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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

Phospho-mutant 6T/S-A NeuroD4 has increased proneural activity relative to WT NeuroD4 (a-g). a Schematic representation of WT NeuroD4 and full phospho-mutant 6T/S-A NeuroD4 protein sequences, indicating the relative positions of the six SP or TP sites that have been mutated to AP sites. b-g Two-cell stage embryos were unilaterally injected with 100 pg of either WT or 6T/S-A NeuroD4 mRNA, and at stage 18, gene expression was assayed by qRT-PCR (b), or by whole mount ISH (d-g) with representative embryo images shown in (c). For qRT-PCR analysis (b) significance is calculated as described in the methods section for phospho-mutant NeuroD4 relative to WT NeuroD4 (blue adjoining lines and stars) and relative to uninjected control embryos (shown with black stars) [n = 5]; (p < 0.05) = *; (p < 0.025) = **; (p < 0.0125) = ***. For ISH analysis, embryos were scored according to the scale described in Additional file 1 for expression of neural-β-tubulin (D [n = 100–115]), p27Xic1 (E [n = 24-33]), xMyt1 (F [n = 26-32]), and xNeuroD1 (G [n = 23-28]). Views in (C [i-iii]) are dorso-ventral (DV); views in (C [iv]) are rostro-caudal (RC) with dorsal surface facing up, trigeminal ganglia indicated by arrows. All images show injected side to the right
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Fig2: Phospho-mutant 6T/S-A NeuroD4 has increased proneural activity relative to WT NeuroD4 (a-g). a Schematic representation of WT NeuroD4 and full phospho-mutant 6T/S-A NeuroD4 protein sequences, indicating the relative positions of the six SP or TP sites that have been mutated to AP sites. b-g Two-cell stage embryos were unilaterally injected with 100 pg of either WT or 6T/S-A NeuroD4 mRNA, and at stage 18, gene expression was assayed by qRT-PCR (b), or by whole mount ISH (d-g) with representative embryo images shown in (c). For qRT-PCR analysis (b) significance is calculated as described in the methods section for phospho-mutant NeuroD4 relative to WT NeuroD4 (blue adjoining lines and stars) and relative to uninjected control embryos (shown with black stars) [n = 5]; (p < 0.05) = *; (p < 0.025) = **; (p < 0.0125) = ***. For ISH analysis, embryos were scored according to the scale described in Additional file 1 for expression of neural-β-tubulin (D [n = 100–115]), p27Xic1 (E [n = 24-33]), xMyt1 (F [n = 26-32]), and xNeuroD1 (G [n = 23-28]). Views in (C [i-iii]) are dorso-ventral (DV); views in (C [iv]) are rostro-caudal (RC) with dorsal surface facing up, trigeminal ganglia indicated by arrows. All images show injected side to the right

Mentions: To determine the activity of NeuroD4 when phosphorylation on SP and TP sites is prevented, we generated a phospho-mutant version where all six SP/TP sites are mutated to alanine-proline (AP), creating 6T/S-A NeuroD4 (Fig. 2a). In order to compare their proneural activity, mRNA encoding WT and 6T/S-A NeuroD4 proteins were injected unilaterally into two-cell stage Xenopus embryos, and neural-β-tubulin expression was assayed at stage 18 (Fig. 2b-d). Consistent with previous reports [17, 23], over-expression of WT NeuroD4 produces a mild to moderate increase in neural-β-tubulin expression, seen in both the neural plate and lateral ectoderm of embryos. 6T/S-A NeuroD4 induces significantly greater ectopic neural-β-tubulin expression than that induced by WT NeuroD4, as judged by in situ hybridisation (ISH) and by qRT-PCR. Hence, preventing modification of SP/TP sites substantially enhances the proneural activity of NeuroD4, similar to our previous findings with Ngn2 and Ascl1 [14–16]. Phosphorylation on multiple SP and TP sites may therefore be a widely conserved mechanism for limiting the activity of bHLH proneural proteins.Fig. 2


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

Hardwick LJ, Philpott A - Neural Dev (2015)

