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.

Characterization of a zinc finger DNA-binding protein expressed specifically in Petunia petals and seedlings.

“…A cluster of basic amino acids which may be a nuclear targeting signal is located just upstream of the serine/threonine-rich sequence. A close association of nuclear targeting sequence and phosphorylation sites has been reported for some DNAbinding proteins (Scheidtmann et al, 1982). Many activator proteins have been demonstrated to have characteristic activation domains.…”

“…Preparation of nuclear extracts, DNase I footprinting and gel shift assays Nuclear extracts from Petunia petal were prepared essentially as described by Green et al (1987) except that the petals were frozen in liquid nitrogen and ground in a prechilled mortar on dry ice by a modification of a procedure used for nuclear run-on assays (Scheidtmann et al, 1982). The use of frozen material is crucial for Petunia because its nuclei are so fragile that they are easily broken when unfrozen material is used.…”

DNA helicase activity of SV40 large tumor antigen.

“…It is known that several subgroups of T antigen exist in lytically infected cells. These subgroups are defined by differential modifications of amino acid side chains including ADP-ribosylation (Goldman et al, 1981) and phosphorylation (Scheidtmann et al, 1982). Furthermore, a substantial fraction of T antigen is converted some time after its synthesis to tetramers or higher aggre- gated forms (Fanning et al, 1981).…”

Regulation of SV40 DNA replication by phosphorylation of T antigen.

“…Peptide mapping has localized phosphorylated residues to two clusters of serine and threonine residues which reside on opposing ends of the linear polypeptide chain. The amino terminal cluster consists of Ser 106,111,112,123,and Thr 124, while the carboxy terminal region includes Ser 639,676,677,679,and Thr 701 (Scheidtmann et al, 1982). Although these phosphoamino acids have been chemically identified, their functional significance remains a mystery, the elucidation of which may very well prove important in understanding the multifunctional nature of this protein.…”