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Mol. Cell. Biol. 29 (18): 4878-4890

Copyright © 2009 by the American Society for Microbiology. All rights reserved.

Time-Dependent Activation of Phox2a by the Cyclic AMP Pathway Modulates Onset and Duration of p27Kip1 Transcription{triangledown} ,{dagger}

Min Hwa Shin,1,3 Nirmala Mavila,1,3 Wen-Horng Wang,1,3 Sasha Vega Alvarez,1,3 Mark C. Hall,2,3, and Ourania M. Andrisani1,3*

Department of Basic Medical Sciences,1 Department of Biochemistry,2 Center for Cancer Research, Purdue University, West Lafayette, Indiana 479073

Received for publication 19 December 2008. Revision received 1 February 2009. Accepted for publication 18 June 2009.

Abstract: In noradrenergic progenitors, Phox2a mediates cell cycle exit and neuronal differentiation by inducing p27Kip1 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 p27Kip1 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 p27Kip1 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 p27Kip1 transcription.


* Corresponding author. Mailing address: Department of Basic Medical Sciences, Purdue University, 625 Harrison Street, West Lafayette, IN 47907-2026. Phone: (765) 494-8131. Fax: (765) 494-0781. E-mail: andrisao{at}purdue.edu

{triangledown} Published ahead of print on 29 June 2009.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.



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