Editors' ChoiceCell Biology

cAMP Signaling Timer

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Science Signaling  08 Sep 2009:
Vol. 2, Issue 87, pp. ec296
DOI: 10.1126/scisignal.287ec296

During neuronal development, precursor cells first cease dividing, then begin the process of differentiation, forming morphologically distinct processes and producing the specific neurotransmitter biosynthetic enzymes that will ultimately define the mature neuron. Tissue culture models using stable cell lines have helped delineate the pathways that contribute to these developmental events in various types of neurons. Shin et al. investigated the mechanisms by which adenosine 3′-5′ monophosphate (cAMP) contributes to differentiation of the catecholaminergic cell line (CAD), which is derived from a mouse brain tumor. The transcription factor Phox2a, which stimulates the expression of the gene encoding the cell cycle inhibitor p27Kip1 and genes involved in norepinephrine biosynthesis, was known to be involved in CAD cell differentiation, as well as differentiation of catecholaminergic neurons in vivo (see Eiden). Mass spectrometry analysis of CAD cells expressing a tagged form of Phox2a revealed that the protein was phosphorylated in unstimulated cells at a serine cluster (Ser202, Ser206, Ser208), with Ser206 the most prominently phosphorylated site. Analysis of the phosphorylation of the Ser206 site (with phosphospecific antibodies in CAD cells expressing tagged wild-type Phox2a under the control of a tetracycline-repressible system) revealed that forskolin, which activates adenylyl cyclase to increase cAMP concentrations, triggered a transient decrease in Ser206 phosphorylation and a similarly timed increase in the mRNA encoding p27Kip1 4 hours after forskolin application, both of which returned to basal amounts by 12 hours. Noting a protein kinase A (PKA) consensus phosphorylation site at position Ser153, the authors tested whether this site was involved in the termination of the Phox2a activity and verified with in vitro kinase assays that this site was phosphorylated by PKA. From analyzing the kinetics of phosphorylation of Phox2a, changes in the mRNA abundance for p27Kip1, and the formation of neurites in response to forskolin, the authors propose that cAMP signaling first triggers dephosphorylation of the Ser206 site, which is necessary for Phox2a to transcriptionally activate p27Kip1; then dephosphorylation of the Ser202 and Ser208 sites allows cAMP-stimulated PKA to phosphorylate the inhibitory site at Ser153, thereby terminating DNA binding and transcriptional activity of Phox2a. Chromatin immunoprecipitation experiments with the various mutants and the wild-type Phox2a were consistent with this model of cAMP-mediated timer that both activates and then deactivates Phox2a to control its activity at the promoter of the gene encoding p27Kip1.

M. H. Shin, N. Mavila, W.-H. Wang, S. V. Alvarez, M. C. Hall, O. M. Andrisani, Time-dependent activation of Phox2a by the cyclic AMP pathway modulates onset and duration of p27Kip1 transcription. Mol. Cell. Biol. 29, 4878–4890 (2009). [Abstract] [Full Text]

L. E. Eiden, Timing the Phox-trot: Duration of Phox2a-dependent transcription is controlled by an intramolecular dephosphorylation/phosphorylation clock. Mol. Cell. Biol. 29, 4875–4877 (2009). [Full Text]

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