Protein kinase A enhances, whereas glycogen synthase kinase-3 beta inhibits, the activity of the exon 2-encoded transactivator domain of heterogeneous nuclear ribonucleoprotein D in a hierarchical fashion.
Heterogeneous nuclear ribonucleoprotein D (hnRNP D) is implicated in transcriptional regulation. Alternative splicing of exons 2 and 7 generates four isoforms of the protein. We report here that only isoforms that contain the product of exon 2 (amino acids 79-97) were able to transactivate. Moreover, the exon 2-encoded protein domain alone was sufficient to drive transcription. TATA-binding protein and p300 interacted with a synthetic peptide corresponding to exon 2, and both proteins co-precipitated with hnRNP D. Stimulation of protein kinase A (PKA) and protein kinase C (PKC) synergistically induced the transactivating ability of hnRNP D, and the exon 2-encoded domain was sufficient for this inducibility. In kinase assays PKA phosphorylated Ser-87 of hnRNP D, whereas glycogen synthase kinase-3 beta (GSK-3 beta) phosphorylated Ser-83, but only if Ser-87 had been pre-phosphorylated by PKA. Phosphorylation of Ser-87 enhanced, whereas phosphorylation of Ser-83 repressed, transactivation. Overexpression of GSK-3 beta inhibited transactivation by hnRNP D, but stimulation of PKC negated the inhibitory effect of GSK-3 beta. We suggest that a hierarchical phosphorylation pathway regulates the transactivating ability of hnRNP D: PKA activates hnRNP D, but at the same time renders it sensitive to inhibition by GSK-3 beta; the latter inhibition can be suspended by inactivating GSK-3 beta with PKC.
Tolnay
Mate
M
Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA. mtolnay@hotmail.com
Juang
Yuang-Taung
YT
Tsokos
George C
GC
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D005091:Exons; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D035181:TATA-Box Binding Protein; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D035181:TATA-Box Binding Protein; D015534:Trans-Activators; D014157:Transcription Factors; D012694:Serine; D038341:Glycogen Synthase Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3