Ronald T. Javier

Interactions of the PDZ-protein MAGI-1 with adenovirus E4-ORF1 and high-risk papillomavirus E6 oncoproteins

The oncoproteins of small DNA tumor viruses promote tumorigenesis by complexing with cellular factors intimately involved in the control of cell proliferation. The major oncogenic determinants for human adenovirus type 9 (Ad9) and high-risk human papillomaviruses (HPV) are the E4-ORF1 and E6 proteins, respectively. These seemingly unrelated viral oncoproteins are similar in that their transforming activities in cells depend, in part, on a carboxyl-terminal PDZ domain-binding motif which mediates interactions with the cellular PDZ-protein DLG. Here we demonstrated that both Ad9 E4-ORF1 and high-risk HPV E6 proteins also bind to the DLG-related PDZ-protein MAGI-1. These interactions resulted in MAGI-1 being aberrantly sequestered in the cytoplasm by the Ad9 E4-ORF1 protein or being targeted for degradation by high-risk HPV E6 proteins. Transformation-defective mutant viral proteins, however, were deficient for these activities. Our findings indicate that MAGI-1 is a member of a select group of cellular PDZ proteins targeted by both adenovirus E4-ORF1 and high-risk HPV E6 proteins and, in addition, suggest that the tumorigenic potentials of these viral oncoproteins depend, in part, on an ability to inhibit the function of MAGI-1 in cells.

Multi-PDZ Domain Protein MUPP1 Is a Cellular Target for both Adenovirus E4-ORF1 and High-Risk Papillomavirus Type 18 E6 Oncoproteins

ABSTRACT A general theme that has emerged from studies of DNA tumor viruses is that otherwise unrelated oncoproteins encoded by these viruses often target the same important cellular factors. Major oncogenic determinants for human adenovirus type 9 (Ad9) and high-risk human papillomaviruses (HPV) are the E4-ORF1 and E6 oncoproteins, respectively, and although otherwise unrelated, both of these viral proteins possess a functional PDZ domain-binding motif that is essential for their transforming activity and for binding to the PDZ domain-containing and putative tumor suppressor protein DLG. We report here that the PDZ domain-binding motifs of Ad9 E4-ORF1 and high-risk HPV-18 E6 also mediate binding to the widely expressed cellular factor MUPP1, a large multi-PDZ domain protein predicted to function as an adapter in signal transduction. With regard to the consequences of these interactions in cells, we showed that Ad9 E4-ORF1 aberrantly sequesters MUPP1 within the cytoplasm of cells whereas HPV-18 E6 targets this cellular protein for degradation. These effects were specific because mutant viral proteins unable to bind MUPP1 lack these activities. From these results, we propose that the multi-PDZ domain protein MUPP1 is involved in negatively regulating cellular proliferation and that the transforming activities of two different viral oncoproteins depend, in part, on their ability to inactivate this cellular factor.

Connexin 43 Interacts with Zona Occludens-1 and -2 Proteins in a Cell Cycle Stage-specific Manner

Gap junction channels play an important role in cell growth control, secretion and embryonic development. Gap junctional communication and channel assembly can be regulated by protein-protein interaction with kinases and phosphatases. We have utilized tandem mass spectrometry (MS/MS) sequence analysis as a screen to identify proteins from cell lysates that interact with the C-terminal cytoplasmic region of connexin 43 (Cx43). MS/MS analysis of tryptic fragments yielded several proteins including zona occludens-1 (ZO-1), a structural protein previously identified to interact with Cx43, and ZO-2, a potential novel interacting partner. We confirmed the interaction of ZO-2 with Cx43 by using a combination of fusion protein “pull down,” co-immunoprecipitation, and co-localization experiments. We show that the C-terminal region of Cx43 is necessary for interaction with the PDZ2 domain of ZO-2. Far Western analysis revealed that ZO-2 can directly bind to Cx43 independent of other interacting partners. Immunofluorescence studies indicate that both ZO-1 and ZO-2 can co-localize with Cx43 within the plasma membrane at apparent gap junctional structures. We examined Cx43 interaction with ZO-1 and ZO-2 at different stages of the cell cycle and found that Cx43 had a strong preference for interaction with ZO-1 during G0, whereas ZO-2 interaction occurred approximately equally during G0 and S phases. Since essentially all of the Cx43 in G0 cells is assembled into Triton X-100-resistant junctions, Cx43-ZO-1 interaction may contribute to their stability.

