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Originally published In Press as doi:10.1074/jbc.M110885200 on February 27, 2002

J. Biol. Chem., Vol. 277, Issue 19, 17231-17238, May 10, 2002
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Insulin Receptor Substrate 1 Translocation to the Nucleus by the Human JC Virus T-antigen*

Adam LassakDagger, Luis Del ValleDagger, Francesca Peruzzi, Jin Ying Wang, Sahnila Enam, Sidney Croul, Kamel Khalili, and Krzysztof Reiss§

From the Center for Neurovirology and Cancer Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania 19122

Received for publication, November 13, 2001, and in revised form, February 12, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin receptor substrate 1 (IRS-1) is the major signaling molecule for the insulin and insulin-like growth factor I receptors, which transduces both metabolic and growth-promoting signals, and has transforming properties when overexpressed in the cells. Here we show that IRS-1 is translocated to the nucleus in the presence of the early viral protein-T-antigen of the human polyomavirus JC. Nuclear IRS-1 was detected in T-antigen-positive cell lines and in T-antigen-positive biopsies from patients diagnosed with medulloblastoma. The IRS-1 domain responsible for a direct JC virus T-antigen binding was localized within the N-terminal portion of IRS-1 molecule, and the binding was independent from IRS-1 tyrosine phosphorylation and was strongly inhibited by IRS-1 serine phosphorylation. In addition, competition for the IRS-1-T-antigen binding by a dominant negative mutant of IRS-1 inhibited growth and survival of JC virus T-antigen-transformed cells in anchorage-independent culture conditions. Based on these findings, we propose a novel role for the IRS-1-T-antigen complex in controlling cellular equilibrium during viral infection. It may involve uncoupling of IRS-1 from its surface receptor and translocation of its function to the nucleus.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin receptor substrate 1 (IRS-1)1 is a 160-kDa cytosolic protein implicated in insulin and IGF-I signal transduction. IRS-1 plays an essential role in IGF-I-mediated cell proliferation (1, 2), and has transforming properties when overexpressed in different cell types (3, 4). The structure of IRS-1 reveals two conserved regions within the N-terminal portion of the protein (5, 6). The first one is called PH for its similarity to a pleckstrin homology domain (7), and the second shows similarity to a putative phosphotyrosine-binding (PTB) domain present in Shc and other proteins (6). The PTB domain recognizes phosphorylated tyrosine within NPXY motifs, providing a mechanism to couple IRS-1 with the Tyr950 in the juxtamembrane region of the IGF-IR (8). PH domains contain a positively charged binding pocket that may mediate interaction with phospholipids (9) and with proteins containing acidic motifs (10). Following activation, over 20 phosphorylation sites on the IRS-1 docking molecule can recruit a variety of proteins equipped with Src homology domains (11). Independent from its tyrosine phosphorylation, IRS-1 interacts with alpha vbeta 3 (12) and alpha 5beta 1 (13, 14) integrins, with typical nuclear proteins such as the SV40 large T-antigen (3) and nucleolin (10) and is constitutively phosphorylated in v-Src transformed cells (15). Transforming properties of IRS-1 were suspected for quite some time even before the first convincing evidence was furnished by utilizing R- cells (3T3-like fibroblasts derived from mice with targeted disruption of IGF-IR gene) (3, 4). Although R- cells are remarkably resistant to transformation (15, 16), co-expression of IRS-1 and SV40 T-antigen induced R- transformation, a phenotype efficiently reversed by antisense IRS-1 mRNA (4). Importantly, overexpression of either IRS-1 or T-antigen alone did not transform R- cells, suggesting a functional relationship between these two proteins in transformation (4). Other reports have confirmed the transforming potential of IRS-1. Elevated levels of IRS-1 have been detected in human pancreatic cancer, in early stages of hepatocellular carcinomas, in MCF-7 breast cancer cell lines, and in medulloblastomas (17-20). Finally, NIH 3T3 fibroblasts, which possess functional IGF-IR, are easily transformed by IRS-1 overexpression alone (4). The aforementioned interaction between IRS-1 and the simian virus 40 T-antigen prompted us to investigate whether human counterpart of the SV40 T-antigen, JCV T-antigen, interacts with IRS-1 and whether such an interaction may actually contribute to the transformation in humans.

The human neurotropic polyomavirus JC (JCV) asymptomatically infects nearly 90% of the human population (21). The early genome of this virus encodes the regulatory protein JCV T-antigen, which has transforming properties in vitro, induces tumors of neuronal origin in experimental animals (22-24), and has been detected in a variety of human brain tumors including medulloblastomas (25). In this article we report for the first time immunocytochemical detection of nuclear IRS-1 in JCV T-antigen-positive murine medulloblastoma cell lines, in fibroblasts stably transfected with JCV T-antigen cDNA, and in living cells expressing a GFP-IRS-1 fusion protein. Importantly, IRS-1 co-precipitates with the JCV T-antigen, and the interaction is independent of IRS-1 tyrosine phosphorylation and is strongly inhibited by IRS-1 serine phosphorylation. We further show that the IRS-1-JCV T-antigen binding is direct, that it maps within the N-terminal portion of IRS-1, and that the N-terminal fragment (PH/PTB) efficiently competes for the T-antigen binding and inhibits anchorage-independent growth of JCV T-antigen-positive medulloblastoma cell lines. Importantly, nuclear IRS-1 was detected in human medulloblastoma biopsies that were also positive for anti-JCV T-antigen immunostaining. Based on these findings, we postulate that the interaction with T-antigen may uncouple IRS-1 from its surface receptors initiating series of nuclear events, which in turn could affect JCV T-antigen-mediated cellular transformation.

