The retinoblastoma protein alters the phosphorylation state of polyomavirus large T antigen in murine cell extracts and inhibits polyomavirus origin DNA replication

Abstract

The retinoblastoma tumor suppressor protein (pRb) can associate with the transforming proteins of several DNA tumor viruses, including the large T antigen encoded by polyomavirus (Py T Ag). Although pRb function is critical for regulating progression from G1 to S phase, a role for pRb in S phase has not been demonstrated or excluded. To identify a potential effect of pRb on DNA replication, pRb protein was added to reaction mixtures containing Py T Ag, Py origin-containing DNA (Py ori-DNA), and murine FM3A cell extracts. We found that pRb strongly represses Py ori-DNA replication in vitro. Unexpectedly, however, this inhibition only partially depends on the interaction of pRb with Py T Ag, since a mutant Py T Ag (dl141) lacking the pRb interaction region was also significantly inhibited by pRb. This result suggests that pRb interferes with or alters one or more components of the murine cell replication extract. Furthermore, the ability of Py T Ag to be phosphorylated in such extracts is markedly reduced in the presence of pRb. Since cyclin-dependent kinase (CDK) phosphorylation of Py T Ag is required for its replication function, we hypothesize that pRb interferes with this phosphorylation event. Indeed, the S-phase CDK complex (cyclin A-CDK2), which phosphorylates both pRb and Py T Ag, alleviates inhibition caused by pRb. Moreover, hyperphosphorylated pRb is incapable of inhibiting replication of Py ori-DNA in vitro. We propose a new requirement for maintaining pRb phosphorylation in S phase, namely, to prevent deleterious effects on the cellular replication machinery.

FIG. 1

pRb, but not p53, inhibits the replication of Py T Ag in vitro. (A) Increasing concentrations of pRb were added to replication mixtures containing FM3A extract (300 μg), Py ori-DNA (0.2 μg), and Py T Ag (0.6 μg). After a 3-h incubation at 33°C, 5-μl aliquots were acid precipitated and counted by scintillation. A background of 0.5 pmol has been subtracted. (B) DNA replication products were purified from the replication mixtures, linearized, digested with DpnI, and analyzed on 1% agarose gels followed by autoradiography. Linear DNA (form I) is indicated on the left. (C) Replication mixtures containing FM3A extract (300 μg), Py ori-DNA (0.2 μg), and Py T Ag (0.6 μg) received increasing concentrations of pRb or p53 purified from insect cells or from bacteria (GST-Rb, GST-p53, or GST). pRb¹ and pRb³ are different preparations of pRb. 1 (0.5:1), 2 (1:1), and 3 (2:1) denote the ratios of pRb or p53 to Py T Ag. After a 3-h incubation at 33°C, the DNA products were analyzed as described for panel B. Form I and form II DNAs are indicated on the left.

FIG. 2

pRb inhibits the replication activities of wild-type Py T Ag and a mutant Py T Ag (dl141) that does not bind to pRb. (A and B) Insect cells were coinfected with recombinant baculoviruses expressing either wild-type Py T Ag (w; lanes 1, 2, 7, 9, 10, and 15) or mutant Py T Ag dl141 (m; lanes 3, 4, 8, 11, 12, and 16) and pRb (lanes 1 to 6 and 9 to 14). Extracts from the coinfected insect cells were immunoprecipitated with Py T Ag-specific antibody PAb F5 (A, lanes 1 to 8). The supernatants from those immunoprecipitates were reimmunoprecipitated with a pRb-specific antibody IF-8 (lanes 9 to 16). In panel B, the extracts were first immunoprecipitated with the pRb-specific antibody (lanes 1 to 8) and then reimmunoprecipitated with the Py T Ag-specific antibody (lanes 9 to 16). The immunoprecipitates were analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with IF-8 (A) or PAbF5 (B). Sizes are indicated in kilodaltons. (C) Increasing concentrations of pRb were added to mixtures containing FM3A extract (300 μg), Py ori-DNA (0.2 μg), and wild-type (wt) or mutant (mt; dl141) Py T Ag (0.6 μg). After a 3-h incubation at 33°C, 5-μl aliquots were acid precipitated and counted by scintillation. A background of 0.5 pmol has been subtracted.

