The phosphorylation sites of simian virus 40 large T antigen were determined within the primary structure of the molecule. Exhaustive digestion of 32P-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 aminoterminal fragment of 17,000 daltons and overlapping fragments from the carboxyterminal region ranging in

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... 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 aminoterminal 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 Cyslo5 and Lys127. This segment contained five serines and two threonines. Among these, Ser,06, Ser123, and Thr124 were identified as phosphorylated residues; in addition, either one or both of Ser1ll and Ser,12 were phosphorylated. The neighboring residues, Ser123 and Thr124, were found in three different phosphorylation states in that either Ser123 or Thr,24 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 Thr701. 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 Ser639 located next to a cluster of six acidic residues. In phosphopeptide 5, a large peptide ranging from Asn653 to Arg691, 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. The simian virus 40 (SV40) large T antigen (large T) is a phosphoprotein of 82,000 daltons (82K) (36, 46). It plays a central role in the growth cycle of the virus as well as in virusinduced malignant transformation. During lytic infection large T initiates replication and regulates transcription of the viral genome (reviewed in reference 49). In vitro, large T binds to DNA, specifically to the regulatory region on the SV40 genome that includes the origin of DNA replication (19, 40, 45, 47) ; moreover, it is associated with an ATPase and a protein kinase activity (1, 17, 48) . How these different functions are exerted by a single protein is not understood, but it is possible that large T can assume different functional forms which are interconvertible by phosphor-ylation. Compatible with this possibility are the following findings: large T is phosphorylated at multiple sites (52) in a reversible fashion (9, 38); subclasses of large T can be separated by isoelectric focusing (15); and correlations exist between the degree of phosphorylation of large T on one hand and its degree of oligomerization (10, 16) or its affinity for DNA (31) on the other. However, the functional significance of these quantitative differences in the phosphorylation state is not yet clear. Montenarh and Henning reported that highly phosphorylated large T exhibits the strongest affinity for (calf thymus) DNA (31), whereas Shaw and Tegtmeyer found no difference between phosphorylated and dephosphorylated large T in its capacity to bind to the replication origin of the SV40 genome (41). 116 on May 8, 2020 by guest