title: Phosphorylation status of human RNA-binding protein 8A in cells and its inhibitory regulation by Magoh.
abstract: The RNA-binding protein 8A (RBM8A)-mago-nashi homolog, proliferation-associated (Magoh) complex is a component of the exon junction complex (EJC) required for mRNA metabolism involving nonsense-mediated mRNA decay (NMD). RBM8A is a phosphorylated protein that plays some roles in NMD. However, the detailed status and mechanism of the phosphorylation of RBM8A is not completely understood. Therefore, in this study, we analyzed in detail RBM8A phosphorylation in human cells. Accordingly, analysis of the phosphorylation status of RBM8A protein in whole-cell lysates by using Phos-tag gels revealed that the majority of endogenous RBM8A was phosphorylated throughout the cell-cycle progression. Nuclear and cytoplasmic RBM8A and RBM8A in the EJC were also found to be mostly phosphorylated. We also screened the phosphorylated serine by mutational analysis using Phos-tag gels to reveal modifications of serine residues 166 and 168. A single substitution at position 168 that concomitantly abolished the phosphorylation of serine 166 suggested the priority of kinase reaction between these sites. Furthermore, analysis of the role of the binding protein Magoh in RBM8A phosphorylation revealed its inhibitory effect in vitro and in vivo. Thus, we conclude that almost all synthesized RBM8A proteins are rapidly phosphorylated in cells and that phosphorylation occurs before the complex formation with Magoh.
authors.affiliation: Medical Research Institute, Kanazawa Medical University, Uchinada, Kahoku, Ishikawa 920-0293, Japan ishigaki@kanazawa-med.ac.jp.
authors.forename: Yasuhito
authors.initials: Y
authors.lastname: Ishigaki
authors.affiliation: Medical Research Institute, Kanazawa Medical University, Uchinada, Kahoku, Ishikawa 920-0293, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Nakamura
authors.affiliation: Medical Research Institute, Kanazawa Medical University, Uchinada, Kahoku, Ishikawa 920-0293, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Tatsuno
authors.affiliation: Medical Research Institute, Kanazawa Medical University, Uchinada, Kahoku, Ishikawa 920-0293, Japan.
authors.forename: Shaofu
authors.initials: S
authors.lastname: Ma
authors.affiliation: Medical Research Institute, Kanazawa Medical University, Uchinada, Kahoku, Ishikawa 920-0293, Japan Medical Care Proteomics Biotechnology Co., Ltd., Uchinada-machi, Kahoku 920-0293, Japan.
authors.forename: Naohisa
authors.initials: N
authors.lastname: Tomosugi
mesh_terms: D002478:Cells, Cultured; D005260:Female; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D009687:Nuclear Proteins / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D002583:Uterine Cervical Neoplasms / Q000378:metabolism* / Q000473:pathology
chemical_list: C491494:MAGOH protein, human; D009687:Nuclear Proteins; C406243:RBM8A protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012694:Serine; C087728:SRPK1 protein, human; D017346:Protein Serine-Threonine Kinases
keywords: Magoh; Phos-tag gel; RBM8A(Y14); mRNA; phosphorylation