Novel binding of GTP to the phosphoprotein (P) of vesicular stomatitis virus.
The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the RNA polymerase (L) that transcribes the negative strand genome RNA into mRNAs both in vitro and in vivo. We have previously shown that the P protein of VSV, expressed in E. coli, is biologically inactive unless phosphorylated at specific serine residues by cellular casein kinase II (CKII). In the present study we present evidence that the P protein, in addition to being phosphorylated, binds covalently to GTP only when it is phosphorylated. Competition experiments show that ATP, ADP, GTP, and GDP can compete for the binding site(s) of GTP but not AMP, GMP, CTP, or UTP. Interestingly, once GTP is bound to P protein it cannot be displaced by unlabeled GTP. The GTP binding site has been mapped within the domain where the phosphorylation of P protein by CKII occurs. Finally, we show that phosphorylation negative P mutants P3A (P60A, P62A, P64A), P3E (P60E, P62E, P64E), and P3R (P60R, P62R, P64R) failed to bind to GTP, indicating that phosphorylation of P is indeed essential for binding to GTP. Although the precise role of binding of GTP to P is unclear, it appears that phosphorylation of P may initiate a structural change within the P protein allowing GTP to bind, thus manifesting biological function to the transcription factor.
Mathur
Manjula
M
Department of Virology, Lerner Research Institute, The Cleveland Clinic Foundation, OH 44195, USA.
Banerjee
Amiya K
AK
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D001665:Binding Sites; D047390:Casein Kinase II; D002460:Cell Line; D004926:Escherichia coli / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007313:Insecta; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015678:Viral Structural Proteins / Q000737:chemistry* / Q000378:metabolism*
C081250:P protein, Vesicular stomatitis virus; D010750:Phosphoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D012313:RNA; D006160:Guanosine Triphosphate; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases