©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Binding of an Interferon-inducible Protein (p202) to the Retinoblastoma Protein (*)

(Received for publication, September 28, 1994; and in revised form, December 19, 1994)

Divaker Choubey (1) (2) Peter Lengyel (1)(§)

From the  (1)Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520 and the (2)Department of Clinical Immunology and Biological Therapy, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Many of the antimicrobial, immunomodulatory, and cell growth regulatory activities of the interferons are mediated by interferon-inducible proteins. One family of such murine proteins is encoded by six or more adjacent and structurally related genes (gene 200 cluster). Two homologous human genes have also been reported. p202, encoded by the Ifi202 gene in the gene 200 cluster, is a 52-kDa nuclear phosphoprotein. Constitutive overexpression of p202 in transfected cells is growth-inhibitory. We report here that p202 binds the cell growth regulatory retinoblastoma protein (pRb) in vitro and in vivo. The binding is due to direct interaction between the two proteins. p202 has two nonoverlapping segments for binding pRb, and pRb has two nonoverlapping segments (one of them including the pocket region) for binding p202. The hypophosphorylated form of pRb binds to p202. p202 is the first interferon-inducible protein found to bind pRb.


INTRODUCTION

The various activities of the interferons (IFNs) (^1)are mediated by IFN-inducible ``effector'' proteins(1, 2, 3, 4) . One family of IFN-inducible proteins (including p202, p203, p204, and D3) is encoded by six or more structurally related murine genes at the q21-q23 region of chromosome 1 (gene 200 cluster)(5, 6, 7, 8) . Two homologous human genes (MNDA and IFI16) have also been described (9, 10) . All the proteins encoded by these genes share one or two homologous (partially conserved) 200-amino acid-long segments. Three proteins (p202, p204, and MNDA) are nuclear(9, 11, 12) .

p202 is a 52-kDa phosphoprotein. Its amino acid sequence contains potential sites for phosphorylation by several kinases including p34(12) . The level of p202 is increased in various cultured cells 15-20-fold after treatment with IFN. After exposing cells to IFN, p202 accumulates first in the cytoplasm and moves to the nucleus only after a delay of 36 h. In metaphase cells, p202 appears to be associated with chromatin. p202 is not released from nuclei (isolated from IFN-treated cells) by DNase I or low salt treatment. This suggests that the association of p202 with the nucleus is not (or not only) due to binding to DNA, but may be a consequence of binding to proteins. Constitutive overexpression of p202 in transfected cells is growth-inhibitory(12) . This activity of p202, together with the occurrence in p202 of the amino acid sequence LXCXE, also present in several (although not all) proteins (13) binding the retinoblastoma protein (pRb), prompted us to test for an interaction between p202 and pRb.

The 105-kDa pRb is encoded by the retinoblastoma tumor suppressor gene and is a negative growth regulator(14, 15, 16) . The functioning of pRb is regulated by phosphorylation in a cell cycle-dependent manner(17) . pRb is phosphorylated during the late G(1) and S phases and is dephosphorylated at the end of mitosis(18, 19, 20) . Growth arrest caused by deprivation of growth factors, high cell density, induction of differentiation, or senescence is associated with the disappearance of the hyperphosphorylated form of pRb(19, 20, 21, 22) . The hypophosphorylated form of pRb retains the cells in the G(0)/G(1) phase of the cell cycle(18, 23) . Several cellular proteins interact with pRb including the transcription factors E2F and DP1(24) .

IFNs can inhibit the growth of various cultured cells by prolonging all phases of the cell cycle or by arresting the growth at the G(0)/G(1) phase, e.g. in the case of sensitive hematopoietic cells(25) . IFNs were also reported to suppress phosphorylation of pRb in such hematopoietic cells(26) .

Here we report experiments demonstrating that the IFN-inducible p202 protein binds pRb both in vitro and in vivo. The binding is a consequence of direct p202-pRb interaction. p202 contains two nonoverlapping segments for binding pRb, and pRb contains two nonoverlapping segments (one of them including the pocket region) for binding p202. The hypophosphorylated (growth-inhibitory) form of pRb is bound by p202.


MATERIALS AND METHODS

Cell Lines, Growth Conditions, and Treatment with IFN

Murine AKR-2B cloned embryo cells were cultured according to (11) . Murine NS1 myeloma and human J82 bladder carcinoma cells, human U2OS osteosarcoma and SAOS-2 osteosarcoma cells (from American Type Culture Collection), and human HeLa cervical carcinoma cells (from G. Sen) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. When indicated, the cells were treated with recombinant human IFN-alpha(2)/alpha(1)-(1-83) at 1000 units/ml for 36 h (27) .

Antibodies

Monoclonal anti-human pRb antibodies, clone G3-245, and clone G99-549 were from Pharmingen. The monoclonal anti-human pRb antibodies linked to agarose beads (Ab1-clone 36) were from Oncogene Science Inc. A polyclonal rabbit anti-human pRb antiserum (C-15) was from Santa Cruz Biotechnology Inc. All these antibodies cross-react with murine pRb. The polyclonal rabbit anti-p202 antiserum was described earlier(12) .