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INTRODUCTION
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Cell Lines-- We used four cell lines Bs-1a, BsB8, BsB13, and BsB14 derived from cerebellar medulloblastoma of transgenic mice expressing the early genome of the archetype form JC virus (23, 26). Bs-1a cells are JCV T-antigen-negative, and BsB8, BsB13, and BsB14 are JCV T-antigen-positive. R600 embryo fibroblasts (27, 28) were developed from R- cells, which originated from mouse embryos with a targeted disruption of the IGF-IR gene (29). R600 cells were transiently transfected with PCDNA3zeo/JCVT plasmid by calcium phosphate reagents from Promega. Culture conditions and characterization of LNCaP cells, which are IRS-1-negative prostate cancer cells, and LNCaP cells stably expressing different mutants of IRS-1 are described in our previous work (13). Finally, anchorage-independent growth of BsB8 and Bs-1a cells was evaluated in 35-mm culture dishes covered with PolyHema (Aldrich) by the methodology routinely used in our laboratory (20, 27, 30).

Immunohistochemistry-- We employed thin paraffin sections and an avidin-biotin-peroxidase complex system (Vectastain Elite ABC peroxidase kit; Vector Laboratories). The sections were processed according to the protocol previously described (20, 25). We used rabbit anti-IRS-1 antibodies that recognize either C-terminal portion of IRS-1 (Upstate Biotechnology, Inc., Lake Placid, NY) or N-terminal IRS-1 (A-19; Santa Cruz) and rabbit anti-SV40 T-antigen (Oncogene) that cross-reacts with JCV T-antigen (25) as primary antibodies. Biotin-conjugated goat anti-rabbit IgG (Vector) was used as a secondary antibody. For monolayer cultures, we used rabbit secondary antibodies conjugated to fluoresceine (Jackson ImmunoResearch) and rhodamine (Vector). To avoid cross-reactivity between secondary antibodies, the cells were first incubated with anti-T-antigen antibody, and with the anti-mouse fluorescein isothiocyanate-conjugated secondary antibody. Following intensive washing, the cells were incubated with anti-IRS-1 antibody and with corresponding anti-rabbit rodamine-conjugated secondary antibody. For all immunocyto- and immunohistochemical staining, we performed routine controls by omitting primary antibody or by using irrelevant, anti-bromodeoxyuridine antibody instead of primary antibodies.

Western Blot Analyses and Immunoprecipitation-- We used rabbit anti-SV40 T-antigen (Ab-2; Oncogene) antibody that recognizes JCV T-antigen for both Western blot detection and immunoprecipitation of JCV T-antigen. For IRS-1 immunoprecipitation and Western blot, we used anti-rabbit IRS-1 antibody that recognizes the C-terminal portion of the IRS-1 (Upstate Biotechnology). To detect the PH/PTB mutant, we employed rabbit antibody that recognizes epitope mapping at the N terminus of IRS-1 (A-19, Santa Cruz). To determine tyrosine phosphorylation of IRS-1, quiescent cells (48 h in serum-free medium) were stimulated with IGF-I (50 ng ml-1), or were left without stimulation (hypophosphorylated IRS-1), and total proteins were extracted 15 min later. IRS-1 was immunoprecipitated from 500 µg of protein extract with rabbit anti-IRS-1 antibody (Transduction Laboratories) and agarose-conjugated protein A (Calbiochem). Corresponding blots were developed first with anti-phosphotyrosine antibody (PY20; Transduction Laboratories) and reprobed with N-terminal anti-IRS-1 antibody (Santa Cruz). We induced serine phosphorylation of IRS-1 by incubating BsB8 cells with 2 µM okadaic acid (Sigma) for 40 min and monitored the effectiveness of the procedure by mobility shift (13).

