Mechanisms of simian virus 40 T-antigen activation by phosphorylation of threonine 124.
Previous studies have shown that phosphorylation of simian virus 40 (SV40) T antigen at threonine 124 enhances the binding of T antigen to the SV40 core origin of replication and the unwinding of the core origin DNA via hexamer-hexamer interactions. Here, we report that threonine 124 phosphorylation enhances the interaction of T-antigen amino acids 1 to 259 and 89 to 259 with the core origin of replication. Phosphorylation, therefore, activates the minimal DNA binding domain of T antigen even in the absence of domains required for hexamer formation. Activation is mediated by only one of three DNA binding elements in the minimal DNA binding domain of T antigen. This element, including amino acids 167, 215, and 219, enhances binding to the unique arrangement of four pentanucleotides in the core origin but not to other pentanucleotide arrangements found in ancillary regions of the SV40 origin of replication. Interestingly, the same four pentanucleotides in the core origin are necessary and sufficient for phosphorylation-enhanced DNA binding. Further, we show that phosphorylation of threonine 124 promotes the assembly of high-order complexes of the minimal DNA binding domain of T antigen with core origin DNA. We propose that phosphorylation induces conformational shifts in the minimal DNA binding domain of T antigen and thereby enhances interactions among T-antigen subunits oriented by core origin pentanucleotides. Similar subunit interactions would enhance both assembly of full-length T antigen into binary hexamer complexes and origin unwinding.
McVey
D
D
Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, 11794-5222, USA.
Woelker
B
B
Tegtmeyer
P
P
D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004261:DNA Replication; D010766:Phosphorylation; D013539:Simian virus 40 / Q000276:immunology* / Q000502:physiology; D013912:Threonine / Q000378:metabolism*; D019065:Virus Assembly; D014779:Virus Replication
D000952:Antigens, Polyomavirus Transforming; D013912:Threonine; D004247:DNA