Ki-1/57 interacts with PRMT1 and is a substrate for arginine methylation.
The human 57 kDa Ki-1 antigen (Ki-1/57) is a cytoplasmic and nuclear protein, associated with Ser/Thr protein kinase activity, and phosphorylated at the serine and threonine residues upon cellular activation. We have shown that Ki-1/57 interacts with chromo-helicase DNA-binding domain protein 3 and with the adaptor/signaling protein receptor of activated kinase 1 in the nucleus. Among the identified proteins that interacted with Ki-1/57 in a yeast two-hybrid system was the protein arginine-methyltransferase-1 (PRMT1). Most interestingly, when PRMT1 was used as bait in a yeast two-hybrid system we were able to identify Ki-1/57 as prey among 14 other interacting proteins, the majority of which are involved in RNA metabolism or in the regulation of transcription. We found that Ki-1/57 and its putative paralog CGI-55 have two conserved Gly/Arg-rich motif clusters (RGG/RXR box, where X is any amino acid) that may be substrates for arginine-methylation by PRMT1. We observed that all Ki-1/57 protein fragments containing RGG/RXR box clusters interact with PRMT1 and are targets for methylation in vitro. Furthermore, we found that Ki-1/57 is a target for methylation in vivo. Using immunofluorescence experiments we observed that treatment of HeLa cells with an inhibitor of methylation, adenosine-2',3'-dialdehyde (Adox), led to a reduction in the cytoplasmic immunostaining of Ki-1/57, whereas its paralog CGI-55 was partially redistributed from the nucleus to the cytoplasm upon Adox treatment. In summary, our data show that the yeast two-hybrid assay is an effective system for identifying novel PRMT arginine-methylation substrates and may be successfully applied to other members of the growing family of PRMTs.
Passos
Dario O
DO
Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, Campinas, Brazil.
Bressan
Gustavo C
GC
Nery
Flavia C
FC
Kobarg
Jörg
J
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D002460:Cell Line; D017124:Conserved Sequence; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D018006:Myogenic Regulatory Factors / Q000378:metabolism*; D011237:Predictive Value of Tests; D025941:Protein Interaction Mapping; D011484:Protein-Arginine N-Methyltransferases / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques
C500946:HABP4 protein, human; D018006:Myogenic Regulatory Factors; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C428129:SERBP1 protein, human; D001120:Arginine; C099988:PRMT1 protein, human; D011484:Protein-Arginine N-Methyltransferases