Expression Vectors and Cloning Strategies-- We cloned JCV T-antigen cDNA (31) (PCR-amplified) into the KpnI/EcoRI site of pcDNA3zeo expression vector (Invitrogen). The start primer was 5'-CGGggtaccATGGACAAAGTGCTGAATAG-3', and the end primer was 5'-CCGgaattcGCTTTACTTAACAGTTGCAG-3'. Lowercase letters correspond to the KpnI and EcoRI sites, respectively. GFP-IRS-1 fusion protein was prepared by cloning PCR-amplified IRS-1 cDNA (13) into HindIII site of pLEGFP-N1 retroviral expression system (Promega). The start primer was 5'-TCACCCaagcttATGGCGAGCCCTCCGGATAC-3', and the end primer was 5'-TCACCCaagcttTTGACGATCCTCTGGCTGC-3'. Lowercase letters correspond to the HindIII restriction site. In an attempt to prepare IRS-1 cDNA fragments with the GST sequence at the 5' end, the cDNA inserts were produced by PCR with a pCMV/IRS-1 plasmid as a DNA template (13). The following primers were used to amplify IRS-1 truncation mutants: IRS-1 (1-300), start primer, 5'-CGggatccTCATGGCGAGCCCTCCGGATAC-3', and end primer, 5'-CGgaattcAAGTTCGAGATCTCCGAGTCAG-3'; IRS-1 (212-529), start primer, 5'-CGggatccTCTGGATGCAAGTGGATGAC-3', and end primer, 5'-CGgaattcAAATGGTAGGAGATGTGCCTG-3'; IRS-1 (462-740), start primer, 5'-CGggatccCTGCATGGGTGGCAAGGG-3', and end primer, 5'-CGgaattcAGCTGCTGGTGTTGGAATCTC-3'; IRS-1 (701-1000), start primer, 5'-ATAAGAATgcggccgcACCATACTCATGCCCTTCC-3', and end primer, 5'-ATAAGAATgcggccgcTCATGGTGAGGTATCCACATAGC-3'; and IRS-1 (931-1233), start primer, 5'-CGggatccCTGGCTCGGAAGAGTACATG-3', and end primer, 5'-CGgaattcATTGACGATCCTCTGGCTGC-3'. With the exception of the IRS-1 (701-1000) mutant, all of the start primers contain BamHI restriction sites, and end primers contain EcoRI restriction sites in the overhangs, which are shown in lowercase letters. Because IRS-1 (701-1000) mutant contains an internal EcoRI site, a NotI restriction site was introduced instead. The correct size products were excised from the gel and purified with a gel extraction kit (Qiagen, Santa Clarita, CA), following the manufacturer's recommendations. Purified PCR products were digested with BamHI and EcoRI restriction enzymes, ligated into BamHI/EcoRI cloning sites of pGEX-5X-3, and sequenced to check for eventual misincorporations. IRS-1 (701-1000) mutant was digested with NotI and cloned into the NotI site of pGEX-5X-3.

GST Pull-down Assay-- We expressed and purified GST-IRS-1 fusion proteins according to previously described protocol (31). In brief, radiolabeled JCV T-antigen was synthesized with the transcription and translation-coupled wheat germ extract system according to the manufacturer's recommendations (Promega). For the in vitro binding assays, we used 3 µl of 35S-labeled, in vitro translated JCV T-antigen (95 kDa) and 5 µg of GST or appropriate GST fusion proteins coupled to glutathione-Sepharose. The bound proteins were eluted with Laemmli sample buffer and separated by SDS/PAGE.

Clinical Samples-- We obtained a total of 17 human medulloblastoma samples from the pathology archives (Medical College of Pennsylvania-Hahnemann University, Philadelphia, PA). Formalin-fixed, paraffin-embedded surgical resections were histologically classified according to the World Health Organization Classification of Tumors of the Nervous System.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nuclear Translocation of IRS-1 in Cells Expressing JCV T-antigen-- To investigate a potential involvement of IRS-1 in JCV T-antigen-associated transformation, we analyzed the levels of the expression and the localization of IRS-1 in JCV T-antigen-negative (Fig. 1, a-c) and JCV T-antigen-positive (Fig. 1, d-f) medulloblastomas cell lines. The cell lines derived from transgenic mice expressing the early genome of the archetype form of the JC virus (22). These mice develop cerebellar tumors characterized by the presence of JCV T-antigen-positive and -negative cells. On this basis we have developed both JCV T-antigen-positive and -negative medulloblastoma cell lines (23). As shown in Fig. 1b at low magnification and in Fig. 1c at higher magnification, JCV T-antigen-negative cells (Bs-1a) are characterized by a typical cytoplasmic IRS-1 immunostaining. In contrast, JCV T-antigen-positive cells (BsB8) have a strong nuclear and much less apparent cytoplasmic immunostaining for IRS-1 (Fig. 1, e and f).


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  		Fig. 1.   Immunocytochemical detection of IRS-1. Monolayer cultures of JCV T-antigen-negative (Bs-1a) (panels a-c) and JCV T-antigen-positive (BsB8) (panels d-f) mouse medulloblastoma cell lines were fixed and immunolabeled with anti-IRS-1 antibody (Upstate Biotechnology, Inc.) that recognizes C-terminal fragment of the IRS-1 molecule. Note an apparent nuclear localization of IRS-1 exclusively in T-antigen-positive cells depicted at low (panel e) and high (panel f) magnification. Panels a, b, d, and e, original magnification × 20; panels c and f, original magnification × 100.

