Simian Virus 40 Large T Antigen Is Phosphorylated at Multiple Sites Clustered in Two Separate Regions

Abstract

The phosphorylation sites of simian virus 40 large T antigen were determined within the primary structure of the molecule. Exhaustive digestion of 32 P-labeled large T antigen with trypsin generated six major phosphopeptides which could be separated in a newly developed isobutyric acid-containing chromatography system. By partial tryptic digestion, large T antigen was cleaved into an amino-terminal fragment of 17,000 daltons and overlapping fragments from the carboxy-terminal region ranging in size between 71,000 and 13,000 daltons. The location of the phosphopeptides was then determined by fingerprint analyses of individual fragments. Their physical properties were analyzed by sizing on polyacrylamide gels and by sequential digestion and peptide mapping; their amino acid composition was determined by differential labeling with various amino acids. The amino-terminal 17,000-dalton fragment gave rise to only one phosphopeptide (phosphopeptide 3) that contained half of the phosphate label incorporated into large T antigen. It contained phosphoserine and phosphothreonine sites, all of which were clustered within a small segment between Cys 105 and Lys 127 . This segment contained five serines and two threonines. Among these, Ser 106 , Ser 123 , and Thr 124 were identified as phosphorylated residues; in addition, either one or both of Ser 111 and Ser 112 were phosphorylated. The neighboring residues, Ser 123 and Thr 124 , were found in three different phosphorylation states in that either Ser 123 or Thr 124 or both were phosphorylated. Phosphopeptides 1, 2, 4, 5, and 6 were all derived from a single fragment extending 26,000 daltons upstream from the carboxy terminus of large T antigen. Phosphopeptide 6 was identical with the previously determined phosphothreonine peptide phosphorylated at Thr 701 . Phosphopeptides 1, 2, 4, and 5 contained only serine-bound phosphate. Phosphopeptides 1, 2, and 4 represented overlapping peptides, all of which were phosphorylated at Ser 639 located next to a cluster of six acidic residues. In phosphopeptide 5, a large peptide ranging from Asn 653 to Arg 691 , at least two of seven serines were phosphorylated. Thus, large T antigen contains at least eight phosphorylation sites. Their clustering within two separate regions might correlate with structural and functional domains of this protein.