PR48, a novel regulatory subunit of protein phosphatase 2A, interacts with Cdc6 and modulates DNA replication in human cells

Abstract

Initiation of DNA replication in eukaryotes is dependent on the activity of protein phosphatase 2A (PP2A), but specific phosphoprotein substrates pertinent to this requirement have not been identified. A novel regulatory subunit of PP2A, termed PR48, was identified by a yeast two-hybrid screen of a human placental cDNA library, using human Cdc6, an essential component of prereplicative complexes, as bait. PR48 binds specifically to an amino-terminal segment of Cdc6 and forms functional holoenzyme complexes with A and C subunits of PP2A. PR48 localizes to the nucleus of mammalian cells, and its forced overexpression perturbs cell cycle progression, causing a G(1) arrest. These results suggest that dephosphorylation of Cdc6 by PP2A, mediated by a specific interaction with PR48, is a regulatory event controlling initiation of DNA replication in mammalian cells.

FIG. 1

Identification of human PR48 from a yeast two-hybrid screen. (A) Schematic representation of Cdc6 segments used as bait proteins. Amino acid positions are numbered relative to the translation initiation codon. Boxes represent highly conserved sequence blocks shared among Cdc6 and ORC1 proteins from different species. The nucleotide binding motifs are shaded. Open circles depict the positions of CDK phosphorylation sites. (B) Two-hybrid assay with pairwise combinations of PR48 (fused to GAL4AD) and various regions of Cdc6 (fused to GAL4BD) in which growth on media lacking histidine (−His) reflects a physical interaction between the two proteins, as seen for PR48 plus Cdc6N and PR48 plus Cdc6F. Negative controls include PR48 plus vector alone or PR48 plus lamin C. The previously described interaction between p53 and SV40 large T antigen provides a positive control.

FIG. 2

Comparison of PR48 to related B" subunits of PP2A and homologous sequences in GenBank. (A) Amino acid sequence alignment of PR48 with previously reported B" proteins and homologous sequences in GenBank. Identical residues are shaded. (B) Dendrogram from analysis by DNASTAR indicating the relative similarities between PR48 and related proteins from nematode, plant, and mammalian species. Units on the horizontal axis indicate the number of substitution events.

FIG. 2

Comparison of PR48 to related B" subunits of PP2A and homologous sequences in GenBank. (A) Amino acid sequence alignment of PR48 with previously reported B" proteins and homologous sequences in GenBank. Identical residues are shaded. (B) Dendrogram from analysis by DNASTAR indicating the relative similarities between PR48 and related proteins from nematode, plant, and mammalian species. Units on the horizontal axis indicate the number of substitution events.

FIG. 2

Comparison of PR48 to related B" subunits of PP2A and homologous sequences in GenBank. (A) Amino acid sequence alignment of PR48 with previously reported B" proteins and homologous sequences in GenBank. Identical residues are shaded. (B) Dendrogram from analysis by DNASTAR indicating the relative similarities between PR48 and related proteins from nematode, plant, and mammalian species. Units on the horizontal axis indicate the number of substitution events.

FIG. 3

Interaction of PR48 and Cdc6 in vitro. PR48 and firefly luciferase (luc) proteins were translated in rabbit reticulocyte lysates and labeled with [³⁵S]methionine (lanes 4 and 5). PR48 copurified with GST-Cdc6 (lane 2) but not with GST (lane 3), as assessed by binding to proteins coupled to glutathione-agarose beads. Luciferase failed to interact with GST-Cdc6 (lane 1), serving as a negative control. Proteins were separated by SDS-PAGE and visualized by autoradiography.

FIG. 4

Interaction of PR48 and Cdc6 in vivo. C2C12 cells were cotransfected with Cdc6–c-Myc and HA-PR48 or with Cdc6–c-Myc and HA–MNF-α. Proteins were immunoprecipitated from whole-cell extracts (lanes 1 and 2) with anti-Myc antibody and separated by SDS-PAGE, and immunoblots were probed with anti-HA antibody (lane 3 and 4). The loading in lane 1 and 2 is equivalent to the cell extracts used for coimmunoprecipitation and subsequent immunoblotting (lane 3 and 4).

FIG. 5

Subcellular localization of PR48. (A) Immunoblot of nuclear and cytosolic proteins from human HeLa cells transfected to express HA-Cdc6 (lanes 1 and 4) and HA-PR48 (lanes 2 and 5). Immunodetection of Sp1 and immunodetection of actin provide controls for the cell fractionation procedure and protein loading (30 μg per lane), respectively. (B) Fluorescence of GFP, PR48-GFP, or GFP-Cdc6 after transfection of HeLa cells.

FIG. 5

Subcellular localization of PR48. (A) Immunoblot of nuclear and cytosolic proteins from human HeLa cells transfected to express HA-Cdc6 (lanes 1 and 4) and HA-PR48 (lanes 2 and 5). Immunodetection of Sp1 and immunodetection of actin provide controls for the cell fractionation procedure and protein loading (30 μg per lane), respectively. (B) Fluorescence of GFP, PR48-GFP, or GFP-Cdc6 after transfection of HeLa cells.

FIG. 6

PR48 forms a PP2A holoenzyme complex. (A) Schematic depiction of the subunit composition of PP2A. (B) Two-hybrid assay with pairwise combinations of PR48 or PR130C (fused to GAL4AD) and Aα (fused to GAL4BD) reveals a physical interaction between PR48 or PR130C with the Aα subunit of PP2A, as indicated by induction of β-galactosidase activity. Negative controls include Aα plus vector or Aα plus SV40 large T antigen. The interaction between p53 and SV40 large T antigen provides a positive control (Promega).

FIG. 7

Reconstitution of an active PP2A holoenzyme containing PR48. (A) PP2A holoenzymes were isolated from Sf9 cells that had been triply infected with baculoviruses expressing hexahistidine-tagged Aα subunit, the C subunit, and either HA-PR48 or the Bα subunit. The complexes were resolved by SDS-PAGE and stained with Coomassie brilliant blue (CBB) or immunoblotted with antibodies against the HA epitope (anti-HA), the Bα subunit (anti-Bα), the Aα subunit (anti-Aα), or the catalytic subunit (anti-C); 3 μg of each preparation was loaded onto the gels for Coomassie blue staining, while 0.5 μg was used for immunoblotting. Also shown are Coomassie blue-stained markers (MW) whose molecular masses (in kilodaltons) are indicated on the left. The mobilities of Aα, HA-PR48, Bα, and C are indicated by lines starting at the right. (B) Phosphatase activity of purified PP2A holoenzymes. The purified holoenzymes shown in panel A were assayed for phosphatase activity, using 2 μM ³²P-labeled myosin light chain as described in Materials and Methods. Phosphatase activity is expressed as picomoles of phosphate released per minute per milligram of purified enzyme. Background was determined in parallel incubations in which enzyme was omitted and was subtracted from values obtained in the presence of enzyme.

FIG. 8

Overexpression of PR48 alters cell cycle progression. Flow cytometry profiles of DNA content (propidium iodide fluorescence) and estimates of the percentage of cells in G₁, S, and G₂/M stages of the cell cycle in asynchronously growing HeLa cells under control conditions and following transfection to express either GFP, PR48-GFP, Bα-GFP, or B′α1-GFP. Profiles of cell number versus DNA content (DNA) were constructed from the cells transfected with an empty vector, pTarget (Vector), or selectively within the transfected cell population as identified by direct (GFP) fluorescence.