Nuclear localization IRS-1 in JCV T-antigen-positive cells prompted us to confirm this unexpected finding by other methodologies. Within 48 h after retroviral delivery of GFP fused in frame with IRS-1 cDNA, we detected a strong nuclear fluorescence from living cultures of T-antigen-positive BsB8 cells (Fig. 2, e and f) and a predominant cytoplasmatic fluorescence in T-antigen-negative Bs-1a cells (Fig. 2, b and c). Fig. 2 (c and f) shows the exact localization of the IRS-1-associated green fluorescence in monolayer cultures by superimposing the phase contrast images (Fig. 2, a and d) with corresponding fluorescent images (Fig. 2, b and e). Therefore, by employing two independent experimental techniques, immunocytochemistry and the expression of GFP-IRS-1 fusion protein, we show that cells expressing JCV T-antigen translocate substantial amounts of IRS-1 from cytoplasm to the nuclear compartment of the cell.


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  		Fig. 2.   Fluorescent images of cells expressing GFP-IRS-1 fusion protein. Exponentially growing monolayer cultures of JCV T-antigen-negative (Bs-1a) (panels a-c) and JCV T-antigen-positive (BsB8) (panels d-f) medulloblastoma cell lines were examined under fluorescent microscope within 48 h after retroviral delivery of the GFP-IRS-1 fusion protein. Corresponding phase contrast images are depicted in panels a and d, respectively. Panels c and f show exact localization of the IRS-1-associated green fluorescence in monolayer cultures by superimposing the phase contrast images (panels a and d) with corresponding fluorescent images (panels b and e). All panels show the original magnification × 20.

IRS-1-JCV T-antigen Nuclear Co-localization-- The availability of JCV T-antigen-positive cell lines that express relatively high levels of the IRS-I protein (20) allowed us to determine whether JCV T-antigen and IRS-1 co-localize within the nucleus. BsB8 cells were double stained with anti-IRS-1 and anti-T-antigen antibodies (Fig. 3, a-c). The majority of BsB8 cells in the field showed a typical nuclear T-antigen staining visualized as green fluorescence (Fig. 3a). The cells showed a weak cytoplasmic and strong nuclear immunoreactivity with anti-IRS-1 antibody as red fluorescence (Fig. 3b). Finally, by superimposing Fig. 3a with Fig. 3b, we visualized (in yellow) nuclear co-localization of IRS-1 and JCV T-antigen (Fig. 3c). Using the same approach, IRS-1 was detected exclusively in the cytoplasm of the T-antigen-negative Bs-1a cells (Fig. 3, d-f).


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  		Fig. 3.   Nuclear co-localization of IRS-1 and JCV T-antigen. Panels a-c, JCV T-antigen-positive BsB8 cells; panels d-f, JCV T-antigen-negative Bs-1a cells; panels g-i, R600 fibroblast transiently transfected with JCV-T-antigen containing expression vector. All cells were double-labeled with anti-T-antigen and anti-IRS-1 antibodies. Nuclear green fluorescence corresponds to T-antigen immunostaining. Both cytoplasmic and nuclear read fluorescence corresponds to IRS-1 immunostaining. Superimposition of the images depicted in panels a, d, and g with images from panels b, e, and h, respectively, show in yellow nuclear co-localization between IRS-1 and JCV T-antigen. Shown is the original magnification × 100.

To determine whether T-antigen-mediated IRS-1 translocation to the nucleus is cell type-specific, R600 mouse embryo fibroblasts (27) were transfected with an expression vector containing JCV T-antigen cDNA (pcDNA3/zeo/JCVT). Within 48 h after transfection, three of 10 cells depicted in the field were positive for T-antigen (Fig. 3g). Only these three cells showed IRS-1 in the nucleus (Fig. 3h) and its nuclear co-localization with JCV T-antigen (Fig. 3i). The other seven cells, which do not express T-antigen, demonstrated that IRS-1 localized exclusively in the cytoplasm. This observation further verifies that IRS-1 translocation to the nucleus is associated with JCV T-antigen and that the process is not limited to cells of nervous system lineage.

Characterization of the IRS-1-JCV T-antigen Binding-- A possible molecular interaction between IRS-1 and JCV T-antigen was characterized by immunoprecipitation/Western blot analysis in three T-antigen-positive medulloblastoma cell lines (Fig. 4a, lanes 2-4). As expected, the immunocomplex was not detected in T-antigen-negative Bs-1a cells (lane 1) or in control samples tested with irrelevant primary antibody (not shown). The association between IRS-1 and JCV T-antigen was then confirmed, in reverse, by initially immunoprecipitating T-antigen and then developing the blot with anti-IRS-1 antibody (Fig. 4a, lower panel).


