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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.


Similar levels of proneural activity are seen amongst paired site mutants (a-d). a Schematic representation of the paired site phospho-mutant NeuroD4 constructs. Two-cell stage embryos were unilaterally injected with 100 pg mRNA of the respective NeuroD4 construct and assayed at stage 18 for expression of neural-β-tubulin by qRT-PCR (B[n = 3]), or whole embryo ISH (C[n = 62-79]) with representative images shown in (d). Views are dorso-ventral with injected side to the right; (p < 0.05) = *; (p < 0.025) = **; (p < 0.0125) = ***
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Fig4: Similar levels of proneural activity are seen amongst paired site mutants (a-d). a Schematic representation of the paired site phospho-mutant NeuroD4 constructs. Two-cell stage embryos were unilaterally injected with 100 pg mRNA of the respective NeuroD4 construct and assayed at stage 18 for expression of neural-β-tubulin by qRT-PCR (B[n = 3]), or whole embryo ISH (C[n = 62-79]) with representative images shown in (d). Views are dorso-ventral with injected side to the right; (p < 0.05) = *; (p < 0.025) = **; (p < 0.0125) = ***

Mentions: To distinguish between these alternative possibilities, a second panel of phospho-mutant constructs were made in which pairs of adjacent SP or TP sites were mutated together (Fig. 4a) and assayed in Xenopus embryos as described above. All three paired mutants produce a similar six to seven fold increase in neural-β-tubulin expression that is significantly higher than that induced by WT NeuroD4, but still substantially lower than that induced by the full phospho-mutant 6T/S-A NeuroD4 (Fig. 4b-e). Therefore, mutation of paired residues enhances NeuroD4 proneural activity compared to single site mutations, but the location of these paired sites does not influence the level of NeuroD4 activity; collective mutation of all six sites is required for the full neurogenic activity.Fig. 4


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

Hardwick LJ, Philpott A - Neural Dev (2015)

Similar levels of proneural activity are seen amongst paired site mutants (a-d). a Schematic representation of the paired site phospho-mutant NeuroD4 constructs. Two-cell stage embryos were unilaterally injected with 100 pg mRNA of the respective NeuroD4 construct and assayed at stage 18 for expression of neural-β-tubulin by qRT-PCR (B[n = 3]), or whole embryo ISH (C[n = 62-79]) with representative images shown in (d). Views are dorso-ventral with injected side to the right; (p < 0.05) = *; (p < 0.025) = **; (p < 0.0125) = ***
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Similar levels of proneural activity are seen amongst paired site mutants (a-d). a Schematic representation of the paired site phospho-mutant NeuroD4 constructs. Two-cell stage embryos were unilaterally injected with 100 pg mRNA of the respective NeuroD4 construct and assayed at stage 18 for expression of neural-β-tubulin by qRT-PCR (B[n = 3]), or whole embryo ISH (C[n = 62-79]) with representative images shown in (d). Views are dorso-ventral with injected side to the right; (p < 0.05) = *; (p < 0.025) = **; (p < 0.0125) = ***
Mentions: To distinguish between these alternative possibilities, a second panel of phospho-mutant constructs were made in which pairs of adjacent SP or TP sites were mutated together (Fig. 4a) and assayed in Xenopus embryos as described above. All three paired mutants produce a similar six to seven fold increase in neural-β-tubulin expression that is significantly higher than that induced by WT NeuroD4, but still substantially lower than that induced by the full phospho-mutant 6T/S-A NeuroD4 (Fig. 4b-e). Therefore, mutation of paired residues enhances NeuroD4 proneural activity compared to single site mutations, but the location of these paired sites does not influence the level of NeuroD4 activity; collective mutation of all six sites is required for the full neurogenic activity.Fig. 4

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.