Interaction of human p57 with PCNA. (A) Yeast HF7c cells were simultaneously transformed with a plasmid expressing a GAL4^bd fusion protein and a plasmid expressing a GAL4^ad fusion protein as indicated. Cells were streaked on nonselective medium with histidine (-Leu, -Trp), selective medium without histidine (-Leu, -Trp, -His), and selective medium without histidine but containing 5 mM 3-amino-1,2, 3-triazole (-Leu, -Trp, -His, 3-AT). Staining for β-galactosidase expression, activated from an independent GAL4 responsive promoter, is shown (β-gal, Lower Right). The C-terminal domain of p21 possesses as a trans-activating activity (self-activation) when expressed as a fusion protein with the GAL4 DNA binding domain. (B) Equal amounts of histidine-tagged human p57C (lane 1), mouse p57 (lane 2), human p27 (lane 3), full-length human p57 (lane 4), or human p21 (lane 5) were incubated with 2 μg purified human PCNA protein. Mixtures were recovered on Ni-Sepharose beads, resolved by SDS/PAGE, and stained with Coomassie blue. (C) Exponentially growing Sf9 cells were singly infected with baculoviruses expressing the histidine-tagged C-terminal domain of human p57 (His-p57C), full-length human p57 (hp57), human PCNA, or doubly infected with their combinations as indicated on the top of each lane. Cells were metabolically labeled with [³⁵S]methionine 40 h postinfection and p57 protein complexes were recovered on Ni-Sepharose or immunoprecipitated with an anti-p57 antibody and analyzed by SDS/PAGE and autoradiography. (D) Exponentially growing Sf9 cells were coinfected with baculoviruses expressing various human proteins. CDK4 and cyclin D1 protein complexes were immunoprecipitated with indicated antibodies and analyzed by SDS/PAGE and autoradiography.

p57 and p21 binds to PCNA via similar sequences. (A) A small region of human p21 (residues 139–160) sufficient for binding to PCNA (17–19) contain two critical residues, Met-147 and Phe-150 (boxed), are conserved in both mouse p21 and human p57. Mouse p57 lacks a complete corresponding region and predictably the ability to bind PCNA. A series of wild-type (residues 263–285) and mutant p57 peptides used in this study are listed. The hp57(BWS8) corresponds to a mutant p57 identified in a BWS patient (#8) (11). Comparison of binding between the p57 proteins and PCNA determined from experiments shown in Fig. 2C are shown on the right side and binding efficiency between wild-type p57 protein and PCNA was set at 100%. (B) p21 and p57 bind to PCNA via similar sequences. ³⁵S-labeled PCNA protein (20 μl) was incubated with the various p21 or p57 peptides and then added to an equal amount of GST-p57C or GST-p21C fusion proteins attached to glutathione-agarose beads. GST-fusion proteins and bound PCNA were recovered from the different mixtures and resolved by SDS/PAGE. The gel was stained with Coomassie blue (Lower) to verify equal recovery of the GST fusion protein prior to autoradiography (Upper). (C) Mapping of PCNA binding site in vitro. ³⁵S-labeled PCNA protein (20 μl) was incubated with 2 μg wild-type or various mutant GST-p57C proteins. GST-p57C protein and bound PCNA were recovered from the different mixtures and resolved by SDS/PAGE. (D) Mapping of PCNA binding site in vivo. Yeast HF7c cells were simultaneously transformed with the parental pGAD or plasmid expressing a GAL4^bd fusion protein with a wild-type or a mutant p57 C-terminal domain and with a plasmid expressing GAL4^ad fusion with PCNA protein. Cells were streaked on nonselective medium with histidine (-Leu, -Trp) or selective medium without histidine (-Leu, -Trp, -His).

p57 C-terminal domain mediated inhibition of PCNA-dependent DNA synthesis by DNA polymerase δ. (A) Effects of full-length CDK inhibitor p21 and the p21 peptide containing the PCNA binding activity (residues 139–160) on nucleotide incorporation catalyzed by pol δ. (B) Effects of His-p57C and p57 peptide (residues 263–285) on nucleotide incorporation catalyzed by pol δ. (C) Effects of CDK inhibitors p57 and p21 on the length of the DNA products formed in the pol δ holoenzyme catalyzed reaction. All reactions contained 5 ng PCNA except lanes 16 and 17 where 50 ng PCNA protein was added. The levels of inhibitors used were as follows: lanes 2–4 contained 0.3, 0.12 and 0.06 μM p21, respectively; lanes 5–8, 22.5, 9, 4.5 and 2.25 μM p57C, respectively; lanes 9–12, 16.5, 6.6, 3.3, and 16.5 μM p57 peptide (residues 263–285), respectively; lanes 13 and 14, 0.8 and 0.075 μM p21 peptide (residues 139–160). Lane 15 contained 50 μM of a p57 peptide corresponding to the last 15 amino acid residues of human p57 (residues 303–313) outside the conserved PCNA binding sequence. Lane 16 contained 6.6 μM p57 peptide (residues 263–285). Lanes 1 and 17 contained no inhibitors. The reaction described in lane 17 represents the replication activity observed with excess PCNA.

Inhibition of cell cycle and cell transformation by p57. (A) Saos-2 cells were transiently transfected with 1 μg of pcDNA3-green fluorescent protein and 10 μg of indicated plasmids. The percent distribution in different cell cycle stages was analyzed by flow cytometry. (B) Histogram of the average number of foci per plate counted 12 days posttransfection for two individual experiments combined. Twelve plates (six for each experiment) that were split 1:3 on the day following the transfection were counted for each point. Bars = SD.