Time-dependent activation of Phox2a by the cyclic AMP pathway modulates onset and duration of p27Kip1 transcription.
In noradrenergic progenitors, Phox2a mediates cell cycle exit and neuronal differentiation by inducing p27(Kip1) transcription in response to activation of the cyclic AMP (cAMP) pathway. The mechanism of cAMP-mediated activation of Phox2a is unknown. We identified a cluster of phosphoserine-proline sites in Phox2a by mass spectrometry. Ser206 appeared to be the most prominent phosphorylation site. A phospho-Ser206 Phox2a antibody detected dephosphorylation of Phox2a that was dependent on activation of the cAMP pathway, which occurred prior to neuronal differentiation of noradrenergic CAD cells. Employing serine-to-alanine and serine-to-aspartic acid Phox2a substitution mutants expressed in inducible CAD cell lines, we demonstrated that the transcriptional activity of Phox2a is regulated by two sequential cAMP-dependent events: first, cAMP signaling promotes dephosphorylation of Phox2a in at least one site, Ser206, thereby allowing Phox2a to bind DNA and initiate p27(Kip1) transcription; second, following dephosphorylation of the phosphoserine cluster (Ser202 and Ser208), Phox2a becomes phosphorylated by protein kinase A (PKA) on Ser153, which prevents association of Phox2a with DNA and terminates p27(Kip1) transcription. This represents a novel mechanism by which the same stimulus, cAMP signaling, first activates Phox2a by dephosphorylation of Ser206 and then, after a built-in delay, inactivates Phox2a via PKA-dependent phosphorylation of Ser153, thereby modulating onset and duration of p27(Kip1) transcription.
Shin
Min Hwa
MH
Department of Basic Medical Sciences, Purdue University, West Lafayette, IN 47907-2026, USA.
Mavila
Nirmala
N
Wang
Wen-Horng
WH
Vega Alvarez
Sasha
S
Hall
Mark C
MC
Andrisani
Ourania M
OM
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002454:Cell Differentiation; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007381:Intermediate Filament Proteins / Q000378:metabolism; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D064531:Peripherins; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014158:Transcription, Genetic
D018398:Homeodomain Proteins; D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D064531:Peripherins; D012333:RNA, Messenger; D050760:Cyclin-Dependent Kinase Inhibitor p27; D010768:Phosphoserine; D004247:DNA; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000409:Alanine