Viral oncoprotein-induced mislocalization of select PDZ proteins disrupts tight junctions and causes polarity defects in epithelial cells

The development of human cancers is frequently associated with a failure of epithelial cells to form tight junctions and to establish proper apicobasal polarity. Interestingly, the oncogenic potential of the adenovirus E4-ORF1 protein correlates with its binding to the cellular PDZ proteins MUPP1, MAGI-1, ZO-2 and SAP97, the first three of which assemble protein complexes at tight junctions. Given that E4-ORF1 sequesters these three PDZ proteins in the cytoplasm of fibroblasts, we postulated that E4-ORF1 would inhibit tight junction formation in epithelial cells. Providing further support for this idea, we identified MUPP1-related PATJ, a key component of the tight junction-associated CRB3-PALS1-PATJ polarity complex, as a new PDZ-protein target for both the E4-ORF1 and high-risk human papillomavirus type 18 E6 oncoproteins. Moreover, in epithelial cells, E4-ORF1 blocked the tight junction localization of PATJ and ZO-2, as well as their interacting partners, and disrupted both the tight junction barrier and apicobasal polarity. These significant findings expose a direct link between the tumorigenic potential of E4-ORF1 and inactivation of cellular PDZ proteins involved in tight junction assembly and polarity establishment.

Emerging Theme: Cellular PDZ Proteins as Common Targets of Pathogenic Viruses

ABSTRACT More than a decade ago, three viral oncoproteins, adenovirus type 9 E4-ORF1, human T-lymphotropic virus type 1 Tax, and high-risk human papillomavirus E6, were found to encode a related carboxyl-terminal PDZ domain-binding motif (PBM) that mediates interactions with a select group of cellular PDZ proteins. Recent studies have shown that many other viruses also encode PBM-containing proteins that bind to cellular PDZ proteins. Interestingly, these recently recognized viruses include not only some with oncogenic potential (hepatitis B virus, rhesus papillomavirus, cottontail rabbit papillomavirus) but also many without this potential (influenza virus, Dengue virus, tick-borne encephalitis virus, rabies virus, severe acute respiratory syndrome coronavirus, human immunodeficiency virus). Examination of the cellular PDZ proteins that are targets of viral PBMs reveals that the viral proteins often interact with the same or similar types of PDZ proteins, most notably Dlg1 and other members of the membrane-associated guanylate kinase protein family, as well as Scribble. In addition, cellular PDZ protein targets of viral PBMs commonly control tight junction formation, cell polarity establishment, and apoptosis. These findings reveal a new theme in virology wherein many different virus families encode proteins that bind and perturb the function of cellular PDZ proteins. The inhibition or perturbation of the function of cellular PDZ proteins appears to be a widely used strategy for viruses to enhance their replication, disseminate in the host, and transmit to new hosts.

Discs-Large and Strabismus are functionally linked to plasma membrane formation

During early embryogenesis in Drosophila melanogaster, extensive vesicle transport occurs to build cell boundaries for 6,000 nuclei. Here we show that this important process depends on a functional complex formed between the tumour suppressor and adaptor protein Discs-Large (Dlg) and the integral membrane protein Strabismus (Stbm)/Van Gogh (Vang). In support of this idea, embryos with mutations in either dlg or stbm displayed severe defects in plasma membrane formation. Conversely, overexpression of Dlg and Stbm synergistically induced excessive plasma membrane formation. In addition, ectopic co-expression of Stbm (which associated with post-Golgi vesicles) and the mammalian Dlg homologue SAP97/hDlg promoted translocation of SAP97 from the cytoplasm to both post-Golgi vesicles and the plasma membrane. This effect was dependent on the interaction between Stbm and SAP97. These findings suggest that the Dlg–Stbm complex recruits membrane-associated proteins and lipids from internal membranes to sites of new plasma membrane formation.

The multi‐PDZ domain protein MUPP1 is a cytoplasmic ligand for the membrane‐spanning proteoglycan NG2

A yeast two‐hybrid screen was employed to identify ligands for the cytoplasmic domain of the NG2 chondroitin sulfate proteoglycan. Two overlapping cDNA clones selected in the screen are identical in sequence to a DNA segment coding for the most amino‐terminal of the 13 PDZ domains found in the multi‐PDZ‐protein MUPP1. Antibodies made against recombinant polypeptides representing these two clones (NIP‐2 and NIP‐7) are reactive with the same 250‐kDa molecule recognized by anti‐MUPP1 antibodies, confirming the presence of the NIP‐2 and NIP‐7 sequences in the MUPP1 protein. NIP‐2 and NIP‐7 GST fusion proteins effectively recognize NG2 in pull‐down assays, demonstrating the ability of these polypeptide segments to interact with the intact proteoglycan. The fusion proteins fail to bind NG2 missing the C‐terminal half of the cytoplasmic domain, emphasizing the role of the NG2 C‐terminus in the interaction with MUPP1. The existence of an NG2/MUPP1 interaction in situ is demonstrated by the ability of NG2 antibodies to co‐immunoprecipitate both NG2 and MUPP1 from detergent extracts of cells expressing the two molecules. MUPP1 may serve as a multivalent scaffold that provides a means of linking NG2 with key structural and/or signaling components in the cytoplasm. J. Cell. Biochem. 79:213–224, 2000.

