Polyoma middle-sized T antigen can be phosphorylated on tyrosine at multiple sites in vitro.

Abstract

The polyoma middle‐sized T antigen (MT antigen) is associated with a protein kinase activity which phosphorylates tyrosine residues in polyoma T antigens in vitro. We have studied the sites of tyrosine phosphorylation of MT antigens phosphorylated in immunoprecipitates or in soluble form after partial purification by immunoaffinity chromatography. By analyzing the amino acid sequences of tryptic peptides of MT antigen, and by analyzing deletion mutant MT antigens, we have identified two major sites of phosphorylation in MT antigen, tyrosines 250 and 315. Additional sites were phosphorylated under some conditions. A synthetic peptide (Glu.Glu.Glu.Glu.Tyr.Met.Pro.Met.Glu), corresponding to the sequence around tyrosine 315, was phosphorylated when added to immunoprecipitates containing MT antigen.

An antibody to a synthetic peptide recognizes polyomavirus middle-T antigen and reveals multiple in vitro tyrosine phosphorylation sites.

“…The observation that the middle-T lacking tyrosine 315 retained acceptor activity in the kinase reaction, and the finding that middle-T in immunoprecipitates formed by using antibodies that recognize tyrosine 315 also retains acceptor activity, lead to the suggestion that middle-T is phosphorylated at multiple sites. The results reported here and by Hunter et al (12) suggest that tyrosines 322, 297, and 250 are such additional sites. Transforming deletion mutants that lack tyrosine 322 and 297 have already been described (3,15).…”

“…This peptide is very likely to include tyrosine 250, since it is present in fingerprints of mutant middle-T species that lack other tyrosine residues in the carboxy-terminal half of middle-T, and fingerprints of d18 middle-T contain a peptide of similar but altered mobility. Hunter et al (12) have sequenced a tryptic peptide with similar properties and have found that [32P]phosphate is released at cycle 10. All these data are consistent with the identification of tyrosine 250 as an in vitro phosphorylation site in middle-T.…”

“…Furthermore, our data do not allow us to say whether other tyrosines in middle-T are phosphorylated in vitro. Hunter et al (12) have suggested that tyrosine 297 is phosphorylated based on sequencing data. Examination of the sequences surrounding the sites identified here does not reveal any consensus recognition sequence for the kinase.…”

“…Mapping data indicate that the phosphate present in this peptide is attached to tyrosine 250. Hunter and colleagues (12), using different methods, also concluded recently that tyrosine 250 is phosphorylated in vitro. These data imply that there are multiple in vitro phosphorylation sites on middle-T.…”

Polyoma middle tumor antigen interacts with SHC protein via the NPTY (Asn-Pro-Thr-Tyr) motif in middle tumor antigen.

“…However, two mutants, d123 and d11015, activated tyrosine kinases (6,7) and yet failed to transform, indicating that activation is not sufficient. One explanation that applies to d123 was found in the phosphorylation of MT. In complexes with tyrosine kinases, MT itself became phosphorylated at Tyr-315 and, to a lesser extent, at Tyr-250 and Tyr-322 (8)(9)(10). MTs with mutations at position 315, d123 (11), and Py1178T (12), or position 250, pT250 (13), were transformation defective.…”