Polyomavirus Middle T-Antigen Is a Transmembrane Protein That Binds Signaling Proteins in Discrete Subcellular Membrane Sites

ABSTRACT Murine polyomavirus middle T-antigen (MT) induces tumors by mimicking an activated growth factor receptor. An essential component of this action is a 22-amino-acid hydrophobic region close to the C terminus which locates MT to cell membranes. Here, we demonstrate that this sequence is a transmembrane domain (TMD) by showing that a hemagglutinin (HA) tag added to the MT C terminus is exposed on the outside of the cells, with the N terminus inside. To determine whether this MT TMD is inserted into the endoplasmic reticulum (ER) membrane, we added the ER retention signal KDEL to the MT C terminus (MTKDEL). This mutant protein locates only in the ER, demonstrating that MT does insert into membranes solely at this location. In addition, this ER-located MT failed to transform. Examination of the binding proteins associated with the MTKDEL protein demonstrated that it associates with PP2A and c-Src but fails to interact with ShcA, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ1 (PLC-γ1), despite being tyrosine phosphorylated. Additional mutant and antibody studies show that MT binding to PP2A is probably required for MT to efficiently exit the ER and migrate to the plasma membrane though the TMD also plays a role in this relocation. Overall, these data, together with previous publications, illustrate that MT associates with signaling proteins at different sites in its maturation pathway. MT binds to PP2A in the cytoplasm, to c-Src at the endoplasmic reticulum, and to ShcA, PI3K, and PLC-γ1 at subsequent locations en route to the plasma membrane.

[1]  B. Schaffhausen,et al.  Lessons in Signaling and Tumorigenesis from Polyomavirus Middle T Antigen , 2009, Microbiology and Molecular Biology Reviews.

[2]  B. Griffin,et al.  Polyoma virus and simian virus 40 as cancer models: history and perspectives. , 2009, Seminars in cancer biology.

[3]  T. Roberts,et al.  Lessons from polyoma middle T antigen on signaling and transformation: A DNA tumor virus contribution to the war on cancer. , 2009, Virology.

[4]  Blanche Schwappach,et al.  The GET Complex Mediates Insertion of Tail-Anchored Proteins into the ER Membrane , 2008, Cell.

[5]  S. Colombo,et al.  How tails guide tail-anchored proteins to their destinations. , 2007, Current opinion in cell biology.

[6]  R. Hegde,et al.  Identification of a Targeting Factor for Posttranslational Membrane Protein Insertion into the ER , 2007, Cell.

[7]  B. Schaffhausen,et al.  Genetic Analysis of the Polyomavirus DnaJ Domain , 2005, Journal of Virology.

[8]  S. Dilworth.  Polyoma virus middle T antigen and its role in identifying cancer-related molecules , 2002, Nature Reviews Cancer.

[9]  S. Dilworth,et al.  ShcA tyrosine phosphorylation sites can replace ShcA binding in signalling by middle T‐antigen , 2001, The EMBO journal.

[10]  W. Gullick,et al.  Cell specific transformation by c-fms activating loop mutations is attributable to constitutive receptor degradation , 1999, Oncogene.

[11]  B. Griffin,et al.  Regulation of cytoskeletal association by a basic amino acid motif in polyoma virus middle T antigen , 1998, Oncogene.

[12]  D. Andrews,et al.  At the onset of transformation polyomavirus middle-T recruits shc and src to a perinuclear compartment coincident with condensation of endosomes , 1998, Oncogene.

[13]  D. Andrews,et al.  Evidence for multiple mechanisms for membrane binding and integration via carboxyl-terminal insertion sequences. , 1997, Biochemistry.

[14]  S. Dilworth,et al.  pp60c-src binding to polyomavirus middle T-antigen (MT) requires residues 185 to 210 of the MT sequence , 1997, Journal of virology.

[15]  A. Marti,et al.  Polyomavirus middle-T antigen lacking a membrane anchor sequence accumulates in the nucleus. , 1996, The Journal of general virology.

[16]  R. Schlegel,et al.  E5 oncoprotein retained in the endoplasmic reticulum/cis Golgi still induces PDGF receptor autophosphorylation but does not transform cells. , 1995, The EMBO journal.

[17]  W. Liu,et al.  Association of Polyomavirus Middle Tumor Antigen with Phospholipase C-γ1(*) , 1995, The Journal of Biological Chemistry.

[18]  N. Hynes,et al.  Intracellular expression of single chain antibodies reverts ErbB-2 transformation. , 1994, The Journal of biological chemistry.

