Correction for Shin et al., Mol. Cell. Biol. 29 (18) 4878-4890.
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Molecular and Cellular Biology, September 2009, p. 4875-4877, Vol. 29, No. 18
0270-7306/09/$08.00+0     doi:10.1128/MCB.00972-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

COMMENTARY

Timing the Phox-Trot: Duration of Phox2a-Dependent Transcription Is Controlled by an Intramolecular Dephosphorylation/Phosphorylation Clock{triangledown}

Lee E. Eiden*

Section on Molecular Neuroscience, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892-4090

In this issue, Shin et al. (17) provide an analysis of phosphorylation of three key serine residues on the transcription factor Phox2a that allow the biphasic activation of p27kip1, leading to differentiation of CAD neuronal progenitor cells. The results suggest some interesting predictions about regulation of Phox-dependent differentiation in vivo and add rich detail to the signaling mechanisms underlying the parallel transition of neuronal progenitors from dividing to nondividing cells and from cells without a neurotransmitter identity to cells with a unique and definitive one.

A key feature of nervous system development is the specification of the type of neurotransmitter a given neuron or group of neurons will synthesize and release, contributing to the precise anatomical and neurochemical circuitry that defines functional neurobiological pathways in the brain and peripheral nervous system (7). For example, neurons that synthesize the catecholamine neurotransmitter norepinephrine are concentrated in a small cell group called the locus coeruleus within the brain stem. In the human brain, this group makes up only 13,000 or so (3) of about 100 billion neurons. Nevertheless, release of norepinephrine at the projections from this small group of cells throughout the brain and spinal cord controls and modulates processes as basic as blood pressure regulation and pain gating and as refined as attention shifting in response to the emotional content of visual and auditory inputs to the cerebral cortex (5). How this small group of cells comes to be so precisely specified and localized within the brain during development is therefore a fascinating question, answered in large part when Goridis and coworkers demonstrated in the mouse that the achaete-scute transcription factor, first shown to be important in specifying the noradrenergic lineage in the periphery (9), is also required for the development of noradrenergic neurons in the central nervous system (10) through induction of the paired family homeoprotein transcription factor Phox2a. Phox2a binds to the promoters of the genes encoding the norepinephrine biosynthetic enzymes tyrosine hydroxlyase (TH) and dopamine beta-hydroxylase (DBH), programming these neuronal progenitors as noradrenergic neurons (18).

Transcription factors that determine the chemical coding of neurons also participate in the exit from the cell cycle that is necessary for neuronal differentiation (8). The discovery within the last several years that Phox2a (also called Arix), in addition to binding to the TH and DBH gene promoters (18), controls expression of the cell cycle regulator p27kip1 (14) was of considerable interest in linking these two types of cellular events. The developmentally expressed morphogen BMP2, via induction of achaete-scute homologs (MASH in mice), and cyclic AMP (cAMP) elevation (in response to various Gs-coupled first messengers) synergize to induce transcription of the Phox2a gene (2, 12, 14). Additional cAMP-dependent signaling is then required for the activation of Phox2a to allow its coordinated binding to the promoters of genes for chemical coding, such as the DBH gene, and those for exit from the cell cycle, such as the p27kip1 gene. DBH gene transcription continues in the noradrenergic neuron for the lifetime of the animal. Induction of p27kip1, however, through uncharacterized Phox2a-dependent serine/threonine kinase- and phosphatase-dependent events, is required only transiently for regulation of cell cycle events. The molecular modifications of Phox2a that allow it to simultaneously serve these temporally distinct transcriptional roles of prolonged neurotransmitter production and transient cell cycle control have been an intriguing puzzle. The report by Shin et al. (17) provides an important clue as to how this occurs by demonstrating sequential regulated serine modifications of the Phox2a molecule in CAD cells, a cell line derived from the locus coeruleus that serves as a model for noradrenergic progenitor cells (15).

The Andrisani laboratory has previously developed a CAD cell line in which Phox2a is expressed under the control of a chemically inducible promoter. In this way, Phox2a expression can be controlled independently from the BMP2- and cAMP-dependent signaling that allows it to be expressed in neuronal progenitor cells in the first place. Thus, signaling effects of cAMP on the Phox2a protein itself can be elucidated independently of Phox2a transcription. Additionally, multiple Phox2a phosphorylation events and their effects on control of p27kip1 gene transcription could be analyzed by expression of Phox2a mutant proteins with phosphoacceptor sites selectively removed or rendered constitutively active, either singly or in combination. A major phosphoserine cluster at serines 202, 206, and 208 in Phox2a was identified by mass spectrometry, with S206 the most strongly phosphorylated residue. An antibody directed to phosphoserine 206 in Phox2a was then generated. Immunohistochemical detection of Phox2a phosphorylation at position 206, in conjunction with mutants of Phox2a without a serine phosphoacceptor residue at the 202 and 208 sites, revealed that cAMP signaling leads paradoxically (through activation of an as yet unidentified protein phosphoserine phosphatase) to dephosphorylation of S206. In this form (Figure 1), Phox2a binds to the p27kip1 gene promoter, enhancing p27kip1 gene transcription. Phox2a lacking a phosphate group at position 206 becomes a substrate for phosphorylation at yet another site within the Phox2a molecule, serine 153, but only after additional dephosphorylation of S202 and S208 within the cluster. The S153-phosphorylated, S202/S206/S208-dephosphorylated form of Phox2a is a molecule no longer capable of association with DNA and therefore no longer able to transactivate the p27kip1 promoter. This finding is consistent with previous work indicating that cAMP induces Phox2a binding to the p27kip1 promoter, transcription of the p27kip1 gene, and CAD and neural crest cell differentiation (4, 14). Mapping of cAMP, protein kinase A (PKA), and protein phosphatase regulation of Phox2a activation and p27kip1 gene transcription to specific serine residues on Phox2a has allowed a processive model for sequential activation and built-in deactivation of Phox2a regulation of the p27kip1 gene promoter to emerge (Fig. 1). This model provides an understanding of how cAMP signaling can turn on and then turn off the transcription of a gene responsible for neuronal progenitor differentiation, via the intrinsic properties of a single protein.