FIG. 2

pRb inhibits the replication activities of wild-type Py T Ag and a mutant Py T Ag (dl141) that does not bind to pRb. (A and B) Insect cells were coinfected with recombinant baculoviruses expressing either wild-type Py T Ag (w; lanes 1, 2, 7, 9, 10, and 15) or mutant Py T Ag dl141 (m; lanes 3, 4, 8, 11, 12, and 16) and pRb (lanes 1 to 6 and 9 to 14). Extracts from the coinfected insect cells were immunoprecipitated with Py T Ag-specific antibody PAb F5 (A, lanes 1 to 8). The supernatants from those immunoprecipitates were reimmunoprecipitated with a pRb-specific antibody IF-8 (lanes 9 to 16). In panel B, the extracts were first immunoprecipitated with the pRb-specific antibody (lanes 1 to 8) and then reimmunoprecipitated with the Py T Ag-specific antibody (lanes 9 to 16). The immunoprecipitates were analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with IF-8 (A) or PAbF5 (B). Sizes are indicated in kilodaltons. (C) Increasing concentrations of pRb were added to mixtures containing FM3A extract (300 μg), Py ori-DNA (0.2 μg), and wild-type (wt) or mutant (mt; dl141) Py T Ag (0.6 μg). After a 3-h incubation at 33°C, 5-μl aliquots were acid precipitated and counted by scintillation. A background of 0.5 pmol has been subtracted.

FIG. 3

pRb changes the phosphorylation state of Py T Ag in extracts of FM3A cells. (A) Py T Ag (0.6 μg) was incubated in complete Py ori-DNA replication reaction mixtures at 33°C for 3 h in the presence (+) or absence (−) of pRb (1.2 μg). Two different preparations of FM3A extract (1 and 2, 300 μg each) were used. The total reaction volume (50 μl) was analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with Py T Ag-specific antibody PAb F5. (B) Py T Ag (0.6 μg) was incubated in a complete replication reaction including [γ-³²P]ATP at 33°C for 3 h with (+) or without (−) pRb (1.2 μg). Py T Ag was immunoprecipitated with Py T Ag-specific antibody PAb F5 and analyzed by SDS-PAGE. The Western blot (Western) was probed with PAb F5 and IF-8 (specific for pRb) before being subjected to autoradiography (Autorad). Molecular masses of marker proteins are indicated in kilodaltons on the left; the positions of Py T Ag and pRb are indicated on the right.

FIG. 4

pRb does not affect the phosphorylation state of a prelabeled Py T Ag. Increasing concentrations of pRb were added to complete replication reactions containing ³²P-labeled Py T Ag. After 3 h of incubation at 33°C, the mixtures were split to two. (A) One half was analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted by PAb F5 (Western) prior to autoradiography (Autorad). (B) From the other half (25 μl), DNA was purified from the replication mixtures, linearized, digested with DpnI, and examined on 1% agarose gel and autoradiography. Form I DNA is indicated on the right; sizes of markers are indicated in kilodaltons on the left.

FIG. 5

pRb inhibits phosphorylation of Py T Ag by cyclin A-CDK2. (A) Py T Ag (0.1 μg; lanes 1 to 8), pRb (0.8 μg; lanes 5 to 12), and increasing concentrations of lysates of Sf9 cells coinfected with cyclin (Cyc) A- and CDK2-expressing baculoviruses (0 μl [lanes 1, 5, and 9], 0.5 μl [lanes 2, 6, and 10], 1 μl [lanes 3, 7, and 11], and 2 μl [lanes 4, 8, and 12]) were incubated in kinase buffer at room temperature for 30 min with 1 mM ATP and [γ-³²P]ATP. The phosphorylated proteins were analyzed by SDS-PAGE. (B) Py T Ag (0.15 μg) was incubated with increasing concentrations of pRb (1 [0.35 μg], 2 [0.7 μg], and 3 [1.4 μg]) in complete replication reactions at 33°C for 3 h. Then increasing concentrations of cyclin A-CDK2 (0 μl [lanes a to e], 0.5 μl [lane f], 1 μl [lane g], and 2 μl [lane h]) were added to the reactions containing the most inhibitory levels of pRb. The DNA products were purified from the reactions, linearized, digested with DpnI, and analyzed on 1% agarose gels and by autoradiography. Form I and form II DNAs are indicated on the left.

FIG. 6

Hyperphosphorylated pRb does not inhibit Py ori-DNA replication. (A) High 5 (lanes 1, 3, 4, and 5) or Sf9 (lane 2) cells were infected with baculoviruses expressing His-pRb (lanes 1 to 5) either alone or along with cyclin A- and HA-CDK2-expressing baculoviruses (lanes 3 to 5). pRb proteins were purified on Ni-NTA columns and analyzed by SDS-PAGE. The molecular masses of marker proteins are indicated at the left. The arrow at the left points to the underphosphorylated form of pRb. (B) Underphosphorylated (from lane 1 in panel A; white bars) and hyperphosphorylated (from lane 5 in panel A; black bars) pRb proteins at the indicated amounts were added to replication mixtures (as described in the legend to Fig. 1 and in Materials and Methods). The incorporation of [α-³²P]dAMP into acid-insoluble DNA products was measured by scintillation counting.

FIG. 7

pRb inhibits Py ori-DNA replication by interfering with CDK phosphorylation of Py T Ag. Cyc, cyclin.