Plasmids

The GST-202-(19-445) expression plasmid (hereafter indicated as GST-202) was prepared in two steps. The HincII fragment from 202 cDNA (7) was inserted into the SmaI site of the GST-1N vector (Pharmacia Biotech Inc.). The plasmid obtained was cleaved with EcoRI (the resulting short fragment was discarded), and the resulting long fragment was ligated to the small EcoRI fragment that had been excised from 202 cDNA. GST-202-(58-291), GST-202-(255-445), and GST-202-(295-445) were generated by ligating restriction fragments from 202 cDNA (BspHI-EcoRI, BstEII-BamHI, and EcoRI fragments, respectively) into the GST vector. GST-Rb-(237-922), GST-Rb-(1-254), and GST-Rb-(255-922) were generated by ligating restriction fragments from murine retinoblastoma cDNA (PvuII, HindIII-Eco47III, and Eco47III-HindIII fragments, respectively) into the SmaI site of the GST vector (Pharmacia Biotech Inc.).

To obtain retinoblastoma templates for generating transcripts in vitro to be translated into truncated pRb segments, the pRb plasmid (in pGEM3, Promega) was digested with appropriate restriction enzymes (using NdeI for pRb-(1-479), Eco47III for pRb-(1-254), and ClaI for pRb-(1-132)). The plasmid encoding the GST-E1a fusion protein (pGST-E1a) was generated by blunt-end ligating the EcoRI-SalI fragment from E1a cDNA (from J. Germino) into the SmaI site of the GST vector. The plasmid for the mammalian expression of p202 (pCMV-202) was generated by ligating p202 cDNA into the BamHI site of the pCMV vector (Invitrogen). The GST-human retinoblastoma segment fusion proteins (GST-Rb-(379-928) and GST-Rb-(379-928)(706CF)) were from D. M. Livingston and W. G. Kaelin, Jr.(28) .

Expression of GST Fusion Proteins and Loading of Glutathione-Sepharose Beads

The GST fusion proteins were expressed in Escherichia coli DH5alpha and affinity-purified as described by Kaelin et al.(28) . The beads were loaded with 0.5 µg of the indicated GST fusion protein, blocked in blocking buffer (0.25% gelatin, 50 mM KCl, 50 mM HEPES (pH 7.5)) at room temperature for 30 min, washed in binding buffer (150 mM NaCl, 50 mM HEPES (pH 7.5), 0.1% Nonidet P-40), and used for affinity chromatography.

Transcription and Translation in Vitro

Transcripts in vitro from 202 cDNA (in the pBluescript vector, Stratagene) and murine pRb cDNA (in the pGEM3 vector) were translated in a rabbit reticulocyte lysate (Promega) supplemented with [S]methionine(12) .

Preparation of Extracts from Mammalian Cells

Extracts from cultured cells were prepared in ice-cold extraction buffer (50 mM HEPES (pH 7.5), 0.1% Nonidet P-40, 100 mM NaF, 0.2 mM orthovanadate, 100 µg/ml leupeptin (Boehringer Mannheim), 1 mM phenylmethylsulfonyl fluoride (Sigma)) supplemented with 250 mM NaCl. The extracts were diluted 2-fold with (unsupplemented) extraction buffer for affinity chromatography.

Affinity Chromatography of Proteins Translated in Vitro and Mammalian Cell Extracts on Glutathione-Sepharose Beads Loaded with GST Fusion Proteins

Aliquots (10 µl) from the reaction mixtures in which S-labeled proteins had been translated in vitro were incubated with washed glutathione-Sepharose beads (loaded with the indicated GST fusion protein) in binding buffer at room temperature for 30 min. The beads were washed five times in binding buffer, and the bound proteins were released by boiling in sample buffer (29) and subjected to SDS-PAGE and fluorography.

Aliquots from the cell extracts (which had been diluted 2-fold with extraction buffer) were incubated at 4 °C for 90 min with glutathione-Sepharose beads loaded with the indicated GST fusion protein. The beads were washed five times in extraction buffer supplemented with 125 mM NaCl, and the bound proteins were eluted as indicated above by boiling in sample buffer or at room temperature with extraction buffer supplemented with 500 mM NaCl.

Immunoblotting

Far Western blotting (30) was performed using [S]Met-labeled murine pRb. Western blotting with anti-202 antiserum was performed according to a published procedure (12) .

Immunoprecipitation

Extracts from serum-starved murine AKR-2B cells (control or treated with IFN) were incubated at 4 °C for 2 h with anti-Rb antibodies (Ab1-clone C36) that had been conjugated to agarose beads. The beads were washed with extraction buffer five times, and the bound proteins were released in extraction buffer supplemented with 500 mM NaCl and subjected to SDS-PAGE and Western blotting using the anti-202 antiserum.

Expression of Murine pRb and p202 in Transfected Cells

pRb-wt (from J. Sedivy) with murine retinoblastoma cDNA inserted into a pMAMneo-Blue-derived expression vector containing the cytomegalovirus enhancer (CLONTECH) and p202 cDNA in the form of pCMV-202 were transfected into AKR-2B cells using a calcium phosphate-based procedure(31) . Independent G418-resistant clones were picked from the cultures transfected with pCMV-Rb, pCMV-202, or pCMV vector. The three types of clones were pooled separately, and the levels of pRb and p202 expressed were assayed by Western blotting.


RESULTS

Wanting to test the effects of p202 overexpression, we have transfected cultured AKR-2B cells with a plasmid encoding p202 cDNA, driven by a strong enhancer and linked to a drug resistance (G418) marker. Among the fewer than 30 drug-resistant clones obtained, the level of p202 expression was only 2-3-fold above the basal level, and even these grew more slowly than control cells (data not shown; see also (12) ). These findings, obtained repeatedly, together with the fact that transfection of the vector only (with the drug resistance marker) resulted in many more (over 3000) clones, indicate that constitutive overexpression of p202 is growth-inhibitory.