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  		Fig. 4.   Phosphorylation-dependent interaction between IRS-1 and JCV T-antigen. Panel a, immunoprecipitation/Western blot (IP/W) analysis of JCV T-antigen-positive (BsB8, BsB13, and BsB14) and -negative (Bs1a) medulloblastoma cell lines. Cell lysates were immunoprecipitated with anti-RS-1 antibody, and the corresponding blot was developed with anti-T-antigen antibody. In reverse, in the lower portion of panel a, protein lysates were immunoprecipitated (IP) with anti-T-antigen antibody, and the corresponding blot was developed with anti-IRS-1 antibody. Panel b, immunoprecipitation/Western blot analysis of BsB8 cells cultured in serum-free medium only (SFM), in the presence of OKA, or following IGF-I stimulation (IGF-I). Cell lysates were immunoprecipitated with anti-IRS-1 antibody, and the corresponding blots were developed with anti-T-antigen antibody (JCVT), anti-phosphotyrosine antibody (PY), and anti-IRS-1 antibody (IRS-1). Panels c-e, fluorescent images of BsB8 cells expressing GFP-IRS-1 fusion protein at 6 h (panel d) and 12 h (panel e) following the treatment with OKA. Depicted in panel c, cells before the treatment show an apparent nuclear fluorescence.

We next asked whether the IRS-1-JCV T-antigen interaction depends on the state of IRS-1 phosphorylation. This is particularly important, because IRS-1 has multiple tyrosine and serine residues, whose state of phosphorylation regulate IRS-1 function (32). Culturing BsB8 cells in serum-free medium maintains IRS-1 in a hypophosphorylated state. IGF-I treatment induces IRS-1 tyrosine phosphorylation, and treatment with okadaic acid (OKA) induces IRS-1 phosphorylation on serine residues (33). Following immunoprecipitation of IRS-1, the resulting Western blots (Fig. 4b) were processed with anti-T-antigen antibody (top panel), anti-phosphotyrosine antibody (middle panel), and anti-IRS-1 antibody (bottom panel). Both the hypophosphorylated form of IRS-1 and IRS-1 phosphorylated on tyrosine residues bound JCV T-antigen equally well. In contrast, the binding was severely impaired in cells treated with OKA, which induces IRS-1 serine phosphorylation (Fig. 4b, top panel). The middle panel of Fig. 4b shows that following IGF-I stimulation, IRS-1 is heavily phosphorylated on tyrosine residues. The blot was purposely overexposed to visualize residual IRS-1 tyrosine phosphorylation in serum-free medium or following OKA treatment. Finally, the bottom panel of Fig. 4b shows that IRS-1 was immunoprecipitated equally in all three samples. Notably, the band corresponding to IRS-1 after the OKA treatment migrates significantly slower than other two IRS-1 bands on the blot. Because the mobility shift is considered a hallmark of IRS-1 serine phosphorylation (34), this additionally confirmed the effectiveness of the treatment with OKA.

If serine phosphorylation of IRS-1 decreases T-antigen binding, one would expect a gradual loss of nuclear IRS-1 and its reappearance in the cytoplasm in the presence of OKA. Prior to OKA treatment, GFP/IRS fusion protein was detected predominantly in the nuclei of BsB8/GFP-IRS cells (Fig. 4c). Within 6 h after the addition of OKA, cytoplasmic fluorescence became much more intense (Fig. 4d), and after 12 h, the majority of the GFP-IRS-1 was observed in the cytoplasm, and some remained still in the nucleus (Fig. 4e). Because longer treatment with OKA resulted in massive cell death, we were not able to reach the point in which GFP/IRS-1 could be visible exclusively within the cytoplasm. The above results indicate that IRS-1 binding to JCV T-antigen is not only responsible for IRS-1 translocation but also may play a role in maintaining IRS-1 within the nuclear compartment.

Dominant Negative Effects against IRS-1-JCV T-antigen Interaction-- A series of IRS-1 truncation mutants were employed in a GST pull-down assay to characterize the region responsible for the interaction with JCV T-antigen. The strongest T-antigen binding mapped within the first 300-amino acid stretch of IRS-1 (Fig. 5a). This region of IRS-1 contains a PH domain and a PTB domain (6, 32). The other mutant still capable of pulling down T-antigen comprises the region between amino acids 212 and 529. Because this part of IRS-1 overlaps with 97 amino acids of the PTB domain, it is likely that the PTB domain is involved in T-antigen binding. On the other hand, the PH/PTB fragment binds T-antigen more efficiently than the 212-529 fragments, suggesting the possibility of more than one binding site for T-antigen in PH/PTB region. The other mutants spanning the entire C-terminal fragment of IRS-1 did not bind T-antigen.