[19]  T. Roberts,et al.  Polyoma middle tumor antigen interacts with SHC protein via the NPTY (Asn-Pro-Thr-Tyr) motif in middle tumor antigen. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[20]  T. Rapoport,et al.  Evolutionary conservation of components of the protein translocation complex , 1994, Nature.

[21]  Michael D. Jones,et al.  Transformation by polyoma virus middle T-antigen involves the binding and tyrosine phosphorylation of Shc , 1994, Nature.

[22]  Nanxin Li,et al.  Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling , 1993, Nature.

[23]  T. Pawson,et al.  The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1 , 1993, Nature.

[24]  S. Dilworth,et al.  Novel monoclonal antibodies that differentiate between the binding of pp60c-src or protein phosphatase 2A by polyomavirus middle T antigen , 1993, Journal of virology.

[25]  T. Benjamin,et al.  Functional asymmetry of the regions juxtaposed to the membrane-binding sequence of polyomavirus middle T antigen , 1992, Molecular and cellular biology.

[26]  C. Slaughter,et al.  Association of protein phosphatase 2A with polyoma virus medium tumor antigen. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[27]  T. B. Miller,et al.  Polyoma small and middle T antigens and SV40 small t antigen form stable complexes with protein phosphatase 2A , 1990, Cell.

[28]  A. T. Young,et al.  Phosphorylation of middle T by pp60c-src : A switch for binding of phosphatidylinositol 3-kinase and optimal tumorigenesis , 1989, Cell.

[29]  L. Cantley,et al.  Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate , 1988, Nature.

[30]  A. Smith,et al.  Mapping of the amino-terminal half of polyomavirus middle-T antigen indicates that this region is the binding domain for pp60c-src , 1987, Journal of virology.

[31]  W. Welch,et al.  Medium tumor antigen of polyomavirus transformation-defective mutant NG59 is associated with 73-kilodalton heat shock protein , 1987, Journal of virology.

[32]  H. Hanafusa,et al.  Association of the polyomavirus middle-T antigen with c-yes protein , 1987, Nature.

[33]  B. Oostra,et al.  Site-directed mutagenesis of polyomavirus middle-T antigen sequences encoding tyrosine 315 and tyrosine 250 , 1986, Journal of virology.

[34]  B. Oostra,et al.  In vitro mutagenesis of the putative membrane-binding domain of polyomavirus middle-T antigen , 1986, Journal of virology.

[35]  H. Hansson,et al.  Subcellular localisation of the middle and large T‐antigens of polyoma virus. , 1986, The EMBO journal.

[36]  A. Markham,et al.  Mutations around the NG59 lesion indicate an active association of polyoma virus middle‐T antigen with pp60c‐src is required for cell transformation. , 1986, The EMBO journal.

[37]  A. Voronova,et al.  Construction and expression of a recombinant DNA gene encoding a polyomavirus middle-size tumor antigen with the carboxyl terminus of the vesicular stomatitis virus glycoprotein G , 1984, Molecular and cellular biology.

[38]  Alan E. Smith,et al.  Polyoma virus transforming protein associates with the product of the c-src cellular gene , 1983, Nature.

[39]  B. Schaffhausen,et al.  Polyoma virus middle T antigen: relationship to cell membranes and apparent lack of ATP-binding activity , 1982, Molecular and cellular biology.

[40]  M. Yaniv,et al.  Absence of nucleosomes in a histone-containing nucleoprotein complex obtained by dissociation of purified SV40 virions , 1982, Cell.

[41]  G. Carmichael,et al.  Carboxy terminus of polyoma middle-sized tumor antigen is required for attachment to membranes, associated protein kinase activities, and cell transformation. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[42]  B E Griffin,et al.  Monoclonal antibodies against polyoma virus tumor antigens. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[43]  R. Treisman,et al.  Transformation of rat cells by an altered polyoma virus genome expressing only the middle-T protein , 1981, Nature.

[44]  U. Novak,et al.  Requirement for the C-terminal region of middle T-antigen in cellular transformation by virus , 1981 .

[45]  U. Novak,et al.  Requirement for the C-terminal region of middle T-antigen in cellular transformation by polyoma virus. , 1981, Nucleic acids research.

[46]  T. Hunter,et al.  An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates , 1979, Cell.

[47]  E. Soeda,et al.  Sequence from early region of polyoma virus DNA containing viral replication origin and encoding small, middle and (part of) large T antigens , 1979, Cell.

[48]  Y. Ito,et al.  Virus-specific proteins in the plasma membrane of cells lytically infected or transformed by pol-oma virus. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[49]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.