Since, as reported earlier(12) , the association of p202 with isolated nuclei seems to be based primarily on protein-protein interaction, we wish to identify proteins that bind to p202 and, possibly, mediate its growth-inhibitory action. A far Western assay using labeled p202 as the probe for testing an extract from AKR-2B cells revealed the binding of p202 to several proteins (data not shown). The occurrence in the p202 sequence of LXCXE, a motif known to mediate the binding of several proteins to the negative growth regulator protein pRb (13) (Fig. 1), prompted us to examine first whether p202 binds to pRb.


Figure 1: Occurrence of the pRb-binding motif LXCXE in p202. Shown is the alignment of the motif in various pRb-binding proteins. HPV, human papilloma virus.



Binding of p202 to pRb in Vitro

To facilitate the study, we generated a GST-202 fusion protein (GST-202-(19-445)) in E. coli and affinity-purified it. Labeled murine pRb (which had been translated in vitro) bound to GST-202, but not to GST (Fig. 2A, leftpanel). The selectivity of the binding to GST-202 was indicated by the finding that none of several labeled in vitro translated proteins, i.e. luciferase (Promega), three brome mosaic virus-encoded proteins (of 109, 94, and 35 kDa) (Promega), or p204 (a protein with regions similar in sequence to those in p202)(7) , bound to GST-202 or to GST (data not shown). The binding of pRb to GST-202 reached the maximal level within 5 min (data not shown). 15-20% of the input pRb was bound even at 250 mM NaCl or in the presence of 5 mM EDTA. Labeled p202 (which had been translated in vitro) bound to GST-Rb, but not to GST (Fig. 2A, rightpanel).


Figure 2: Binding of pRb to p202 in vitro. A: leftpanel, binding of pRb translated invitro to GST-202. [S]Met-labeled murine pRb was incubated with glutathione-Sepharose beads loaded with either GST (lane2) or GST-202 (lane3). After washing the beads, the bound proteins were eluted and analyzed by SDS-PAGE and fluorography. An aliquot of [S]Met-labeled pRb (IVT-Rb) was run in lane1. The pRb band is indicated by an arrow. Rightpanel, binding of p202 translated in vitro to GST-Rb. [S]Met-labeled p202 was incubated with glutathione-Sepharose beads loaded with either GST (lane2) or GST-Rb (lane3). Processing was as described for the leftpanel. An aliquot of [S]Met-labeled p202 (IVT-202) was run in lane1. The p202 band is indicated by an arrow. B: leftpanel, binding of pRb in cell extracts to GST-202 assayed by Western blotting. Extracts from the indicated murine (lanes 1-4) or human (lanes 5-10) lines were incubated with glutathione-Sepharose beads loaded with either GST (lanes1, 3, 5, 7, and 9) or GST-202 (lanes2, 4, 6, 8, and 10). After washing the beads, the bound protein was released by boiling in SDS sample buffer and was analyzed by SDS-PAGE and Western blotting with the monoclonal anti-Rb antibody G3-245. The pRb band is indicated by an arrow. Rightpanel, binding of p202 in cell extracts to GST-Rb assayed by Western blotting. Extracts from growing AKR-2B cells without (lanes3 and 4) or after (lanes5 and 6) exposure to IFN were incubated with glutathione-Sepharose beads loaded with GST (lanes3 and 5) or GST-Rb (lanes4 and 6). After washing the beads, the proteins were released by boiling in SDS sample buffer and were analyzed by SDS-PAGE and Western blotting with a polyclonal anti-p202 antiserum. Aliquots from extracts of control and IFN-treated AKR-2B cells were also analyzed (lanes1 and 2, respectively). The p202 band is indicated by an arrow. C: binding of pRb translated in vitro to GST-202 assayed by far Western blotting. Affinity-purified GST-202 (2 µg) (lanes 2 and 5), GST (5 µg) (lanes 1 and 4), and unstained protein markers (PM) (myosin heavy chain, 200 kDa; phosphorylase b, 97 kDa; bovine serum albumin, 68 kDa; and ovalbumin, 43 kDa (from Life Technologies, Inc.)) (lanes 3 and 6) were subjected to SDS-PAGE, blotted to a membrane, and stained for proteins with Ponceau S (lanes 1-3) or processed for far Western blotting using labeled pRb translated in vitro (lanes 4-6). The p202 band is indicated by an arrow. D, direct binding of pRb to GST-202 assayed by Western blotting. Purified recombinant pRb was incubated with glutathione-Sepharose beads not loaded (indicated as GA; lane 1) or loaded with GST (lane 2) or GST-202 (lane 3). After washing the beads, the bound protein was released and analyzed by SDS-PAGE and Western blotting using anti-Rb antibodies. The pRb band is indicated by an arrow. For further details, see ``Materials and Methods.''



We proceeded by testing whether pRb in cell extracts (which might be differently phosphorylated from that translated in vitro) also binds to GST-202. As shown in Fig. 2B (leftpanel), both murine pRb from NS1 or AKR-2B cells and human pRb from HeLa or U2OS cells bound to GST-202, but not to GST. (The two faster migrating bands in lane2 correspond to degraded forms of pRb. These could be detected because more pRb could be extracted from the NS1 cell line than from the other lines tested.) No pRb was recovered on GST-202 from an extract of cells of the human osteosarcoma line SAOS-2. This human cell line does not express wild-type pRb; it expresses only a cytoplasmic carboxyl-truncated version of pRb(32) .