Phospho-mutant 6T/S-A NeuroD4 has increased proneural activity relative to WT NeuroD4 (a-g). a Schematic representation of WT NeuroD4 and full phospho-mutant 6T/S-A NeuroD4 protein sequences, indicating the relative positions of the six SP or TP sites that have been mutated to AP sites. b-g Two-cell stage embryos were unilaterally injected with 100 pg of either WT or 6T/S-A NeuroD4 mRNA, and at stage 18, gene expression was assayed by qRT-PCR (b), or by whole mount ISH (d-g) with representative embryo images shown in (c). For qRT-PCR analysis (b) significance is calculated as described in the methods section for phospho-mutant NeuroD4 relative to WT NeuroD4 (blue adjoining lines and stars) and relative to uninjected control embryos (shown with black stars) [n = 5]; (p < 0.05) = *; (p < 0.025) = **; (p < 0.0125) = ***. For ISH analysis, embryos were scored according to the scale described in Additional file 1 for expression of neural-β-tubulin (D [n = 100–115]), p27Xic1 (E [n = 24-33]), xMyt1 (F [n = 26-32]), and xNeuroD1 (G [n = 23-28]). Views in (C [i-iii]) are dorso-ventral (DV); views in (C [iv]) are rostro-caudal (RC) with dorsal surface facing up, trigeminal ganglia indicated by arrows. All images show injected side to the right
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4494719&req=5

Fig2: Phospho-mutant 6T/S-A NeuroD4 has increased proneural activity relative to WT NeuroD4 (a-g). a Schematic representation of WT NeuroD4 and full phospho-mutant 6T/S-A NeuroD4 protein sequences, indicating the relative positions of the six SP or TP sites that have been mutated to AP sites. b-g Two-cell stage embryos were unilaterally injected with 100 pg of either WT or 6T/S-A NeuroD4 mRNA, and at stage 18, gene expression was assayed by qRT-PCR (b), or by whole mount ISH (d-g) with representative embryo images shown in (c). For qRT-PCR analysis (b) significance is calculated as described in the methods section for phospho-mutant NeuroD4 relative to WT NeuroD4 (blue adjoining lines and stars) and relative to uninjected control embryos (shown with black stars) [n = 5]; (p < 0.05) = *; (p < 0.025) = **; (p < 0.0125) = ***. For ISH analysis, embryos were scored according to the scale described in Additional file 1 for expression of neural-β-tubulin (D [n = 100–115]), p27Xic1 (E [n = 24-33]), xMyt1 (F [n = 26-32]), and xNeuroD1 (G [n = 23-28]). Views in (C [i-iii]) are dorso-ventral (DV); views in (C [iv]) are rostro-caudal (RC) with dorsal surface facing up, trigeminal ganglia indicated by arrows. All images show injected side to the right
Mentions: To determine the activity of NeuroD4 when phosphorylation on SP and TP sites is prevented, we generated a phospho-mutant version where all six SP/TP sites are mutated to alanine-proline (AP), creating 6T/S-A NeuroD4 (Fig. 2a). In order to compare their proneural activity, mRNA encoding WT and 6T/S-A NeuroD4 proteins were injected unilaterally into two-cell stage Xenopus embryos, and neural-β-tubulin expression was assayed at stage 18 (Fig. 2b-d). Consistent with previous reports [17, 23], over-expression of WT NeuroD4 produces a mild to moderate increase in neural-β-tubulin expression, seen in both the neural plate and lateral ectoderm of embryos. 6T/S-A NeuroD4 induces significantly greater ectopic neural-β-tubulin expression than that induced by WT NeuroD4, as judged by in situ hybridisation (ISH) and by qRT-PCR. Hence, preventing modification of SP/TP sites substantially enhances the proneural activity of NeuroD4, similar to our previous findings with Ngn2 and Ascl1 [14–16]. Phosphorylation on multiple SP and TP sites may therefore be a widely conserved mechanism for limiting the activity of bHLH proneural proteins.Fig. 2

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