Determinants on simian virus 40 large T antigen are important for recognition and phosphorylation by casein kinase I

Abstract

Casein kinase I has been shown to phosphorylate Ser123 and possibly Thrl24, in simian virus 40 (SV40) large T antigen; the same sites are also modified in cultured cells incubated with 32Pi [Friedrich A. Grlsser, Karl H. Scheidtmann, Polygena T. Tuazon, Jolinda A. Traugh & Gernot Walter (1988) Virology 165, 13 -221. The peptide, A-D-S-Q-H-S-T-P-P, which corresponds to the amino acid sequence 11 8 -125 of SV40 large T antigen, was synthesized together with peptides containing changes in specific amino acid residues on either side of Ser123. These peptides were used as model substrates to determine the amino acids in the SV40 large T antigen important for recognition by casein kinase I. The native peptide identified above, with aspartate at the -4 position, was a poor substrate for casein kinase I in vitro. Peptides with acidic residues added at the -2 and -3 positions, preceding Ser123, were phosphorylated by casein kinase I with apparent K, values around 2 mM and V,,, values up to 500 pmol . min-. ml-'. When acidic residues were added at both sides of the phosphorylatable serine, the peptide had a first-order rate constant over 20-fold higher than peptides with acidic amino acid residues at the N-terminus only; the apparent K, value was 0.65 mM with a V,,, of 2900 pmol . min-' . ml-'. The effects of modifying Serl20 to phosphoserine were examined by addition of a recognition sequence for the CAMP-dependent protein kinase prior to Serl20. Prior phosphorylation of the peptide at Serl20 lowered the apparent K, to 0.061 mM and increased the V,,, to 360 pmol . min-' . ml-', a 50-fold decrease in K , for casein kinase I and a 6-fold increase in V,,, as compared to the non-phosphorylated peptide. This indicates that Serl20, which has been shown to be phosphorylated in vivo, provides an appropriate recognition determinant for casein kinase I.Casein kinase I is a second-messenger-independent protein kinase which phosphorylates serine and threonine residues in an acidic environment [l, 21. Casein kinase I is a monomer and is present in the nucleus, cytoplasm and membrane of all eukaryotic cells examined. The enzyme from reticulocytes has a molecular mass ranging over 34-37 kDa, depending on the Early studies examining phosphorylation of casein variants by casein kinase I, illustrated the effectiveness of glutamate residues at the -2 position preceding the phosphorylatable serine for recognition by casein kinase I [5]. However, the sites phosphorylated on SV40 large T antigen and glycogen synthase by casein kinase I lack an acidic residue at the -2 position. For glycogen synthase, the critical residue has been shown to be serine phosphate at the -3 position relative to the phosphorylatable serine [6]. Phosphorylation In this study, a number of synthetic peptides related to the region containing Serl23 from SV40 large T antigen were examined to further define the determinants recognized by casein kinase I. The kinetics of phosphorylation were studied to determine the relative effectiveness of acidic residues at the -2 an...

Phosphorylation of the Proteins of the Extracellular Matrix of Mineralized Tissues By Casein Kinase-Like Activity

“…The peptide DDDDVASLPGLRRR has been used as a specific CK1 substrate (Flotow et al, 1990). Similar sequences are found in P53, MEESQSDISLELP (Milne et al, 1992), Inhibitor-2, GDDDDAYSDTET (Flotow et al, 1990), and V40 large antigen, ADSQHSTPP (Umphress et al, 1992). Two sequences which represent hierarchical prior phosphorylation by a different kinase are in ox2s-casein, LS(P)TS(P)EENSKK (Meggio et al, 1978), and glycogen synthase, RTLS(P)VASLPGL (Flotow et al, 1990).…”

Control of simian virus 40 DNA replication by the HeLa cell nuclear kinase casein kinase I.

“…Casein kinase I is a serine/threonine kinase which recognizes sites that have several acidic residues amino-terminal to the target residue; for example, serine in the peptide DDDDVASLPGLRRR (2,16). Significantly, phosphoserine in the sequence S(P)XXS [where S(P) is phosphoserine and X is any amino acid] also creates a casein kinase I recognition site, indicating that casein kinase I activity on certain substrates can be regulated by the phosphorylation state of the substrate (14,15,42). Casein kinase I recognition sites in T antigen identified by inspection of the sequence include serine 639 in the sequence DDDDEDS(639) and serine 679 in the sequence SSQS(679).…”

“…Protein phosphorylation has emerged as an important phorylates T antigen on serine 120 and/or 123, as well as regulatory mechanism in nuclear as well as cytoplasmic several carboxy-terminal sites (17,42); no functional effects processes. Cellular protein kinases and phosphatases particof these kinases on T-antigen activity have been demonipate in, among other events, the regulation of differentiastrated.…”

Nuclear protein phosphorylation and growth control

“…Casein kinase I phosphorylates Ser-123 in vitro (Grasser et al, 1988). Phosphorylation at this residue may depend on prior phosphorylation at Ser-120 to generate a recognition determinant (Flotow et al, 1990;Scheidtmann et al, 199 la;Umpress et al, 1992), but the kinase which phosphorylates Ser-120 is not known. Thr-124 is phosphorylated by the cell cycle-regulated kinase cdkl (p34cdc2) in vitro (McVey et al, 1989) and Ser-677 has recently been shown to be phosphorylated in vitro by dsDNA-PK (Lees-Miller et al, 1990;Chen et al, 1991).…”