As expected, p202 from an extract of AKR-2B cells bound to GST-Rb, but not to GST (Fig. 2B, rightpanel, lanes5 and 6). p202 binding to GST-Rb was detected, however, only in an extract from IFN-treated cells and not in that from control cells (compare lanes6 and 4). This is due to the fact that the level of p202 is very low in AKR-2B cells, not detected in our assay, unless the cells are treated with IFN (lanes1 and 2).

We also used the far Western assay for testing whether labeled pRb can bind to p202 specifically. The positive outcome of this test is shown in Fig. 2C. pRb bound to affinity-purified GST-202 (lane5), but not to GST or to a series of protein size markers (lanes4 and 6).

To test whether the binding of p202 to pRb was due to direct interaction between these two proteins, we used purified human recombinant pRb (Canji, Inc.). This was retained on affinity-purified GST-202-Sepharose, but not on GSTSepharose or the glutathione-Sepharose matrix (Fig. 2D).

Binding of p202 to the Hypophosphorylated Form of pRb in Vitro

In the experiments involving the binding of pRb from cell extracts to p202, pRb appeared as a sharp protein band (Fig. 2B, left panel, lanes4, 6, and 10). The possibility that the sharp pRb band may correspond to hypophosphorylated pRb prompted us to examine whether p202 binds preferentially to the hypophosphorylated form of pRb. We prepared an extract from growing (AKR-2B) cells to assure that differently phosphorylated forms of pRb would be present. We incubated aliquots from this extract with immobilized GST, GST-202, and GST-E1a. The bound proteins were eluted, immunoprecipitated by anti-pRb G3-245, and analyzed by SDS-PAGE and Western blotting with a mixture of two monoclonal anti-human pRb antibodies (one of which preferentially recognizes the hypophosphorylated form of pRb) (Fig. 3). As expected, no pRb was retained by GST. Only a fast migrating band of pRb was retained on GST-202. The pRb retained on GST-E1a consisted of a similarly fast moving band together with a slower moving band. This is in line with earlier reports showing that E1a binds both hypo- and hyperphosphorylated forms of pRb(33) . Finally, the pRb bands from the cell extract that was used in the experiment appeared to be similar to those recovered from GST-E1a. These results indicate that from a mixture of differently phosphorylated forms of pRb, p202 selectively, or at least preferentially, binds to the fast migrating hypophosphorylated form.


Figure 3: Binding of the hypophosphorylated form of pRb to GST-202 in vitro. Assay was by Western blotting. Aliquots from an extract of growing AKR-2B cells were incubated with glutathione-Sepharose beads loaded with equal amounts of GST (lane 2), GST-202 (lane 3), or GST-E1a (lane 4). After washing the beads, the proteins were eluted and analyzed by SDS-PAGE and Western blotting using a 1:1 mixture of antibodies to human pRb (clones G3-245 and G99-549). The first of these two antibodies recognizes all forms of pRb; the second antibody has a preference for the hypophosphorylated form. An aliquot of the cell extract was immunoprecipitated with anti-Rb antibodies (G3-245) and analyzed as a control (lane 1). The arrows indicate differently phosphorylated forms of pRb. For further details, see ``Materials and Methods.''



Association of pRb with p202 in Vivo

Since p202 bound the hypophosphorylated form of pRb, we tested for the association of p202 with pRb in vivo using an extract from cells that were serum-starved to increase the proportion of pRb in the hypophosphorylated form. Furthermore, since the level of p202 is very low, barely detectable in AKR-2B cells not treated with IFN, we exposed the cells to IFN for 36 h. Moreover, since the level of pRb in AKR-2B cells is also very low, we transfected the cell line with an expression plasmid (pRb-wt) (34) encoding wild-type murine pRb. This provided us with AKR-2B cells expressing pRb at a 2-3-fold higher level than in untransfected cells. These cells appeared morphologically normal and grew similarly to cells that had been transfected with the vector only (data not shown).

Extracts were prepared from control and IFN-treated cultures of untransfected and serum-starved and of transfected and serum-starved AKR-2B cells. The extracts were used for immunoprecipitation with monoclonal anti-Rb antibodies linked to agarose beads. (These antibodies did not immunoprecipitate p202 that was translated in a reticulocyte lysate, i.e. with no pRb added (data not shown).) The immunoprecipitates from the cell extracts were washed and analyzed by Western blotting using an anti-p202 antiserum. As a size marker, [S]Met-labeled p202 translated in vitro was run (Fig. 4, lane3). A strong p202-specific band was present in the immunoprecipitate from the extract of IFN-treated, pRb-transfected cells (lane2), suggesting that p202 and pRb may be associated in vivo. The p202 band detected in the immunoprecipitate from the extract of untransfected, IFN-treated cells was weak presumably as a consequence of the low level of pRb in AKR-2B cells (data not shown). No p202 was detected in the immunoprecipitates from the extracts of transfected (or untransfected) control cells (lane1). In view of the very low level of p202 under these conditions, this result was expected. We have not succeeded in coprecipitating pRb from a cell extract using our polyclonal antiserum to p202.


Figure 4: Binding of pRb to p202 in vivo. Assay was by coimmunoprecipitation and Western blotting. Extracts from control (lane 1) or IFN-treated, serum-starved (lane 2) AKR-2B cells were immunoprecipitated using monoclonal anti-Rb antibodies (Ab1-clone C36). The immunoprecipitated proteins were analyzed by SDS-PAGE and Western blotting using anti-202 antiserum. [S]Met-labeled p202 translated in vitro (IVT-202) was run as a marker (lane 3). The p202 protein band is indicated by an arrow. For further details, see ``Materials and Methods.''



