Merkel Cell Polyomavirus Small T Antigen Controls Viral Replication and Oncoprotein Expression by Targeting the Cellular Ubiquitin Ligase SCFFbw7

Kwun, Hyun Jin; Shuda, Masahiro; Feng, Huichen; Camacho, Carlos J.; Moore, Patrick S.; Chang, Yuan

doi:10.1016/j.chom.2013.06.008

Cited by 143 publications

(227 citation statements)

References 58 publications

(84 reference statements)

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“…MCV sT, but not SV40 sT expression is sufficient to transform rodent fibroblasts (39,42,52). However, despite the importance of PP2A targeting in defined oncogene transformation studies in human cells (15), MCV sT transformation in rodent cells depends on activities that map to the LSD domain rather than the PP2A targeting domain.…”

Section: Discussionmentioning

confidence: 99%

“…R7 and L142 lie on an exposed ridge that is predicted to facilitate MCV sT binding to PP2A A␣ (Fig. 5B) (42). K118 may be critical for PP2A C docking, although both PP2A A␣ and PP2A C failed to coimmunoprecipitate with this MCV sT mutant, suggesting that it may play a role in proper alignment of both PP2A subunits on sT, as well as direct interaction with PP2C.…”

Section: St Interacts With a And C Subunits Of Pp2a And Inhibits mentioning

confidence: 99%

“…No protein was detected for the H130A substitution mutant. We next determined whether the remaining 23 alanine substitution mutants altered binding to PP2A A␣ as well as induced 4E-BP1 hyperphosphorylation (39) and promoted MCV LT-mediated DNA replication in an MCV origin replication assay (42). Alanine substitution at four destabilizing residues (I122, L126, E146, and Y150) were negative for PP2A binding and 4E-BP1 phosphorylation, presumably due to reduced sT expression but retained activity for MCV DNA replication (Table 2), consistent with our previous report showing that minute amounts of sT coexpression are capable of maximally activating LT-induced DNA replication (42).…”

Section: St Interacts With a And C Subunits Of Pp2a And Inhibits mentioning

confidence: 99%

“…The model of MCV sT structure was generated using the I-TASSER server (http: //zhanglab.ccmb.med.umich.edu/I-TASSER/) using SV 40 sT structure (PDB ID 2PF4 and 2PKG) (42). The figures were generated using PyMOL.…”

Section: Plasmidsmentioning

confidence: 99%

“…This MCV sT function was mapped to amino acids 91 to 95, defined as the large T stabilization domain (LSD) (42), and determined to be essential for rodent cell transformation (42). MCV sT is also required for the hyperphosphorylation-associated inactivation of the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), since expression of a constitutively active 4E-BP1 antagonizes the cell transformation activity of MCV sT (39), but this is independent of Fbw7 targeting (42).…”

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confidence: 99%

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Restricted Protein Phosphatase 2A Targeting by Merkel Cell Polyomavirus Small T Antigen

Kwun

Shuda

Camacho

et al. 2015

J Virol

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Merkel cell polyomavirus (MCV) is a newly discovered human cancer virus encoding a small T (sT) oncoprotein. We performed MCV sT FLAG-affinity purification followed by mass spectroscopy (MS) analysis, which identified several protein phosphatases (PP), including PP2A A and C subunits and PP4C, as potential cellular interacting proteins. PP2A targeting is critical for the transforming properties of nonhuman polyomaviruses, such as simian virus 40 (SV40), but is not required for MCV sT-induced rodent cell transformation. We compared similarities and differences in PP2A binding between MCV and SV40 sT. While SV40 sT coimmunopurified with subunits PP2A A␣ and PP2A C, MCV sT coimmunopurified with PP2A A␣, PP2A A␤, and PP2A C. Scanning alanine mutagenesis at 29 sites across the MCV sT protein revealed that PP2A-binding domains lie on the opposite molecular surface from a previously described large T stabilization domain (LSD) loop that binds E3 ligases, such as Fbw7. MCV sT-PP2A interactions can be functionally distinguished by mutagenesis from MCV sT LSD-dependent 4E-BP1 hyperphosphorylation and viral DNA replication enhancement. MCV sT has a restricted range for PP2A B subunit substitution, inhibiting only the assembly of B56␣ into the phosphatase holoenzyme. In contrast, SV40 sT inhibits the assembly of B55␣, B56␣ and B56 into PP2A. We conclude that MCV sT is required for Merkel cell carcinoma growth, but its in vitro transforming activity depends on LSD interactions rather than PP2A targeting. A pproximately 30 widely expressed serine/threonine (Ser/Thr) protein phosphatases (PPases) have been identified (1, 2). Protein phosphatase 2A (PP2A) is a combinatorial family of Ser/Thr phosphatases that are highly conserved in eukaryotes. The PP2A core enzyme structure is comprised of a 36-kDa catalytic C subunit (PP2A C) bound to a 65-kDa A regulatory subunit (PP2A A). This core heterodimer can be purified, but the PP2A holoenzyme generally contains a third, regulatory B subunit that provides substrate specificity and subcellular localization (3-7). Among the many combinatorial possibilities, there are two C catalytic subunits, at least two 〈 scaffolding subunits, and multiple B substrate recognition subunits (8), which allow for potentially hundreds of different phosphatase specificities and activities. Phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase, c-Myc, and other cancer signaling pathways that impact cellular processes, including cell cycle progression and cellular tumorigenesis are regulated by specific PP2A isoforms (9). Aberrant expression and mutations in PP2A subunits have been implicated in human cancers (8,(10)(11)(12)(13)(14)(15)(16). IMPORTANCE Merkel cell polyomavirus is a newly discovered human cancer virus that promotes cancer, in part, through expression of itsPP2A activity is regulated in part by posttranslational modifications of subunits (17-23). Methylation of PP2A C at Leu309 activates the holoenzyme to allow binding of the B subunit (17), while phosphorylation o...

