The PSF.p54nrb complex is a novel Mnk substrate that binds the mRNA for tumor necrosis factor alpha.
To identify new potential substrates for the MAP kinase signal-integrating kinases (Mnks), we employed a proteomic approach. The Mnks are targeted to the translational machinery through their interaction with the cap-binding initiation factor complex. We tested whether proteins retained on cap resin were substrates for the Mnks in vitro, and identified one such protein as PSF (the PTB (polypyrimidine tract-binding protein)-associated splicing factor). Mnks phosphorylate PSF at two sites in vitro, and our data show that PSF is an Mnk substrate in vivo. We also demonstrate that PSF, together with its partner, p54(nrb), binds RNAs that contain AU-rich elements (AREs), such as those for proinflammatory cytokines (e.g. tumor necrosis factor alpha (TNFalpha)). Indeed, PSF associates specifically with the TNFalpha mRNA in living cells. PSF is phosphorylated at two sites by the Mnks. Our data show that Mnk-mediated phosphorylation increases the binding of PSF to the TNFalpha mRNA in living cells. These findings identify a novel Mnk substrate. They also suggest that the Mnk-catalyzed phosphorylation of PSF may regulate the fate of specific mRNAs by modulating their binding to PSF.p54(nrb).
Buxadé
Maria
M
Division of Molecular Physiology and the Dundee DD1 5EH, United Kingdom; Department of Biochemistry & Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada.
Morrice
Nick
N
Medical Research Council Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.
Krebs
Danielle L
DL
Department of Biochemistry & Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada.
Proud
Christopher G
CG
Division of Molecular Physiology and the Dundee DD1 5EH, United Kingdom; Department of Biochemistry & Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada. Electronic address: cgpr@interchange.ubc.ca.
D020413:3' Untranslated Regions / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D019169:Jurkat Cells; D034521:Nuclear Matrix-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D050810:Octamer Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000072670:PTB-Associated Splicing Factor; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D020413:3' Untranslated Regions; D004268:DNA-Binding Proteins; C083214:NONO protein, human; D034521:Nuclear Matrix-Associated Proteins; D050810:Octamer Transcription Factors; D000072670:PTB-Associated Splicing Factor; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014409:Tumor Necrosis Factor-alpha; D017346:Protein Serine-Threonine Kinases