Two Nonoverlapping Segments of pRb Bind to p202; One of Them Includes the Pocket Region

We tested for interactions between p202 and various truncated and/or mutated forms of murine and human pRb (Fig. 5A). Labeled p202 was retained on two nonoverlapping segments of murine pRb (linked to GST). One of them extended in pRb from amino acids 1 to 254, and the second one from amino acids 255 to 922 (Fig. 5B).


Figure 5: Two nonoverlapping regions of pRb can independently bind p202 in vitro. A, schematic representation of pRb, its segments, and their abilities to bind p202. pRb refers to the murine retinoblastoma protein, and hpRb to the human retinoblastoma protein. The numbers not in parentheses are the NH(2)- and COOH-terminal residues of the segment. (dl702-731) indicates that amino acids 702-731 were deleted (this is also indicated by the thickverticalline; (706,CF) indicates that phenylalanine was substituted for cysteine at position 706. The letters A and B indicate two segments from the pocket region. The extent of p202 binding is indicated in the right-most column: -, no binding; + . . . ++++, weakest to strongest binding. B, binding of p202 to segments of pRb linked to GST. [S]Met-labeled murine p202 translated in vitro was incubated with glutathione-Sepharose beads loaded with GST (lane 2), GST-Rb-(1-254) (lane 3), or GST-Rb-(255-922) (lane 4). After washing the beads, the bound proteins were eluted and analyzed by SDS-PAGE and fluorography. An aliquot of the [S]Met-labeled p202 solution (IVT-202) was run in lane 1. The p202 band is indicated by an arrow. C, binding of murine pRb segments with COOH-terminal truncations to GST-202. [S]Met-labeled pRb (translated in vitro) (lanes 1-3) or pRb segments with COOH-terminal truncations (translated in vitro), i.e. pRb-(1-479) (lanes 4-6), pRb-(1-254) (lanes 7-9), and pRb-(1-132) (lanes 10-12), were incubated with glutathione-Sepharose beads loaded with GST (lanes 2, 5, 8, and 11) or GST-202 (lanes 3, 6, 9, and 12). After washing, the beads were eluted, and the released proteins were analyzed by SDS-PAGE and fluorography. As controls, aliquots of the reaction mixture in which pRb or its segments with COOH-terminal truncations had been translated in vitro were run (IVT; lanes 1, 4, 7, and 10). The bands with the lowest mobilities (marked with open circles) correspond to the pRb segments indicated; the faster moving bands may have arisen in consequence of translation initiation at internal sites of the mRNAs and/or protein degradation. D, binding of p202 to GST-Rb with NH(2)-terminal truncation in the retinoblastoma moiety and an amino acid substitution in its pocket region. [S]Met-labeled p202 translated in vitro was incubated with glutathione-Sepharose beads not loaded (indicated as GA; lane 2) or loaded with GST (lane 3), murine GST-Rb (lane 4), human GST-Rb-(379-928)(706CF) (lane 5), or human GST-Rb-(379-928) (lane 6). After washing the beads, the proteins were eluted and analyzed by SDS-PAGE and a PhosphorImager. As a control, an aliquot of the [S]Met-labeled p202 solution (IVT-202) was run in lane 1. The p202 band is indicated by an arrow. E, binding of pRb with a deletion in the pocket region to GST-E1a and GST-202. Extracts from human HeLa cells carrying wild-type pRb (lanes 1-3) or from human J82 cells carrying a pRb mutant with a deletion from amino acids 702 to 731 (lanes 4-6) were incubated with glutathione-Sepharose beads loaded with GST (lanes 1 and 4), GST-E1a (lanes 2 and 5), or GST-202 (lanes 3 and 6). After washing the beads, the proteins were eluted and analyzed by SDS-PAGE and Western blotting with a polyclonal anti-Rb antiserum. The pRb band is indicated by an arrowhead. For further details, see ``Materials and Methods.''



Experiments involving COOH-terminal deletion mutants of murine pRb confirmed and extended these findings (Fig. 5C). Labeled pRb-(1-922) (lane3), used as a control, and the segments pRb-(1-479) (lane6) and pRb-(1-254) (lane9) were retained on GST-202, although not on GST (lanes2, 5, and 8). The short NH(2)-terminal segment pRb-(1-132) was not retained on GST-202 (lane12) or on GST (lane11).

The NH(2)-terminal truncation mutant of human pRb, i.e. pRb-(379-928), strongly retained labeled p202 (Fig. 5D). The same pRb segment (i.e. pRb-(379-928)), however, carrying an amino acid substitution (Cys to Phe) at position 706 in the pocket region retained p202, but very poorly (Fig. 5D). This result indicates that the pocket region (extending from amino acids 379 to 792) is involved in p202 binding. (In this experiment, a very faint band of p202 retained on GST was detected. This might be a consequence of using a highly sensitive detection device, a PhosphorImager (Molecular Dynamics, Inc.). The conclusion concerning the involvement of the pRb pocket region in p202 binding was further supported by the finding that the natural human pRb mutant in J82 bladder carcinoma cells that harbor a deletion from amino acids 697 to 731 in the pocket region (35) was retained by GST-202 to a much lesser extent than was wild-type pRb from HeLa cells (Fig. 5e, compare lanes3 and 6). In accord with an earlier report(35) , the binding of the mutant pRb from J82 to GST-E1a was also much weaker than that of wild-type pRb (compare lanes2 and 5).