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  		Fig. 5.   Interference with the IRS-1-JCV T-antigen binding. Panel a, GST pull-down assay with in vitro translated JCV T-antigen and GST-IRS-1 truncation mutants. Two mutants, amino acids 1-300 (PH/PTB domain) and amino acids 212-529, are positive for the interaction with JCV T-antigen. All other mutants and GST alone are negative. Panel b, Western blot (W) showing the expression of the PH/PTB mutant in BsB8 cells (BsB8/PH/PTB). IRS-1 negative cells overexpressing PH/PTB domain (LN/PH/PTB) (lane 1) and cells transduced with the empty vector (BsB8/EV) (lane 3) serve as positive and negative controls, respectively. The amount of the endogenous IRS-1 bound to JCV T-antigen evaluated in BsB8/EV and BsB8/PH/PTB cells following immunoprecipitation (IP) with anti-T-antigen antibody (lanes 4 and 5). To determine loading conditions and the efficiency of immunoprecipitation, the blot was stripped and reprobed with anti-T-antigen antibody. Panel c, anchorage-independent growth of Bs-1a and BsB8 cells stably transduced with the EV or with the vector containing PH/PTB cDNA. Cell growth and survival was evaluated on PolyHema-coated dishes in the presence or absence of IGF-I, as previously described (30). Cell number was determined within 1 h after plating (T0), and 48 h later. The results represent the averages of three experiments, each with three plates (n = 9). Panel d, Western blot showing IGF-I-induced IRS-1 tyrosine phosphorylation in BsB8/EV and BsB8/PH/PTB cells. Serum-starved cells were left without treatment (SFM) or were stimulated with IGF-I, and protein extracts were collected at the indicated time points. IRS-1 was immunoprecipitated (IP) from 500 µg of total proteins with anti-IRS-1 antibody (Upstate Biotechnology, Inc.), and Western blot (W) was probed with anti-phosphotyrosine antibody (pY). To monitor loading conditions the same blot was stripped and reprobed with anti-IRS-1 antibody (lower panel).

To test the ability of the PH/PTB domain to function as a dominant negative mutant competing for the IRS-1-T-antigen binding, we prepared protein extracts from BsB8 cells transduced either with the PH/PTB cDNA or with the EV. Following selection, the mixed population of transduced BsB8 cells expressed PH/PTB protein (Fig. 5b, lane 2) as compared with the PH/PTB band from the previously characterized LN/PH/PTB cells (lane 1), which overexpress PH/PTB and do not express wild type IRS-1 (13, 14). We precipitated T-antigen from both BsB8/EV cells (lane 4) and BsB8/PH/PTB cells (lane 5). The apparent weaker binding detected in BsB8/PH/PTB cells suggests a dominant negative action of the PH/PTB fragment against the binding between endogenous IRS-1 and T-antigen. We further evaluated whether expression of the PH/PTB mutant affects growth and survival of T-positive (BsB8) and T-negative (Bs-1a) cells (Fig. 5c). Under anchorage-independent culture conditions, control BsB8 cells expressing the EV showed some ability to proliferate in serum-free medium and strongly responded with cell proliferation following the IGF-I stimulation. Conversely, BsB8/PH/PTB cells become anchorage-dependent and show a substantial loss in the cell number in both serum-free medium and following IGF-I stimulation. Although JCV T-antigen-negative Bs-1a cells do not survive anchorage independence as well as BsB8 cells, they still show restricted abilities to proliferate following IGF-I treatment (20). As shown in Fig. 5c, overexpression of the PH/PTB mutant in Bs-1a cells did not affect their growth capacity in anchorage independence. This indicates that in the absence of JCV T-antigen, the PH/PTB mutant lost its growth repressing function. It is possible, however, that the PH/PTB mutant competes with the endogenous IRS-1 for the receptor binding, decreasing IRS-1 tyrosine phosphorylation and, in turn, affecting IRS-1-mediated growth responses. We have examined this possibility by comparing IGF-I-stimulated IRS-1 tyrosine phosphorylation in BsB8 cells in the presence or absence of PH/PTB domain. As shown in Fig. 5d, levels of IRS-1 tyrosine phosphorylation between BsB8/EV and BsB8/PH/PTB cells were comparable at the indicated time points following IGF-I stimulation. This further indicates that overexpression of the PH/PTB fragment is much more effective on repressing IRS-1-JCV T-antigen binding (Fig. 5b) than on competing with wild type IRS-1 for its phosphorylation (Fig. 5d). This also implies that anchorage-independent growth and survival of cells transformed by JCV T-antigen may involve T-antigen interaction with IRS-1.

By utilizing previously characterized LNCaP cells lacking endogenous IRS-1 (13), we demonstrated that the PH/PTB domain as well as different functional tyrosine mutants of IRS-1 translocate to the nucleus in the presence of JCV T-antigen (Fig. 6). LNCaP cells expressing the PH/PTB domain only, IRS-1 mutants lacking tyrosine residues involved in phosphatidylinositol 3-kinase binding (Y608F and Y939F), a triple tyrosine mutant lacking both phosphatidylinositol 3-kinase and Grb-2 binding (Y608F/Y689F/Y939F), and the mutant with a truncated PH domain were all transiently transfected with the JCV T-antigen expression vector (pcDNA3zeo/JCVT). Within 48 h after transfection, the cell cultures were double immunolabeled with anti-IRS-1 and anti-T-antigen antibodies. T-antigen-positive cells were subsequently examined for subcellular localization of the IRS-1 mutants. The presented results (Fig. 6) indicate that all of the examined IRS-1 mutants, which contain intact PH domains, translocate efficiently to the nucleus in the presence of JCV T-antigen. In contrast, removal of the PH domain prevented IRS-1 translocation to the nucleus, indicating again that the PH domain is involved in this process. This observation opens new possibilities of studying different IRS-1 functional domains in the context of their putative nuclear interactions and their possible association with T-antigen-mediated cellular transformation.


