title: Serine 220 phosphorylation of the Merkel cell polyomavirus large T antigen crucially supports growth of Merkel cell carcinoma cells.
abstract: Merkel cell polyomavirus (MCPyV) is regarded as a major causal factor for Merkel cell carcinoma (MCC). Indeed, tumor cell growth of MCPyV-positive MCC cells is dependent on the expression of a truncated viral Large T antigen (LT) with an intact retinoblastoma protein (RB)-binding site. Here we determined the phosphorylation pattern of a truncated MCPyV-LT characteristically for MCC by mass spectrometry revealing MCPyV-LT as multi-phospho-protein phosphorylated at several serine and threonine residues. Remarkably, disruption of most of these phosphorylation sites did not affect its ability to rescue knockdown of endogenous T antigens in MCC cells indicating that phosphorylation of the respective amino acids is not essential for the growth promoting function of MCPyV-LT. However, alteration of serine 220 to alanine completely abolished the ability of MCPyV-LT to support proliferation of MCC cells. Conversely, mimicking the phosphorylated state by mutation of serine 220 to glutamic acid resulted in a fully functional LT. Moreover, MCPyV-LT(S220A) demonstrated reduced binding to RB in co-immunoprecipitation experiments as well as weaker induction of RB target genes in MCC cells. In conclusion, we provide evidence that phosphorylation of serine 220 is required for efficient RB inactivation in MCC and may therefore be a potential target for future therapeutic approaches.
authors.affiliation: Department of Dermatology, University Hospital Würzburg, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Schrama
authors.affiliation: Department of Dermatology, University Hospital Würzburg, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Hesbacher
authors.affiliation: Department of Dermatology, University Hospital Würzburg, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Angermeyer
authors.affiliation: Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schlosser
authors.affiliation: Department of Dermatology, University of Regensburg, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Haferkamp
authors.affiliation: Department of Dermatology, University Hospital Würzburg, Germany.
authors.forename: Annemarie
authors.initials: A
authors.lastname: Aue
authors.affiliation: Department of Dermatology, University Hospital Würzburg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Adam
authors.affiliation: Department of Dermatology, University Hospital Würzburg, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Weber
authors.affiliation: Department of Dermatology, University Hospital Würzburg, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Schmidt
authors.affiliation: Department of Dermatology, University Hospital Würzburg, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Houben
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D015266:Carcinoma, Merkel Cell / Q000473:pathology*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D006801:Humans; D059965:Merkel cell polyomavirus / Q000276:immunology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D016160:Retinoblastoma Protein / Q000378:metabolism; D012694:Serine; D012878:Skin Neoplasms / Q000473:pathology*
chemical_list: D000952:Antigens, Polyomavirus Transforming; D016160:Retinoblastoma Protein; D012694:Serine
keywords: Large T antigen; merkel cell carcinoma; phosphorylation; polyomavirus