These results indicate that at least two nonoverlapping regions of pRb can bind p202. One of them includes the segment extending from amino acids 1 to 254. The second one is located between amino acids 379 and 928. In this segment, the pocket region is involved in the binding. A summary of the results concerning the binding between pRb mutants and p202 is shown in Fig. 5A.

Two Nonoverlapping Segments of p202 Bind pRb

We generated p202 deletion mutants to identify regions involved in binding pRb. Labeled pRb was bound to the following truncated GST-202 moieties: GST-202-(58-291), GST-202-(255-445), and GST-202-(295-445) (Fig. 6B, lanes 3-5), but not to GST (lane1). These results suggest that p202 contains at least two nonoverlapping segments that bind pRb: one between amino acids 58 and 291 and the second between amino acids 295 and 445. The binding to p202-(58-291) and also to the almost complete p202 (i.e. p202-(19-445)) was pronounced, but much weaker than to COOH-terminal p202-(255-445) or p202-(295-445). It remains to be established whether this is due to a masking in the almost complete p202 of the activity of the stronger binding site in the COOH-terminal segment. This COOH-terminal segment contains a pRb-binding motif, LXCXE. The significance of this motif in the binding of pRb to p202 remains to be tested.


Figure 6: Two nonoverlapping regions of p202 can independently bind pRb in vitro. A, schematic representation of p202 and its segments. The numbers in the structure at the top indicate the amino acid residues at the ends of the LXCXE pRb-binding motif. The lettersa and b are two types of partially conserved 200-amino acid segments. The numbers at the left and right ends of the structures indicate the NH(2)- and COOH-terminal amino acid residues of the p202 moieties. GST was linked to the NH(2) terminus of each of the lower four structures. B, binding of pRb to segments of p202. Glutathione-Sepharose beads were loaded with GST (lane 1), GST-202-(19-445) (lane 2), GST-202-(58-291) (lane 3), GST-202-(255-445) (lane 4), or GST-202-(295-445) (lane 5). The loaded beads were incubated with [S]Met-labeled pRb translated in vitro. After washing the beads, the proteins were eluted and analyzed by SDS-PAGE and fluorography. An aliquot of the [S]Met-labeled pRb preparation was also analyzed (IVT-Rb; lane 6). The pRb band is indicated by an arrow. For further details, see ``Materials and Methods.''




DISCUSSION

The results presented indicate that the IFN-inducible p202 protein binds the pRb protein both in vitro and in vivo. p202 and pRb bind each other directly, as revealed by the association of the purified recombinant proteins produced in bacteria. p202 binds the hypophosphorylated form of pRb.

At least two nonoverlapping segments of p202 (p202-(58-291) and p202-(295-445)) bound pRb. The latter of these contained the pRb-binding motif LXCXE. p204, another protein that is encoded by a gene (Ifi204) from the gene 200 cluster and that is very similar to p202 in its COOH-terminal half(7) , did not bind pRb under conditions in which p202 did.

At least two nonoverlapping segments of pRb (pRb-(1-254) and pRb-(255-922)) bound p202. The second segment included the pocket region. Deletions from the pocket region or an amino acid substitution in it (at position 706) greatly diminished the binding to p202, revealing the role of the pocket region in p202 binding. This is the region to which the DNA tumor virus oncoproteins (adenovirus E1a, simian virus 40 T, and human papilloma virus E7 proteins) (35, 36, 37, 38) as well as the human cytomegalovirus IE2 protein also bind(39) . Furthermore, these oncoproteins bind, at least preferentially, to the hypophosphorylated form of pRb, the same form to which p202 also binds (40) . We used both murine and human pRb in our experiments. The finding that human and murine pRb bound similarly to murine p202 is not unexpected since there is 91% sequence identity between the two pRb proteins(41) .

The effect of p202 binding on pRb function remains to be explored. It is conceivable, for example, that the binding may impair the phosphorylation of pRb (IFN treatment was reported to impair this process) (26) and/or the phosphorylation-dependent release of pRb from its tight association with the nucleus(42, 43) . The finding that p202 also binds to the NH(2)-terminal region of pRb (reported to be required for recognition by certain kinases (44) (and also for the oligomerization of pRb)(45) ) is in line with these possibilities.

The binding of p202 to the pocket region of pRb might account for the fact that p202 also binds to p107 (data not shown). This protein is related in structure to pRb and is also involved in the control of cell proliferation(46, 47) . The two proteins are homologous in sequence in their carboxyl-terminal two-thirds segments, including the pocket region, and the pocket regions of the two proteins bind overlapping, although distinct, sets of proteins.

We detected an association in vivo of p202 with pRb only in an extract from IFN-treated cells. It remains to be established whether this indicates that pRb interacts with p202 only if the level of the latter has been increased by IFN treatment (a 15-20-fold increase in some cell lines)(12) . It might be also be a consequence of the difficulty of the efficient extraction of p202-pRb complexes from cell lysates at low salt concentration. This difficulty may arise from the tight binding of hypophosphorylated pRb and also of p202 to the nuclear fraction(12, 42) . p202 has sequences that might be targets for phosphorylation by p34, a cell cycle-dependent kinase(12) . Such phosphorylation might control the ability of p202 to bind pRb in a cell cycle-dependent manner.

p202 is the first IFN-inducible protein found to bind pRb, a protein with a crucial role in the negative control of cell proliferation. At the same time, at least when constitutively overexpressed, p202 impairs cell proliferation. These facts warrant further studies on the possible role of the binding of these two proteins in the growth-inhibitory activity of IFNs. In considering this problem, it should be noted that p202 binds several proteins in addition to pRb and p107 and that one of these proteins is the transcription factor E2F (data not shown). This transcription factor is involved in the G(1) to S transition, and its activity is controlled in part by pRb and p107(48, 49) .


