The DEAD-box RNA helicase DDX3 interacts with DDX5, co-localizes with it in the cytoplasm during the G2/M phase of the cycle, and affects its shuttling during mRNP export.
DDX3 is involved in RNA transport, translational control, proliferation of RNA viruses, and cancer progression. From yeast two-hybrid screening using the C-terminal region of DDX3 as a bait, the DEAD-box RNA helicase DDX5 was cloned. In immunofluorescence analysis, DDX3 and DDX5 were mainly co-localized in the cytoplasm. Interestingly, cytoplasmic levels of DDX5 increased in the G(2) /M phase and consequently protein-protein interaction also increased in the cytoplasmic fraction. DDX3 was highly phosphorylated at its serine, threonine, and tyrosine residues in the steady state, but not phosphorylated at the serine residue(s) in the G(2) /M phase. DDX5 was less phosphorylated in the G(1) /S phase; however, it was highly phosphorylated at serine, threonine, and tyrosine residues in the G(2) /M phase. PP2A treatment of the cytoplasmic lysate from G(2) /M phase cells positively affected the interaction between DDX3 and DDX5, whereas, PTP1B treatment did not. In an analysis involving recombinant His-DDX3 and His-DDX5, PP2A pretreatment of His-DDX5 increased the interaction with endogenous DDX3, and vice versa. Furthermore, the results of GST pull-down experiments support the conclusion that dephosphorylation of serine and/or threonine residues in both proteins enhanced protein-protein interactions. UV cross-linking experiments showed that DDX3 and DDX5 are involved in mRNP export. Additionally, DDX3 knockdown blocked the shuttling of DDX5 to the nucleus. These data demonstrate a novel interaction between DDX3 and DDX5 through the phosphorylation of both proteins, especially in the G(2) /M phase, and suggest a novel combined mechanism of action, involving RNP remodeling and splicing, for DEAD-box RNA helicases involved in mRNP export.
Department of Biotechnology, Bioenergy Research Center, Chonnam National University, Gwangju 500-757, South Korea.
Yeo-Jin
YJ
Choi
Seong-Gene
SG
Lee
D021581:Active Transport, Cell Nucleus; D002453:Cell Cycle; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry; D053487:DEAD-box RNA Helicases / Q000032:analysis / Q000378:metabolism*; D016195:G2 Phase; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D012261:Ribonucleoproteins / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D012261:Ribonucleoproteins; C026452:messenger ribonucleoprotein; D010749:Phosphoprotein Phosphatases; C119332:DDX3X protein, human; C506749:Ddx5 protein, human; D053487:DEAD-box RNA Helicases