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Multi-site phosphorylation regulates NeuroD4 activity during primary neurogenesis: a conserved mechanism amongst proneural proteins.

Hardwick LJ, Philpott A - Neural Dev (2015)

Bottom Line: We have previously demonstrated that multi-site phosphorylation of two members of the proneural protein family, Ngn2 and Ascl1, limits their ability to drive neuronal differentiation when cyclin-dependent kinase levels are high, as would be found in rapidly cycling cells.Multi-site phosphorylation on serine/threonine-proline pairs is a widely conserved mechanism of limiting proneural protein activity, where it is the number of phosphorylated sites, rather than their location that determines protein activity.Hence, multi-site phosphorylation is very well suited to allow co-ordination of proneural protein activity with the cellular proline-directed kinase environment.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Cambridge, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, Cambridge, CB2 0XZ, UK. ljah2@cam.ac.uk.

ABSTRACT

Background: Basic Helix Loop Helix (bHLH) proneural transcription factors are master regulators of neurogenesis that act at multiple stages in this process. We have previously demonstrated that multi-site phosphorylation of two members of the proneural protein family, Ngn2 and Ascl1, limits their ability to drive neuronal differentiation when cyclin-dependent kinase levels are high, as would be found in rapidly cycling cells. Here we investigate potential phospho-regulation of proneural protein NeuroD4 (also known as Xath3), the Xenopus homologue of Math3/NeuroM, that functions downstream of Ngn2 in the neurogenic cascade.

Results: Using the developing Xenopus embryo system, we show that NeuroD4 is expressed and phosphorylated during primary neurogenesis, and this phosphorylation limits its ability to drive neuronal differentiation. Phosphorylation of up to six serine/threonine-proline sites contributes additively to regulation of NeuroD4 proneural activity without altering neuronal subtype specification, and number rather than location of available phospho-sites is the key for limiting NeuroD4 activity. Mechanistically, a phospho-mutant NeuroD4 displays increased protein stability and enhanced chromatin binding relative to wild-type NeuroD4, resulting in transcriptional up-regulation of a range of target genes that further promote neuronal differentiation.

Conclusions: Multi-site phosphorylation on serine/threonine-proline pairs is a widely conserved mechanism of limiting proneural protein activity, where it is the number of phosphorylated sites, rather than their location that determines protein activity. Hence, multi-site phosphorylation is very well suited to allow co-ordination of proneural protein activity with the cellular proline-directed kinase environment.

No MeSH data available.


Related in: MedlinePlus

NeuroD4 is expressed and phosphorylated during primary neurogenesis (A-C). a Protein sequence alignment for human, mouse and Xenopus NeuroD4 homologues using Clustal W. The conserved bHLH domain is indicated in green and SP/TP sites are highlighted in red. A consensus line is also shown below the alignment to indicate the degree of conservation of amino acids at each position: (*) denotes identical residues in all three sequences; (:) denotes highly conserved residues; (.) denotes weakly conserved residues. b Endogenous expression of NeuroD4 at stage 14 (i) and neural-β-tubulin at stage 17 (ii) was determined by whole mount ISH (white arrows correspond to zones of primary neurogenesis; TG = trigeminal ganglia). Dorso-ventral views, anterior up, stages as indicated. c Western Blot analysis of extracts from stage 13 embryos injected with an HA-tagged wild type (WT) NeuroD4 mRNA, either with or without protein phosphatase treatment. Tubulin was used as a loading control
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Fig1: NeuroD4 is expressed and phosphorylated during primary neurogenesis (A-C). a Protein sequence alignment for human, mouse and Xenopus NeuroD4 homologues using Clustal W. The conserved bHLH domain is indicated in green and SP/TP sites are highlighted in red. A consensus line is also shown below the alignment to indicate the degree of conservation of amino acids at each position: (*) denotes identical residues in all three sequences; (:) denotes highly conserved residues; (.) denotes weakly conserved residues. b Endogenous expression of NeuroD4 at stage 14 (i) and neural-β-tubulin at stage 17 (ii) was determined by whole mount ISH (white arrows correspond to zones of primary neurogenesis; TG = trigeminal ganglia). Dorso-ventral views, anterior up, stages as indicated. c Western Blot analysis of extracts from stage 13 embryos injected with an HA-tagged wild type (WT) NeuroD4 mRNA, either with or without protein phosphatase treatment. Tubulin was used as a loading control

Mentions: bHLH proteins are subdivided into families on the basis of sequence conservation, and NeuroD4 is grouped in the Atonal family, related to the Drosophila proneural gene atonal [18]. Species homologues of each proneural protein often show a high degree of sequence conservation in the bHLH domain that mediates DNA-binding and protein dimerisation, but N and C terminal domains can be significantly divergent in sequence [1]. hese regions are suggested to be unstructured domains and present prime sites for post translational modifications such as phosphorylation [19]. Protein sequence alignment for human (genbank accession number NP 067014.2), mouse (genbank accession number NP 031527.1) and Xenopus (genbank accession number NP 001081213.1) NeuroD4 homologues show not only the strong sequence conservation in the bHLH domain, but also sequence conservation in potential proline-directed kinase sites (serine or threonine followed by a proline, Fig. 1a) that conform to the basic consensus sites for phosphorylation by cyclin dependent kinases (cdks) [20]. In NeuroD4 there are four serine-proline (SP) sites and two threonine-proline (TP) sites; five out of six of these sites are highly conserved with the mouse and human proteins, while the mouse and human proteins have additional non-conserved TP sites in the C terminus that are not shared with the Xenopus protein (Fig. 1a).Fig. 1


Multi-site phosphorylation regulates NeuroD4 activity during primary neurogenesis: a conserved mechanism amongst proneural proteins.