FOOTNOTES

*
This work was supported in part by National Institutes of Health Research Grant R37-AI12320 (to P. L.). Work performed at the University of Texas M. D. Anderson Cancer Center was supported by a grant from the Biomedical Research Foundation (to D. C.) and by the Clayton Foundation for Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Biophysics and Biochemistry, Yale University, P. O. Box 208024, 333 Cedar St., New Haven, CT 06520-8024. Tel.: 203-737-2061; Fax: 203-785-6404.

(^1)
The abbreviations used are: IFNs, interferons; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; pRb, retinoblastoma protein.


ACKNOWLEDGEMENTS

We thank C. Weissmann and H. Weber for recombinant human IFN-alpha(2)/alpha(1)-(1-83); D. M. Livingston and W. G. Kaelin, Jr. for the plasmids pGST-Rb-(379-928) and pGST-Rb-(379-928)(706CF); J. Germino for an E1a plasmid; J. Sedivy for the pRb-wt plasmid; G. Sen for a HeLa strain; and J. Sedivy, M. Solomon, J. U. Gutterman, and B. B. Aggarwal for valuable discussions.


REFERENCES

  1. DeMaeyer, E., and DeMaeyer-Guignard, J. (1988) Interferons and Other Regulatory Cytokines , p. 488, John Wiley & Sons, Inc., New York
  2. Vilcek, J. (1990) in Peptide Growth Factors and Their Receptors (Sporn, M. B., and Roberts, A. B., eds) pp. 3-38, Springer-Verlag, Berlin
  3. Sen, G. C., and Lengyel, P. (1992) J. Biol Chem. 267, 5017-5020 [Free Full Text]
  4. Lengyel, P. (1982) Annu. Rev. Biochem. 51, 251-282 [CrossRef][Medline] [Order article via Infotrieve]
  5. Opdenakker, G., Snoddy, J., Choubey, D., Toniato, E., Pravtcheva, D. D., Seldin, M. F., Ruddle, F. H., and Lengyel, P. (1989) Virology 171, 568-578 [Medline] [Order article via Infotrieve]
  6. Kingsmore, S. F., Snoddy, J., Choubey, D., Lengyel, P., and Seldin, M. F. (1990) Immunogenetics 30, 169-174
  7. Choubey, D., Snoddy, J., Chaturvedi, V., Toniato, E., Opdenakker, G., Thakur, A., Samanta, H., Engel, D., and Lengyel, P. (1989) J. Biol. Chem. 264, 17182-17189 [Abstract/Free Full Text]
  8. Tannenbaum, C. S., Major, J., Ohmori, Y., and Hamilton, T. A. (1993) J. Leukocyte Biol. 53, 563-568 [Abstract]
  9. Briggs, J. A., Burrus, G. R., Stickney, B. D., and Briggs, R. C. (1992) J. Cell. Biochem. 49, 82-92 [Medline] [Order article via Infotrieve]
  10. Trapani, J. A., Browne, K. A., Dawson, M. J., Ramsay, R. G., Eddy, R. L., Shows, T. B., White, P. C., and Dupont, B. (1992) Immunogenetics 36, 369-376 [Medline] [Order article via Infotrieve]
  11. Choubey, D., and Lengyel, P. (1992) J. Cell Biol. 116, 1333-1341 [Abstract]
  12. Choubey, D., and Lengyel, P. (1993) J. Interferon Res. 13, 43-52 [Medline] [Order article via Infotrieve]
  13. Figge, J., Breese, K., Vajda, S., Zhu, Q.-L., Eisele, L., Anderson, T. T., MacColl, R., Friedrich, T., and Smith, T. (1993) Protein Sci. 2, 155-164 [Abstract/Free Full Text]
  14. Friend, S. H., Bernards, R., Rogelj, S., Weinberg, R. A., Rapaport, J. M., Albert, D. M., and Dryja, T. P. (1986) Nature 323, 643-646 [Medline] [Order article via Infotrieve]
  15. Lee, W. H., Bookstein, R., Hong, F., Young, L.-J., Shew, J. Y., and Lee, E. Y.-H. P. (1987) Science 235, 1394-1399 [Medline] [Order article via Infotrieve]
  16. Huang, H. J. S., Yee, J. K., Shew, J. Y., Chen, P. L., Bookstein, R., Friedmann, T., Lee, E. Y., and Lee, W. H. (1988) Science 242, 1563-1566 [Medline] [Order article via Infotrieve]
  17. Weinberg, R. (1991) Science 254, 1138-1146 [Medline] [Order article via Infotrieve]
  18. Buchkovich, K., Duffy, L. A., and Harlow, E. (1989) Cell 58, 1097-1105 [Medline] [Order article via Infotrieve]
  19. DeCaprio, J. A., Ludlow, J. W., Lynch, D., Furukawa, Y., Griffin, J., Piwnica-Worms, H., Huang, C.-M., and Livingston, D. M. (1989) Cell 58, 1085-1095 [Medline] [Order article via Infotrieve]
  20. Chen, P. L., Scully, P., Shew, J.-Y., Wang, J. Y. J., and Lee, W.-H. (1989) Cell 58, 1193-1198 [Medline] [Order article via Infotrieve]
