BRF1 protein turnover and mRNA decay activity are regulated by protein kinase B at the same phosphorylation sites.
BRF1 posttranscriptionally regulates mRNA levels by targeting ARE-bearing transcripts to the decay machinery. We previously showed that protein kinase B (PKB) phosphorylates BRF1 at Ser92, resulting in binding to 14-3-3 and impairment of mRNA decay activity. Here we identify an additional regulatory site at Ser203 that cooperates in vivo with Ser92. In vitro kinase labeling and wortmannin sensitivity indicate that Ser203 phosphorylation is also performed by PKB. Mutation of both serines to alanine uncouples BRF1 from PKB regulation, leading to constitutive mRNA decay even in the presence of stabilizing signals. BRF1 protein is labile because of proteasomal degradation (half-life, <3 h) but becomes stabilized upon phosphorylation and is less stable in PKBalpha(-/-) cells. Surprisingly, phosphorylation-dependent protein stability is also regulated by Ser92 and Ser203, with parallel phosphorylation required at these sites. Phosphorylation-dependent binding to 14-3-3 is abolished only when both sites are mutated. Cell compartment fractionation experiments support a model in which binding to 14-3-3 sequesters BRF1 through relocalization and prevents it from executing its mRNA decay activity, as well as from proteasomal degradation, thereby maintaining high BRF1 protein levels that are required to reinstate decay upon dissipation of the stabilizing signal.
Benjamin
Don
D
Institute for Medical Microbiology, Petersplatz 10, 4003 Basel, Switzerland.
Schmidlin
Martin
M
Min
Lu
L
Gross
Brigitte
B
Moroni
Christoph
C
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D051817:Butyrate Response Factor 1; D045744:Cell Line, Tumor; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D020871:RNA Stability / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D012694:Serine / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D035361:Transcription Factor TFIIIB
D051817:Butyrate Response Factor 1; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D035361:Transcription Factor TFIIIB; C488431:Zfp36l1 protein, mouse; D012694:Serine; D051057:Proto-Oncogene Proteins c-akt; D000409:Alanine