Hardwick LJ, Philpott A - Neural Dev (2015)

NeuroD4 is expressed and phosphorylated during primary neurogenesis (A-C). a Protein sequence alignment for human, mouse and Xenopus NeuroD4 homologues using Clustal W. The conserved bHLH domain is indicated in green and SP/TP sites are highlighted in red. A consensus line is also shown below the alignment to indicate the degree of conservation of amino acids at each position: (*) denotes identical residues in all three sequences; (:) denotes highly conserved residues; (.) denotes weakly conserved residues. b Endogenous expression of NeuroD4 at stage 14 (i) and neural-β-tubulin at stage 17 (ii) was determined by whole mount ISH (white arrows correspond to zones of primary neurogenesis; TG = trigeminal ganglia). Dorso-ventral views, anterior up, stages as indicated. c Western Blot analysis of extracts from stage 13 embryos injected with an HA-tagged wild type (WT) NeuroD4 mRNA, either with or without protein phosphatase treatment. Tubulin was used as a loading control
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494719&req=5

Fig1: NeuroD4 is expressed and phosphorylated during primary neurogenesis (A-C). a Protein sequence alignment for human, mouse and Xenopus NeuroD4 homologues using Clustal W. The conserved bHLH domain is indicated in green and SP/TP sites are highlighted in red. A consensus line is also shown below the alignment to indicate the degree of conservation of amino acids at each position: (*) denotes identical residues in all three sequences; (:) denotes highly conserved residues; (.) denotes weakly conserved residues. b Endogenous expression of NeuroD4 at stage 14 (i) and neural-β-tubulin at stage 17 (ii) was determined by whole mount ISH (white arrows correspond to zones of primary neurogenesis; TG = trigeminal ganglia). Dorso-ventral views, anterior up, stages as indicated. c Western Blot analysis of extracts from stage 13 embryos injected with an HA-tagged wild type (WT) NeuroD4 mRNA, either with or without protein phosphatase treatment. Tubulin was used as a loading control
Mentions: bHLH proteins are subdivided into families on the basis of sequence conservation, and NeuroD4 is grouped in the Atonal family, related to the Drosophila proneural gene atonal [18]. Species homologues of each proneural protein often show a high degree of sequence conservation in the bHLH domain that mediates DNA-binding and protein dimerisation, but N and C terminal domains can be significantly divergent in sequence [1]. hese regions are suggested to be unstructured domains and present prime sites for post translational modifications such as phosphorylation [19]. Protein sequence alignment for human (genbank accession number NP 067014.2), mouse (genbank accession number NP 031527.1) and Xenopus (genbank accession number NP 001081213.1) NeuroD4 homologues show not only the strong sequence conservation in the bHLH domain, but also sequence conservation in potential proline-directed kinase sites (serine or threonine followed by a proline, Fig. 1a) that conform to the basic consensus sites for phosphorylation by cyclin dependent kinases (cdks) [20]. In NeuroD4 there are four serine-proline (SP) sites and two threonine-proline (TP) sites; five out of six of these sites are highly conserved with the mouse and human proteins, while the mouse and human proteins have additional non-conserved TP sites in the C terminus that are not shared with the Xenopus protein (Fig. 1a).Fig. 1

Bottom Line: We have previously demonstrated that multi-site phosphorylation of two members of the proneural protein family, Ngn2 and Ascl1, limits their ability to drive neuronal differentiation when cyclin-dependent kinase levels are high, as would be found in rapidly cycling cells.Multi-site phosphorylation on serine/threonine-proline pairs is a widely conserved mechanism of limiting proneural protein activity, where it is the number of phosphorylated sites, rather than their location that determines protein activity.Hence, multi-site phosphorylation is very well suited to allow co-ordination of proneural protein activity with the cellular proline-directed kinase environment.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Cambridge, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, Cambridge, CB2 0XZ, UK. ljah2@cam.ac.uk.

ABSTRACT

Background: Basic Helix Loop Helix (bHLH) proneural transcription factors are master regulators of neurogenesis that act at multiple stages in this process. We have previously demonstrated that multi-site phosphorylation of two members of the proneural protein family, Ngn2 and Ascl1, limits their ability to drive neuronal differentiation when cyclin-dependent kinase levels are high, as would be found in rapidly cycling cells. Here we investigate potential phospho-regulation of proneural protein NeuroD4 (also known as Xath3), the Xenopus homologue of Math3/NeuroM, that functions downstream of Ngn2 in the neurogenic cascade.

Results: Using the developing Xenopus embryo system, we show that NeuroD4 is expressed and phosphorylated during primary neurogenesis, and this phosphorylation limits its ability to drive neuronal differentiation. Phosphorylation of up to six serine/threonine-proline sites contributes additively to regulation of NeuroD4 proneural activity without altering neuronal subtype specification, and number rather than location of available phospho-sites is the key for limiting NeuroD4 activity. Mechanistically, a phospho-mutant NeuroD4 displays increased protein stability and enhanced chromatin binding relative to wild-type NeuroD4, resulting in transcriptional up-regulation of a range of target genes that further promote neuronal differentiation.

Conclusions: Multi-site phosphorylation on serine/threonine-proline pairs is a widely conserved mechanism of limiting proneural protein activity, where it is the number of phosphorylated sites, rather than their location that determines protein activity. Hence, multi-site phosphorylation is very well suited to allow co-ordination of proneural protein activity with the cellular proline-directed kinase environment.

No MeSH data available.


Related in: MedlinePlus