Role of the LXCXE binding site in Rb function

Abstract

Oncoproteins from DNA tumor viruses such as adenovirus E1a, simian virus 40 T antigen, and human papillomavirus E7 contain an LXCXE sequence, which they use to bind the retinoblastoma protein (Rb) and inhibit its function. Cellular proteins such as histone deacetylases 1 and 2 (HDAC1 and -2) also contain an LXCXE-like sequence, which they use to interact with Rb. The LXCXE binding site in Rb was mutated to assess its role in Rb function. These mutations inhibited binding to HDAC1 and -2, which each contain an LXCXE-like sequence, but had no effect on binding to HDAC3, which lacks an LXCXE-like sequence. Mutation of the LXCXE binding site inhibited active transcriptional repression by Rb and prevented it from effectively repressing the cyclin E and A gene promoters. In contrast, mutations in the LXCXE binding site did not prevent Rb from binding and inactivating E2F. Thus, the LXCXE mutations appear to separate Rb's ability to bind and inactivate E2F from its ability to efficiently recruit HDAC1 and -2 and actively repress transcription. In transient assays, several of the LXCXE binding site mutants caused an increase in the percentage of cells in G(1) by flow cytometry, suggesting that they can arrest cells. However, this effect was transient, as none of the mutants affected cell proliferation in longer-term assays examining bromodeoxyuridine incorporation or colony formation. Our results then suggest that the LXCXE binding site is important for full Rb function. Mutation of the LXCXE binding site does not inhibit binding of the BRG1 ATPase component of the SWI/SNF nucleosome remodeling complex, which has been shown previously to be important for Rb function. Indeed, overexpression of BRG1 and Rb in cells deficient for the proteins led to stable growth inhibition, suggesting a cooperative role for SWI/SNF and the LXCXE binding site in efficient Rb function.

FIG. 1

Diagram of the LXCXE binding site derived from cocrystallization of the Rb pocket with an LXCXE peptide (26). Tyr 709, Lys 713, Tyr 756, and Asn 757 are conserved amino acids in the Rb pocket that appear to make important contacts with the backbone of the LXCXE peptide. Each of these amino acids was mutated to alanine either individually or in combinations.

FIG. 2

Mutations in the LXCXE binding site of Rb inhibit interaction with adenovirus E1a and HDAC1. A coimmunoprecipitation assay was used to assess the effect of LXCXE binding site mutations on the binding of Rb to E1a. An expression vector for the large pocket of Rb (amino acids 379 to 928) (WT [wild type]) (44) or the indicated mutants were transfected into C33a cells along with an expression vector for E1a (43). E1a was immunoprecipitated (I.P.), and associated Rb was detected by Western blotting. “Control” indicates that an irrelevant antibody (to HPV E7) was used for immunoprecipitation.

FIG. 3

Mutation of the LXCXE binding site in Rb does not affect interaction with or inactivation of E2F-1. (A) Expression vectors for E2F-1 and either wild-type (WT) or mutant Rb large pocket were cotransfected into C33a cells, and interaction was followed by coimmunoprecipitation (I.P.) as in Fig. 2. (B) The E2F-CAT reporter plasmid (44), which contains E2F sites upstream of a TATA box, was transfected into CV-1 cells. Expression vectors for wild-type or mutant Rb large pocket (4, 44) were cotransfected as indicated to determine the effect of LXCXE binding site mutations on the ability of Rb to inhibit E2F activity. (C) E1a cannot block Rb inhibition of E2F when the LXCXE binding site is mutated. Wild-type Rb and LXCXE binding site mutant expression vectors were transfected as in panel B along with an expression vector for an E1a mutant where amino acids 2 to 36 are deleted (removes the p300/CBP binding domain, leaving the Rb binding domain intact) (43). CAT activity is representative of five independent experiments, each done in duplicate.

FIG. 4

Rb LXCXE binding site mutants show decreased binding to HDAC1 and -2 but retain binding to HDAC3. (A) E1a competes for binding of HDAC1 to Rb. An expression vector for wild-type (WT) or mutant Rb large pocket was cotransfected into Rb⁻ C33a cells along with expression vectors for Flag-tagged HDAC1 and, where indicated, E1a. Association of Rb and HDAC1 was detected by coimmunoprecipitation (I.P.) as in Fig. 2. (B) C33a cells were cotransfected with expression vectors for wild-type Rb large pocket or the indicated mutants and HDAC1 containing a Flag tag (28). Association of Rb and HDAC1 was assessed by coimmunoprecipitation as indicated. “758” indicates a control Ser-to-Leu mutation at amino acid 758; this amino acid is adjacent to the LXCXE binding site in the crystal structure, but it does not contact the LXCXE (26). (C) Wild-type Rb large pocket or Rb mutant and Flag-tagged HDAC2 expression vectors were transfected into C33a cells. Interaction between Rb and HDAC2 was determined by coimmunoprecipitation. (D) C33a cells were cotransfected with LexA-tagged HDAC3 and Rb or Rb mutant expression vectors. Binding to Rb was assessed by coimmunoprecipitation.

FIG. 5

Mutation of Rb's LXCXE binding site results in abrogation of Rb's ability to actively repress. (A) A reporter (MLPCAT) containing the adenovirus MLP with Gal4 DNA binding sites upstream (28) was cotransfected into CV-1 cells along with expression vectors for wild-type (WT) Rb large pocket or Rb large pocket mutants fused to the DNA binding domain of Gal4 as indicated to assess the effect of LXCXE binding site mutations on active transcriptional repression. As a control, an expression vector for Gal4-Mad (28) was cotransfected. The HDAC inhibitor trichostatin A (TSA) was added to the transfected cells as described previously (28). (B) The cycA-luc reporter was transfected into U2OS cells, along with wild-type Rb large pocket or Rb mutant expression vectors, and luciferase activity was measured. Luciferase activities are plotted relative to reporter alone activity, which is indicated as 100%. Transfection assays are representative of five independent experiments, each done in duplicate.

FIG. 6

Mutation of the LXCXE binding site and growth suppression by Rb. (A) Wild-type (WT) Rb large pocket or Rb mutant expression vectors were cotransfected with an expression vector for CD20 into Rb⁻ Saos-2 cells. Cells were harvested 36 h later, and CD20⁺ cells were analyzed by flow cytometry for DNA content. (B) Expression vectors for wild-type Rb large pocket or large pocket mutants were cotransfected into Rb⁻ Saos-2 cells along with an expression vector for neomycin resistance. Cells were selected in G418 for 3 weeks; colonies were stained with crystal violet and counted.

FIG. 7

BRG1 facilitates growth suppression by Rb and does not require the LXCXE binding site for interaction with Rb. (A) Rb LXCXE binding site mutants still bind to BRG1. Expression vectors for Flag-tagged BRG1 and either wild-type (WT) or mutant Rb large pocket were cotransfected into C33a cells, and association between BRG1 and Rb was assessed by coimmunoprecipitation (I.P.). (B) Wild-type Rb large pocket was transfected into Rb⁻, BRG1/BRM-deficient C33a cells along with an expression vector for neomycin resistance. Cells were selected in G418 for 2 weeks; colonies were stained with crystal violet and counted. Saos-2 cells were selected in G418 for 3 weeks. (C) C33a cells or Saos-2 cells were transfected with the indicated expression vectors and a vector expressing a puromycin resistance gene. Cells resistant to puromycin were examined 5 days after transfection for BrdU incorporation.