  21. Mihara, K., Cao, X.-R., Yen, A., Chandler, S., Driscoll, B., Murphree, A. L., T'ang, A., and Fung, Y.-K. T. (1989) Science 246, 1300-1303 [Medline] [Order article via Infotrieve]
  22. Stein, G. H., Beeson, M., and Gordon, L. (1990) Science 249, 666-669 [Medline] [Order article via Infotrieve]
  23. Goodrich, D. W., Wang, N. P., Qian, Y. W., Lee, E. Y.-H. P., and Lee, W.-H. (1991) Cell 67, 293-302 [Medline] [Order article via Infotrieve]
  24. Ewen, M. E. (1994) Cancer Metastasis Rev. 13, 45-66 [Medline] [Order article via Infotrieve]
  25. Einat, M., Resnitzky, D., and Kimchi, A. (1985) Nature 313, 597-600 [Medline] [Order article via Infotrieve]
  26. Resnitzky, D., Tiefenbrun, N., Berissi, H., and Kimchi, A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 402-406 [Abstract]
  27. Weber, H., Valenzuela, D., Lujber, G., Gubler, M., and Weissmann, C. (1987) EMBO J. 6, 591-598 [Abstract]
  28. Kaelin, W. G., Jr., Pallas, D. C., DeCaprio, J. A., Kaye, F. J., and Livingston, D. M. (1991) Cell 64, 521-532 [Medline] [Order article via Infotrieve]
  29. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  30. Ray, S. K., Arroyo, M., Bagchi, S., and Raychaudhuri, P. (1992) Mol. Cell. Biol. 12, 4327-4333 [Abstract]
  31. Barbacid, M. (1981) J. Virol. 37, 518-523 [Medline] [Order article via Infotrieve]
  32. Shew, J.-Y., Lin, B. T.-Y., Chen, P.-L., Tseng, B. Y., Yang-Feng, T. L., and Lee, W.-H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6-10 [Abstract]
  33. Herrmann, C. H., Su, L. K., Whyte, P., Buchkovich, K., and Harlow, E. (1991) J. Virol. 65, 5848-5859 [Medline] [Order article via Infotrieve]
  34. Karantza, V., Maroo, A., Fay, D., and Sedivy, J. M. (1993) Mol. Cell. Biol. 13, 6640-6652 [Abstract]
  35. Horowitz, J. M., Yandell, D. W., Park, S.-H., Canning, S., Whyte, P., Buchkovich, K., Harlow, E., Weinberg, R. A., and Dryja, T. P. (1989) Science 243, 937-940 [Medline] [Order article via Infotrieve]
  36. Whyte, P., Buchkovich, K. J., Horowitz, J. M., Friend, S. H., Raybuck, M., Weinberg, R. A., and Harlow, E. (1988) Nature 334, 124-129 [CrossRef][Medline] [Order article via Infotrieve]
  37. DeCaprio, J. A., Ludlow, J. W., Figge, J., Shew, J.-Y., Huang, C.-M., Lee, W.-H., Marsilio, E., Paucha, E., and Livingston, D. M. (1988) Cell 54, 275-283 [Medline] [Order article via Infotrieve]
  38. Dyson, N., Howley, P. M., Munger, K., and Harlow, E. (1989) Science 243, 934-937 [Medline] [Order article via Infotrieve]
  39. Hagemeier, C., Caswell, R., Hayhurst, G., Sinclair, J., and Kouzarides, T. (1994) EMBO J. 13, 2897-2903 [Abstract]
  40. Sherr, C. J. (1994) Trends Cell Biol. 4, 15-18 [CrossRef]
  41. Bernards, R., Schackleford, G. M., Gerber, M. R., Horowitz, J. M., Friend, S. H., Schartl, M., Bogenmann, E., Rapaport, J. M., McGee, T., Dryja, T. P., and Weinberg, R. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6474-6478 [Abstract]
  42. Mittnacht, S., Lees, J. A., Desai, D., Harlow, E., Morgan, D. O., and Weinberg, R. A. (1994) EMBO J. 13, 118-127 [Abstract]
  43. Templeton, D. J. (1992) Mol. Cell. Biol. 12, 435-443 [Abstract]
  44. Qian, Y., Luckey, C., Horton, L., Esser, M., and Templeton, D. J. (1992) Mol. Cell. Biol. 12, 5363-5372 [Abstract]
  45. Hensey, C. E., Hong, F., Durfee, T., Qian, Y. W., Lee, E. Y.-H. P., and Lee, W.-H. (1994) J. Biol. Chem. 269, 1380-1387 [Abstract/Free Full Text]
  46. Ewen, M. E., Xing, Y., Lawrence, J. B., and Livingston, D. M. (1991) Cell 66, 1155-1164 [Medline] [Order article via Infotrieve]
  47. Zhu, L., van den Heuvel, S., Helin, K., Fattaey, A., Ewen, M., Livingston, D. M., Dyson, N., and Harlow, E. (1993) Genes & Dev. 7, 1111-1125
  48. Schwarz, J. K., Devoto, S. H., Smith, E. J., Chellappan, S. P., Jakoi, L., and Nevins, J. R. (1993) EMBO J. 12, 1013-1020 [Abstract]
  49. Helin, K., Wu, C.-L., Fattaey, A. R., Lees, J. A., Dynlacht, B. D., Ngwu, C., and Harlow, E. (1993) Genes & Dev. 7, 1850-1861

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