Complexes of polyoma virus medium T antigen and cellular proteins.

Abstract

Antibodies against synthetic peptides corresponding to the carboxyl-terminal six amino acids, Lys-ArgSer-Arg-His-Phe (KF), and an internal region, Glu-Glu-GluGlu-Tyr-Met-Pro-Met-Glu (EE), of polyoma virus medium T antigen were used successively to purify medium T antigen by affinity chromatography. Medium T antigen from cell extracts was first bound to anti-KF antibodies and released from the immune complex with excess KF peptide; then it was bound to anti-EE antibodies and released with excess EE peptide. Two proteins, pp6OCSrC and a new protein of "61,000 Da (61-kDa protein), were copurified because they formed complexes with medium T antigen. The 61-kDa protein-medium T antigen complex was detected in extracts from wild-type-infected and transformed cells but not from cells infected with NG59 virus, which has a mutation in the medium T gene and is transformation defective. Instead, NG59 medium T antigen formed a complex with another cellular protein of =72,000 Da.The transforming protein of polyoma virus, medium T antigen, is a membrane protein (1-3) associated with a tyrosine-specific protein kinase activity (4-6). Biochemical studies have indicated that this activity is not intrinsic to medium T antigen (7,8) but instead resides in a minor fraction sedimenting with a size of 200,000 Da, whereas the bulk of medium T antigen is monomeric and kinase inactive (9). It was shown subsequently that the kinase activity is due to an association between medium T antigen and pp6O1(src, the cellular homolog of the transforming protein of Rous sarcoma virus, pp6OVSrC (10). The interaction between medium T antigen and pp60'-sr may be facilitated by the fact that both proteins are membrane associated and, therefore, come into close contact. In a similar fashion, medium T antigen might interact with other neighboring membrane proteins. We asked how one might identify such proteins without having a functional assay or a specific antiserum available. In the present paper, we demonstrate that in addition to pp60O-s>, medium T antigen also forms a complex with a cellular protein of -61,000 Da. This was achieved by affinity chromatography with two anti-peptide antibodies directed against different regions of medium T antigen.

Association between src-kinases and the polyoma virus oncogene middle T-antigen requires PP2A and a specific sequence motif

“…If we assume there is only one form of complex in these lysates, the most likely interpretation is that the complex consists of one molecule each of MT, a srcfamily kinase, PP2A.A, and PP2A.C. This agrees well with the 200 ± 220 kDa size of the kinase active fraction of MT observed by sedimentation analysis (Courtneidge and Smith, 1983;Grussenmeyer et al, 1985;Walter et al, 1982). This makes it unlikely that a complex between MT and a src-kinase exists without PP2A.…”

Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells

“…Both the PDK1 and ΔPH-PKB proteins were N-terminally glu-glu-tagged, and were purified using a glu-glu antibody generated from mouse ascites, and eluted using an EYMPME peptide [22]. 150 ng of WT PDK1 or 500 ng PDK1 L159G were used.…”

Protein phosphatase 1 interacts with p53BP2, a protein which binds to the tumour suppressor p53

“…Hybridoma cells expressing monoclonal antibodies against a polyoma medium T epitope, which recognise the sequence EFMPME, were a kind gift from Professor Gernot Walter, University of California, San Diego, USA [24]. The protein in the supernatant from the hybridoma cell culture was precipitated in 50% ammonium sulphate and the precipitate redissolved in phosphate buffered saline to one tenth of the original volume and dialysed against the same buffer.…”