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  		Fig. 6.   JCV T-antigen-mediated nuclear translocation of the IRS-1 mutants. Parental LNCaP cells (LNCaP) and LNCaP stable clones expressing different IRS-1 mutants were double immunolabeled with anti-IRS-1 and anti-Tantigen antibodies. LNCaP 2 point mutation, IRS-1 mutant lacking tyrosine residues 608 and 939; LNCaP 3 point mutation, mutant lacking tyrosine residues 608, 689, and 939; LNCaP PH/PTB, PH/PTB fragment; LNCaP delta PH, mutant lacking the entire PH domain. Green fluorescence indicates presence of JCV T-antigen nuclear immunolabeling; red fluorescence reflects nuclear and/or cytoplasmic presence of IRS-1, and finally co-localization of T-antigen and IRS-1 is demonstrated as a yellow fluorescence (double labeling). All of the mutants except the PH/PTB domain were labeled with anti-IRS-1 antibody that recognizes the C-terminal portion of the IRS-1, and the PH/PTB mutant was immunolabeled with the antibody that recognizes IRS-1 N terminus (see "Experimental Procedures"). Shown for all images is the original magnification × 40.

IRS-1-JCV T-antigen Interaction in Medulloblastoma Biopsies-- To investigate the potential involvement of IRS-1 in JCV-associated transformation in humans, we analyzed the levels of expression and the localization of IRS-1 in JCV T-antigen-positive (Fig. 7, c and d) and JCV T-antigen-negative (Fig. 7, a and b) human medulloblastoma biopsies. As determined by anti-IRS-1 immunostaining, tumor cells show a typical cytoplasmic localization of IRS-1 (Fig. 7b) in a JCV T-antigen-negative biopsy (Fig. 7a). Cells with nuclear staining for IRS-1 (Fig. 7d) were abundant in the biopsy that was T-antigen-positive (Fig. 7c). Routine control samples, in which primary antibodies, both anti-T-antigen and anti-IRS-1, were replaced with the irrelevant antibody, were negative (not shown). We further analyzed a collection of 17 medulloblastoma biopsies for the subcellular localization of IRS-1, the results of which are shown in Table I. Although most of medulloblastoma cases were positive for anti-IRS-1 staining (12 of 17), only JCV T-antigen-positive tumors showed IRS-1 immunoreactivity in both nuclear and cytoplasmic compartments. In all JCV T-antigen-negative cases, IRS-1 was found exclusively in the cytoplasm. These findings provide a strong correlation between the presence of JCV T-antigen and the nuclear localization of IRS-1 in human medulloblastomas.


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  		Fig. 7.   Immunohistochemical detection and localization of IRS-1 in medulloblastoma biopsies. Analysis of JCV T-antigen-negative (panels a and b) and positive (panels c and d) cerebellar biopsies from two representative patients diagnosed with medulloblastoma. Paraffin sections were stained with anti-T-antigen antibody (panels a and c) and with anti-IRS-1 antibody (panels b and d). Note an apparent difference in subcellular distribution of IRS-1 immunolabeling between JCV T-antigen-positive and -negative biopsies. Shown for all images is the original magnification × 100.

                              
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Table I
Clinical and immunocytochemical analysis of human medulloblastomas
Diagnosis of the tumor is based on the World Health Organization criteria for medulloblastomas. The anatomical location of the tumors, the age at the time of tumor resection, and the gender of each patient are shown. Presence (+) or absence (-) of immunohistochemical staining for JCV T-antigen and IRS-1 was determined by utilizing rabbit anti-SV40 T-antigen antibody (Ab-2; Oncogene) that recognizes JCV T-antigen and rabbit anti-IRS-1 antibody that recognizes the C-terminal portion of the IRS-1 (Upstate Biotechnology, Inc.). Subcellular localization of the immunostaining was determined under a bright light microscope.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IRS-1 is the major cytoplasmic component of the insulin and IGF-I signaling pathways. It transduces metabolic signals from the insulin receptor (35), plays an essential role in IGF-I-mediated cell proliferation (1, 2), and has transforming properties by itself or in combination with other growth promoting factors (3, 4). Here we present several lines of evidence demonstrating nuclear translocation of IRS-1 in the presence of the human polyomavirus early protein JCV T-antigen. Initially, we detected nuclear IRS-1 both in T-antigen-positive murine medulloblastoma cell lines and in fibroblasts stably transfected with JCV T-antigen cDNA. We have confirmed the significance of this finding by detecting nuclear IRS-1 in a collection of biopsies from patients diagnosed with medulloblastoma. In addition, we observed nuclear translocation of GFP-IRS-1 fusion protein in living cultures of JCV T-antigen-positive cells and predominant cytoplasmic fluorescence in JCV T-antigen-negative cells. Because the binding site for JCV T-antigen was mapped within the N-terminal portion of the IRS-1 molecule, we utilized this N-terminal IRS-1 fragment (PH/PTB domain) to compete for T-antigen binding with the endogenous IRS-1. Finally, our cell culture experiments showed that the PH/PTB mutant attenuates the ability of T-antigen-positive medulloblastoma cells to grow and survive in anchorage independence.