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Section: Discussionmentioning

confidence: 99%

Section: St Interacts With a And C Subunits Of Pp2a And Inhibits mentioning

confidence: 99%

Section: St Interacts With a And C Subunits Of Pp2a And Inhibits mentioning

confidence: 99%

Section: Plasmidsmentioning

confidence: 99%

mentioning

confidence: 99%

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Restricted Protein Phosphatase 2A Targeting by Merkel Cell Polyomavirus Small T Antigen

Kwun

Shuda

Camacho

et al. 2015

J Virol

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Viral oncoprotein antibodies as a marker for recurrence of Merkel cell carcinoma: A prospective validation study

Paulson

Lewis

Redman

et al. 2016

Cancer

136

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Background Merkel cell carcinoma (MCC) is an aggressive skin cancer with a recurrence rate of >40%. Of the 2000 MCC cases/year in the USA, most are caused by the Merkel cell polyomavirus (MCPyV). Antibodies to MCPyV-oncoprotein (T-antigens) have been correlated with MCC tumor burden. We prospectively validated the clinical utility of MCPyV oncoprotein antibody titers for MCC prognostication and surveillance. Methods MCPyV-oncoprotein antibody detection was optimized in a clinical laboratory. A cohort of 219 patients with newly-diagnosed MCC were followed prospectively (median follow-up 1.9 years). Among seropositive patients, antibody titer and disease status were serially tracked. Results Antibodies to MCPyV-oncoproteins were rare among healthy individuals (1%) but present in most MCC patients (114 of 219, 52%, p<0.01). Seropositivity at diagnosis independently predicted decreased recurrence risk (HR=0.58; p=0.04) in multivariate analyses adjusted for age, sex, stage, and immunosuppression. Following initial treatment, seropositive patients whose disease did not recur had rapidly falling titers that became negative by a median of 8.4 months. Among seropositive patients who underwent serial evaluation (71 patients; 282 timepoints), an increasing oncoprotein titer had a positive predictive value of 66% for clinically evident recurrence while a decreasing titer had a negative predictive value of 97%. Conclusions Determination of oncoprotein antibody titer assists in the clinical management of newly diagnosed MCC patients by stratifying them into a higher risk seronegative cohort in whom radiologic imaging may play a more prominent role, and into a lower-risk seropositive cohort whose disease status can be tracked in part via oncoprotein antibody titer.

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Spontaneous lung metastasis formation of human Merkel cell carcinoma cell lines transplanted into scid mice

Knips

Czech-Sioli

Spohn

et al. 2017

Intl Journal of Cancer

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Merkel cell carcinoma (MCC) is an aggressive skin cancer entity that frequently leads to rapid death due to its high propensity to metastasize. The etiology of most MCC cases is linked to Merkel cell polyomavirus (MCPyV), a virus which is monoclonally integrated in up to 95% of tumors. While there are presently no animal models to study the role of authentic MCPyV infection on transformation, tumorigenesis or metastasis formation, xenograft mouse models employing engrafted MCC-derived cell lines (MCCL) represent a promising approach to study certain aspects of MCC pathogenesis. Here, the two MCPyV-positive MCC cell lines WaGa and MKL-1 were subcutaneously engrafted in scid mice. Engraftment of both MCC cell lines resulted in the appearance of circulating tumor cells and metastasis formation, with WaGa-engrafted mice showing a significantly shorter survival time as well as increased numbers of spontaneous lung metastases compared to MKL-1 mice. Interestingly, explanted tumors compared to parental cell lines exhibit an upregulation of MCPyV sT-Antigen expression in all tumors, with WaGa tumors showing significantly higher sT-Antigen expression than MKL-1 tumors. RNA-Seq analysis of explanted tumors and parental cell lines furthermore revealed that in the more aggressive WaGa tumors, genes involved in inflammatory response, growth factor activity and Wnt signalling pathway are significantly upregulated, suggesting that sT-Antigen is the driver of the observed differences in metastasis formation.

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Merkel Cell Polyomavirus Small T Antigen Controls Viral Replication and Oncoprotein Expression by Targeting the Cellular Ubiquitin Ligase SCFFbw7

Cited by 143 publications

References 58 publications

Restricted Protein Phosphatase 2A Targeting by Merkel Cell Polyomavirus Small T Antigen

Restricted Protein Phosphatase 2A Targeting by Merkel Cell Polyomavirus Small T Antigen

Viral oncoprotein antibodies as a marker for recurrence of Merkel cell carcinoma: A prospective validation study

Spontaneous lung metastasis formation of human Merkel cell carcinoma cell lines transplanted into scid mice

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