Ki‐1/57 interacts with PRMT1 and is a substrate for arginine methylation

• Published in: The FEBS journal Vol. 273; no. 17; pp. 3946 - 3961
• Main Authors: Passos, Dario O., Bressan, Gustavo C., Nery, Flavia C., Kobarg, Jörg
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.09.2006
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Antigens; Arginine - metabolism; Binding sites; Cell Line; cellular localization; Conserved Sequence; Deoxyribonucleic acid; Dimerization; DNA; Enzymes; Fluorescence; HeLa Cells; Humans; mapping; Methylation; Molecular Sequence Data; Myogenic Regulatory Factors - metabolism; post‐translational modification; Predictive Value of Tests; protein arginine methylation; Protein Interaction Mapping; Protein-Arginine N-Methyltransferases - metabolism; Proteins; regulatory protein; Repressor Proteins - metabolism; RNA-Binding Proteins - metabolism; Substrate Specificity; Substrates; Two-Hybrid System Techniques
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2006.05399.x

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.