It has been reported that the simian counterpart of JCV T-antigen, SV40 T-antigen, co-precipitates with IRS-1 and that simultaneous overexpression of these two proteins induces transformation of cells with a targeted disruption of the IGF-IR gene (3, 4). A similar protein-protein interaction involving JCV T-antigen may contribute to cellular transformation in humans. Although the involvement of polyomaviruses in human tumors remains controversial (25), a growing line of evidence shows unequivocally that viral proteins have transforming properties in vitro and are highly tumorogenic in animals (22, 36-38). Interestingly, recent studies reveal an association of the JCV genome with spontaneous medulloblastomas in humans (25, 26) and the expression of JCV T-antigen in some human tumor cells (23). Considering the fact that the JC virus asymptomatically infects more than 90% of the human population (21), it is relevant to understand whether and how JCV T-antigen causes cellular transformation. In this respect our recent work supports the concept of a potential synergy between the IGF system and JCV T-antigen in both experimental and human medulloblastomas (20). Elevated expression of IRS-1 in medulloblastomas, in combination with the increasing number of reports demonstrating the presence of the human neurotropic JC virus in these cerebellar tumors (26, 36), suggests that JCV T-antigen and IRS-1 may cooperate in the process of malignant transformation. One should also ask whether this interaction between IRS-1 and JCV T-antigen plays a significant role in the process of cellular transformation. It is well documented that transforming properties of polyomavirus T-antigens are mediated, at least partially, by interaction with and subsequent inactivation of p53 and the pRb family members (39). Is there a biological function for IRS-1 in this scenario, or do the results presented in our report reflect an interaction between two "sticky" molecules that occurs when cellular compartments are disintegrated in the lysis buffer? A partial answer to this question comes from experiments with R- cells, which are 3T3-like fibroblasts isolated from mice with a targeted disruption of the IGF-IR gene (29). Although p53 and the pRb proteins are present, the R- cells are completely resistant to transformation mediated either by SV40 T-antigen (40, 41), or by JCV T-antigen.2 In addition, a mutant of SV40 T-antigen lacking a nuclear localization sequence maintains its transforming properties in vitro (42), and transgenic mice expressing this cytoplasmic form of T-antigen develop spontaneous brain tumors (43). These multiple studies indicate that in addition to its well documented nuclear interactions with p53 and pRb, T-antigen-mediated cellular transformation may require additional components.

Although all known functions of IRS-1 are associated with its cytoplasmic localization, two earlier reports demonstrate that IRS-1 may also interact with nuclear proteins. These include binding of IRS-1 to nucleolin (10) and co-immunoprecipitation of IRS-1 with SV40 T-antigen (3). Because both nucleolin and SV40 T-antigen may also be found in the cytoplasm (44) or in association with the plasma membrane (45), the possibility that IRS-1 actually functions within the nucleus has not been explored (3, 10). Based on the findings reported here, we postulate a new sequence of molecular interactions during JCV T-antigen-mediated cellular transformation. The sequence involves T-antigen binding to IRS-1, translocation of the complex to the nucleus, and subsequent as yet uncharacterized nuclear events. This pathway may serve to establish new roles for IRS-1 as a nuclear protein and a role for the IRS-1-T-antigen complex in cancerogenesis.

    ACKNOWLEDGEMENT

We gratefully acknowledge Dr. Renato Baserga for valuable advice and discussion.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant PO1 NS 36466.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger These authors contributed equally to this work.

§ To whom correspondence should be addressed: Center for Neurovirology and Cancer Biology, College of Science and Technology, Temple University, 1900 North 12th St., Biology Life Science Bldg., Rm. 238, Philadelphia, PA 19122. Tel.: 215-204-0620; Fax: 215-204-0679; E-mail: kreiss@astro.temple.edu.

Published, JBC Papers in Press, February 27, 2002, DOI 10.1074/jbc.M110885200

2 A. Lassak, L. Del Valle, F. Peruzzi, J. Y. Wang, S. Enam, S. Croul, K. Khalili, and K. Reiss, unpublished results.

    ABBREVIATIONS

The abbreviations used are: IRS-1, insulin receptor substrate 1; IGF-I, insulin-like growth factor I; IGF-IR, IGF-I receptor; PH, pleckstrin homology domain; PTB phosphotyrosine binding domain, GFP; green fluorescent protein, OKA, okadaic acid; JCV, JC virus; EV, empty retroviral vector.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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