Novel monoclonal antibodies that differentiate between the binding of pp60c-src or protein phosphatase 2A by polyomavirus middle T antigen.
Fourteen pGEX plasmids that express defined regions of polyomavirus middle T antigen in bacteria have been constructed. These polypeptides have been used to generate 18 new monoclonal antibodies directed against the unique portion of middle T and to map the approximate position of the antibody recognition sites onto the protein sequence. All of the antibodies effectively immunoprecipitate middle T and the associated 60- and 35-kDa components of protein phosphatase 2A. Four of the antibodies, however, do not react with middle T when it is bound to pp60c-src. These four probably bind to amino acids 203 to 218 of the middle T protein sequence, which are encoded by the mRNA immediately 3' to the splice junction that creates the C-terminal unique region. This suggests that additional middle T sequences are required for middle T's interaction with pp60c-src than are needed for its binding to protein phosphatase 2A. The antibodies localize this extra region and provide a means of distinguishing between these two associations.
Dilworth
S M
SM
Department of Chemical Pathology, Royal Postgraduate Medical School, Hammersmith Hospital, London, United Kingdom.
Horner
V P
VP
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology*; D000952:Antigens, Polyomavirus Transforming / Q000276:immunology* / Q000378:metabolism; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D000939:Epitopes; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010446:Peptide Fragments / Q000276:immunology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D011120:Polyomavirus / Q000276:immunology*; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D054648:Protein Phosphatase 2; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D016415:Sequence Alignment
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000952:Antigens, Polyomavirus Transforming; D000939:Epitopes; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D010750:Phosphoproteins; D011494:Protein Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2