SD (error bars) from four independent experiments.

Figure 2

Effect of γ-irradiation on G₁ cdk activity and pRB phosphorylation. (A) Normalized protein extracts from untreated or γ-irradiated (18 hr) cells were precleared with protein A beads, immunoprecipitated with the indicated antibodies, and then assayed for kinase activity by incubation with an excess of [γ-³²P]ATP and substrate (histone H1 or a C-terminal fragment of pRB). (B) SDS/PAGE analysis of [³²P]pRB from wild-type and p21−/− cells 18 hr after irradiation. (C) Western blot analysis of [³²P]pRB with an α-pRB mAb.

Figure 3

Two-dimensional phosphotryptic mapping of pRB derived from wild-type or p21−/− irradiated cells. pRB was immunoprecipitated from either wild-type or p21−/− irradiated cells and subjected to trypsin digestion. The resulting phosphopeptides were resolved by electrophoresis and ascending chromatography and visualized by autoradiography. ∗ denotes phosphopeptides that were present in maps from Rb−/− cells and therefore are not derived from pRB.

Figure 4

Phosphorylation status of cdk2-specific and cdk4-specific pRB phosphorylation sites. Immunoblot analysis of immunporecipitated pRB with α-P-S780 (cdk4 site), α-P-S811 (cdk2 site), and α-P-T350 (cdk2 site).

Figure 5

Model of DNA damage-induced G₁ arrest.