Peptides Containing Cyclin/Cdk-Nuclear Localization Signal Motifs Derived from Viral Initiator Proteins Bind to DNA When Unphosphorylated

Abstract

A single phosphorylation event at T-antigen residue Thr124 regulates initiation of simian virus 40 DNA replication. To explore this regulatory process, a series of peptides were synthesized, centered on Thr124. These peptides contain a nuclear localization signal (NLS) and a recognition site for cyclin/Cdk kinases. When unphosphorylated, the "CDK/NLS" peptides inhibit T-antigen assembly and bind non-sequence specifically to DNA. However, these activities are greatly reduced upon phosphorylation of Thr124. Similar results were obtained by using peptides derived from the CDK/NLS region of bovine papillomavirus E1. Related studies indicate that residues in the NLS bind to DNA, whereas those in the CDK motif regulate binding. These findings are discussed in terms of the control of T-antigen double hexamer assembly and initiation of viral replication.Cell cycle-dependent phosphorylation events regulate the initiation of DNA synthesis in eukaryotes (18,33,50). At the molecular level, however, relatively little is known about these processes. Therefore, model systems, such as that based on simian virus 40 (SV40), are being used to gain insights into phosphorylation-dependent regulation of DNA replication. Initiation of SV40 DNA replication depends upon the binding of the virally encoded T-antigen (T-ag) to the SV40 origin (reviewed in references 3, 17, 18, and 58). Upon binding to the origin, T-ag monomers assemble sequentially into hexamers and then double hexamers (13,15,42,(66)(67)(68). When assembled into a double hexamer, T-ag becomes a DNA helicase that is able to unwind the SV40 origin (4,14,25,59,62,71).In the regulation of SV40 replication, phosphorylation of T-ag on Thr124 is the sole posttranslational modification required for initiation of replication (44,57). Formation of the first hexamer is independent of the phosphorylation status of Thr124; therefore, the phosphorylation of Thr124 is required at a point subsequent to initial hexamer formation (1,45,47,55). Consistent with these studies, on subfragments of the core origin that support double-hexamer formation (35, 61), phosphorylation of Thr124 selectively promotes the formation of the second hexamer (1). The region of T-ag flanking Thr124 contains additional residues that play regulatory roles during SV40 DNA replication. For instance, T-ag residues 126 to 132 contain the nuclear localization signal (NLS) used for nuclear translocation (reviewed in references 22 and 27). Furthermore, the T-ag NLS is flanked by residues that serve as the recognition site for cyclin/Cdk kinases (16, 48), an arrangement found in many other proteins (reviewed in references 26 and 28)To further explore the regulation of T-ag assembly and initiation of viral replication, a series of peptides derived from the CDK/NLS region of T-ag, centered on Thr124, were synthesized. Initial studies revealed that certain of these peptides bind to DNA in a phosphate-dependent manner and interfered with the formation of T-ag hexamers and double hexamers. To establish whether these observatio...

Quantitative Analysis of the Binding of Simian Virus 40 Large T Antigen to DNA

“…This prompted Kim et al (26) to investigate if peptides containing Thr124 could bind DNA. Strikingly, peptides spanning aa 118 to 130 were found to bind nonspecifically to DNA and inhibit double hexamer assembly in vitro, but only when nonphosphorylated (26). These results provided the first evidence that the N-terminal domain may influence the affinity of the protein for DNA.…”

Stability and Function of JC Virus Large T Antigen and T′ Proteins Are Altered by Mutation of Their Phosphorylated Threonine 125 Residues

“…Kim and coworkers (12) proposed that during replication initiation, TAg utilizes an unphosphorylated CDK/NLS motif to bind the core replication origin and form the first of two hexamer structures. The CDK consensus signal in this motif includes the T124 residue.…”

“…However, mutation of this residue to alanine (T124A) disrupts formation of the second SV40 TAg hexamer and origin unwinding without preventing specific DNA binding activity (1,(17)(18)(19)38). Furthermore, mutation of T124 to aspartic acid (T124D) does not affect double-hexamer formation, but DNA binding of such as mutant is inefficient (12). These results suggest that T124 phosphorylation leads to changes in TAg-DNA interactions to facilitate structural changes required for hexamer formation, origin unwinding, and DNA replication (12).…”

“…Furthermore, mutation of T124 to aspartic acid (T124D) does not affect double-hexamer formation, but DNA binding of such as mutant is inefficient (12). These results suggest that T124 phosphorylation leads to changes in TAg-DNA interactions to facilitate structural changes required for hexamer formation, origin unwinding, and DNA replication (12).…”

“…Cell lysates were prepared at 0, 3,6,12,24, and 36 h, using EBC buffer containing protease and phosphatase inhibitors. TAg was immunoprecipitated with PAb 962 and electrophoresed in an SDS-15% polyacrylamide gel.…”