Link of the unique oncogenic properties of adenovirus type 9 E4‐ORF1 to a select interaction with the candidate tumor suppressor protein ZO‐2

Adenovirus type 9 (Ad9) is distinct among human adenoviruses because it elicits solely mammary tumors in animals and its primary oncogenic determinant is the E4 region‐encoded ORF1 (E4‐ORF1) protein. We report here that the PDZ domain‐containing protein ZO‐2, which is a candidate tumor suppressor protein, is a cellular target for tumorigenic Ad9 E4‐ORF1 but not for non‐tumorigenic wild‐type E4‐ORF1 proteins encoded by adenovirus types 5 and 12. Complex formation was mediated by the C‐terminal PDZ domain‐binding motif of Ad9 E4‐ ORF1 and the first PDZ domain of ZO‐2, and in cells this interaction resulted in aberrant sequestration of ZO‐2 within the cytoplasm. Furthermore, transformation‐defective Ad9 E4‐ORF1 mutants exhibited impaired binding to and sequestration of ZO‐2 in cells, and overexpression of wild‐type ZO‐2, but not mutant ZO‐2 lacking the second and third PDZ domains, interfered with Ad9 E4‐ORF1‐induced focus formation. Our results suggest that the select capacity to complex with the candidate tumor suppressor protein ZO‐2 is key to defining the unique transforming and tumorigenic properties of the Ad9 E4‐ORF1 oncoprotein.

The ESEV PDZ-Binding Motif of the Avian Influenza A Virus NS1 Protein Protects Infected Cells from Apoptosis by Directly Targeting Scribble

ABSTRACT The NS1 protein from influenza A viruses contains a four-amino-acid sequence at its carboxyl terminus that is termed the PDZ-binding motif (PBM). The NS1 PBM is predicted to bind to cellular PDZ proteins and functions as a virulence determinant in infected mice. ESEV is the consensus PBM sequence of avian influenza viruses, while RSKV is the consensus sequence of human viruses. Currently circulating highly pathogenic H5N1 influenza viruses encode an NS1 protein with the ESEV PBM. We identified cellular targets of the avian ESEV PBM and identified molecular mechanisms involved in its function. Using glutathione S-transferase (GST) pull-down assays, we found that the ESEV PBM enables NS1 to associate with the PDZ proteins Scribble, Dlg1, MAGI-1, MAGI-2, and MAGI-3. Because Scribble possesses a proapoptotic activity, we investigated the interaction between NS1 and Scribble. The association between NS1 and Scribble is direct and requires the ESEV PBM and two Scribble PDZ domains. We constructed recombinant H3N2 viruses that encode an H6N6 avian virus NS1 protein with either an ESEV or mutant ESEA PBM, allowing an analysis of the ESEV PBM in infections in mammalian cells. The ESEV PBM enhanced viral replication up to 4-fold. In infected cells, NS1 with the ESEV PBM relocalized Scribble into cytoplasmic puncta concentrated in perinuclear regions and also protected cells from apoptosis. In addition, the latter effect was eliminated by small interfering RNA (siRNA)-mediated Scribble depletion. This study shows that one function of the avian ESEV PBM is to reduce apoptosis during infection through disruption of Scribbles proapoptotic function.

Oncogenic function for the Dlg1 mammalian homolog of the Drosophila discs-large tumor suppressor.

The fact that several different human virus oncoproteins, including adenovirus type 9 E4‐ORF1, evolved to target the Dlg1 mammalian homolog of the membrane‐associated Drosophila discs‐large tumor suppressor has implicated this cellular factor in human cancer. Despite a general belief that such interactions function solely to inactivate this suspected human tumor suppressor protein, we demonstrate here that E4‐ORF1 specifically requires endogenous Dlg1 to provoke oncogenic activation of phosphatidylinositol 3‐kinase (PI3K) in cells. Based on our results, we propose a model wherein E4‐ORF1 binding to Dlg1 triggers the resulting complex to translocate to the plasma membrane and, at this site, to promote Ras‐mediated PI3K activation. These findings establish the first known function for Dlg1 in virus‐mediated cellular transformation and also surprisingly expose a previously unrecognized oncogenic activity encoded by this suspected cellular tumor suppressor gene.