Figure 1
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  		FIG. 1. A Phox2a phosphorylation/dephosphorylation clock is one way in which Phox2a phosphorylation events might act as a timer for the duration of p274kip1 transcription during neuronal progenitor differentiation to noradrenergic neurons, as reported by Shin et al. (17).

Several questions about the role of cAMP signaling to Phox2a remain, and several new questions arise, with the model proposed by Shin and coworkers (17). First, although PKA is implicated strongly in the initial S206 dephosphorylation step, additional cAMP sensors may yet be involved in the overall process. cAMP-dependent gene regulation mediated by PKA generally waxes and wanes within minutes to an hour (1, 11), while forskolin-induced Ser206 dephosphorylation and p27kip1 transcriptional activation occur only after 4 hours. cAMP-dependent, PKA-independent signaling leading to neuronal differentiation over a several-hour period has been observed in the PC12 cell model (6, 16). The S206, S202, and S208 Phox2a phosphatases, as well as the protein kinases downstream of PKA (if not PKA itself) that mediate the actual phosphorylation of these serines as well as S153, have not yet been identified. What are the cell states during development in which Phox2a phosphorylation at S206 is not constitutive? What is the susceptibility of the Phox2a molecule with phosphoserine at 153 (Fig. 1) to further phosphorylation within the 202-to-208 cluster, and what would the effects of hyperphosphorylation on DNA binding and p27kip1 transcription be? Finally, how is the regulation of Phox2a phosphorylation at S206 relevant to Phox2a binding to the promoters of the genes, including the TH and DBH genes, that specify the noradrenergic neurotransmitter phenotype?

A number of laboratories have explored the biological relevance of phosphosignaling duration in the cell physiological sequelae of extracellular signal-regulated kinase signaling, for example; however, this involves interaction with a second signaling molecule, c-Fos (13). The report of Shin et al. (17) reveals a novel mechanism of phosphosignaling through a processive ‘intramolecular signaling pathway’ in which the duration of a cellular event, in this case p27kip1 gene transcription, is ‘timed’ by the kinetics of sequential phosphorylation/dephosphorylation events in a single protein. This leads to a final mechanistic question: what is the step in the Phox2a cAMP-dependent phosphorylation ‘cycle’ that determines the interval of p27kip1 transcription? Shin et al. hypothesize that this interval is set by the time of onset of S153 phosphorylation after initial dephosphorylation of S206. But what determines the interval between S206 dephosphorylation and S153 phosphorylation? Although there are several possibilities (Fig. 1), Shin et al. (17) favor a scenario in which the rate of S202/S208 dephosphorylation is controlled by isomerization of proline within the SPRLSPSPLP motif (S206 is in boldface); this is supported by evidence that serine phosphorylation of proline-rich motifs is influenced by the cis/trans proline conformation. In addition, the prolyl isomerase Pin1 preferentially recognizes phospho-Ser/Thr-Pro, rather than Ser-Thr-Pro as a substrate, and protein phosphatases such as PP2A exhibit cis/trans preference for proline-rich phospho-Ser substrates. This hypothesis is additionally attractive because Pin1 activity is highly cell cycle dependent, so that dynamic regulation of p27kip1 by Phox2a would cease after neuronal progenitor differentiation is complete.

The most pressing question, of course, is whether or not this processive mechanism is operative in primary neural crest or central noradrenergic progenitor cells in vivo. It can be anticipated that an answer will soon emerge. Model organisms with specific Phox2a phosphorylation mutants knocked in should subsequently provide important details in the overall picture of Phox2a regulation of the chemical coding of neurotransmission. In the meantime, the speculative mechanism shown in Fig. 1 and less speculatively in Fig. 9 of Shin et al. (17) raises interesting issues about the processive nature of second messenger signaling via protein phosphorylation. Pharmacological approaches to signal transduction that focus on when, and not only on if, signaling requires a given kinase or phosphatase will be useful in obtaining additional clues about how signaling molecules activated for different durations can "thread the needle" between proliferation and transformation or differentiation and programmed cell death by pulsatile activation/deactivation of transcription factors like Phox2a that control both cell-specific genes like the DBH gene and cell cycle regulators like p27kip1.


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FOOTNOTES
 
* Mailing address: Section on Molecular Neuroscience, 36 Convent Drive, MSC 4090, 9000 Rockville Pike, Bethesda, MD 20892-4090. Phone: (301) 496-4110. Fax: (301) 402-1748. E-mail: eidenl{at}mail.nih.gov Back

FOOTNOTES

{triangledown} Published ahead of print on 27 July 2009. Back

The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.

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AUTHOR BIOS

{zmb0180982680002}Lee E. Eiden, Ph.D., is the chief of the Section on Molecular Neuroscience, Laboratory of Cellular and Molecular Regulation, in the National Institute of Mental Health Intramural Research Program and a member of the editorial and bioinformatics boards of Science Signaling.

Molecular and Cellular Biology, September 2009, p. 4875-4877, Vol. 29, No. 18
0270-7306/09/$08.00+0     doi:10.1128/MCB.00972-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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