A tumor necrosis factor alpha- and interleukin 6-inducible protein that interacts with the small subunit of DNA polymerase delta and proliferating cell nuclear antigen

Abstract

A cDNA encoding a protein of 36 kDa, polymerase delta-interacting protein 1 (PDIP1), that interacts with the small subunit (p50) of DNA polymerase delta (pol delta) was identified in a two-hybrid screen of a HepG2 cDNA library by using p50 as bait. The interaction of PDIP1 with p50 was confirmed by pull-down assays, and a similar assay was used to demonstrate that PDIP1 interacts directly with the proliferating cell nuclear antigen (PCNA). PCNA and p50 bound to PDIP1 simultaneously, and PDIP1 stimulated pol delta activity in vitro in the presence, but not the absence, of PCNA, suggesting that PDIP1 also interacts functionally with both p50 and PCNA. Subcellular localization studies demonstrated that PDIP1 is a nuclear protein that colocalizes with PCNA at replication foci. A putative PCNA-binding motif was identified within the C terminus of PDIP1, and a synthetic peptide containing this PCNA-binding motif was shown to bind PCNA by far-Western analysis. Northern analysis demonstrated that PDIP1 mRNA is present in a wide variety of human tissues. PDIP1 was found to be highly homologous to a previously identified protein, B12 [Wolf, F. W., Marks, R. M., Sarma. V., Byers, M. G., Katz, R. W., Shows, T. B. & Dixit, V. M. (1992) J. Biol. Chem. 267, 1317-1326], one of the early response genes induced by tumor necrosis factor alpha. PDIP1 synthesis can also be induced by tumor necrosis factor alpha and by IL-6, cytokines essential for liver regeneration after loss of hepatic tissue. It is suggested that PDIP1 provides a link between cytokine activation and DNA replication in liver as well as in other tissues.

Figure 1

Nucleotide and deduced amino acid sequences of PDIP1 cDNA. The nucleotide (left) and amino acid (right) numberings are indicated. The amino acid sequence begins with the first in-frame methionine. The stop codon is indicated with an asterisk. Two AUUUA mRNA destabilization signals, which begin at nucleotides 1380 and 1583 in the 3′-untranslated region, are underlined, and the polyadenylation signal is in bold. The PCNA-binding motif (amino acids 249–255) is also in bold.

Figure 2

Analysis of PDIP1 mRNA expression by multiple-tissue Northern blotting. A preblotted membrane with mRNAs from nine different human tissues was probed with a 1.6-kb PDIP1 cDNA, randomly labeled with [α-³²P]dCTP as described in Materials and Methods. After stripping, the membrane was reprobed with a 2.0-kb cDNA of a β-actin gene fragment. PDIP1 mRNA levels were normalized to β-actin mRNA and quantitated by using National Institutes of Health IMAGE version 1.62 software.

Figure 3

Interaction of PDIP1 with p50. The indicated amounts of purified GST or GST-PDIP1 were mixed with 1 μg of p50 and processed as described in Materials and Methods. p50 was detected by Western blotting with rabbit anti-p50 as primary antibody. Lanes 1–3, GST; lanes 4–6, GST-PDIP1.

Figure 4

Interaction of PDIP1 with PCNA. (A) Pull-down assay. GST or GST-PDIP1 was mixed with human PCNA and processed as described in Materials and Methods. PCNA was detected with an anti-PCNA monoclonal antibody (PC10). Lane 1, PCNA control; lane 2, GST + PCNA; lane 3, GST-PDIP1 + PCNA. (B) Far-Western analysis. Peptides or proteins bound to poly(vinylidene difluoride) membranes were overlayed with PCNA as described in Materials and Methods. The binding buffer used for the membrane on the left contained 0.25% Tween-20, and the binding buffer used for the membrane on the right contained 0.25% Nonidet P-40. Slot 1, 30 μg of wild-type PDIP1 peptide; slot 2, 30 μg of mutated PDIP1 peptide; slot 3, 30 μg of wild-type p21 peptide; slot 4, 6 μg of wild-type p21 peptide; slot 5, 500 ng of p21 protein; slot 6, 30 ng of PCNA. Bound PCNA was detected with PC10.

Figure 5

PDIP1 simultaneously binds to PCNA and p50. PCNA and p50 were mixed with GST-PDIP1 or GST and processed as described in Materials and Methods. Lane 1, PCNA control; lane 2, p50 control; lane 3, GST, p50, and PCNA; lane 4, GST-PDIP1, p50, and PCNA. PCNA was detected with PC10 as primary antibody, and p50 was detected with rabbit anti-p50 as primary antibody.

Figure 6

Nuclear localization of PDIP1 and colocalization with PCNA at replication foci. (A) MCF7 cells were grown on glass coverslips, fixed, and permeabilized as described in Materials and Methods. The cells were then incubated with anti-PDIP1 antibody for 1 h, washed, incubated with rhodamine-conjugated goat anti-rabbit IgG for 30 min, and visualized by confocal microscopy. (B) MCF7 cells grown on glass coverslips were extracted with 0.1% Triton X-100 before fixation. Primary antibodies were rabbit anti-PIDP1 and human anti-PCNA; secondary antibodies were rhodamine-conjugated goat anti-rabbit IgG and FITC-conjugated goat anti-human IgG. Red, PDIP1; green, PCNA; yellow, merged image.

Figure 7

PDIP1 stimulates PCNA-dependent pol δ activity. BSA, GST, or GST-PDIP1 was preincubated with 0.3 unit of calf thymus pol δ at 4°C for 30 min, then assayed for DNA polymerase activity as described in Materials and Methods. Gray bars, activity in the absence of PCNA; black bars, activity in the presence of PCNA.

Figure 8

Induction of PDIP1 expression in HepG2 cells by TNF-α and IL-6. (A) HepG2 cells were treated with 20 ng/ml TNF-α (in the presence of 10 μg/ml cycloheximide) for the indicated times. Total RNA was isolated and subjected to Northern blotting as described in Materials and Methods and in the legend of Fig. 2. Thirty micrograms of total RNA was loaded for each lane. (B) HepG2 cells were treated with recombinant human IL-6 at a final concentration of 500 units/ml for the indicated times. Cells were lysed, and 30 μg of total protein from each sample was loaded for SDS/PAGE followed by Western blotting. PDIP1 and β-actin were detected with a rabbit anti-PDIP1 and a monoclonal antibody to β-actin, respectively. Protein and mRNA levels were normalized by using β-actin as standard and quantitated by using National Institutes of Health IMAGE version 1.62 software. The fold change in PDIP1 mRNA and protein levels relative to those of β-actin are given at each time point.