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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.


Cumulative mutation of phosphorylation sites creates step-wise increases in proneural activity (a-d). a Schematic representation of the phospho-mutant series of constructs demonstrating the SP and/or TP sites that are mutated to AP sites in each. The series consists of cumulative mutation of sites, working from N to C termini. 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 = 59-77]) 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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Fig5: Cumulative mutation of phosphorylation sites creates step-wise increases in proneural activity (a-d). a Schematic representation of the phospho-mutant series of constructs demonstrating the SP and/or TP sites that are mutated to AP sites in each. The series consists of cumulative mutation of sites, working from N to C termini. 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 = 59-77]) 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: Our data is therefore consistent with a model in which proneural activity varies with the number rather than location of sites phosphorylated. However, the data presented above do not distinguish between either a linear model, whereby mutation of each additional phosphorylation site additively increases the neurogenic activity of NeuroD4, or a threshold model, whereby mutation of a particular number of sites is enough to trigger a dramatic increase in activity. In order to investigate these two possibilities, a further panel of cumulative mutants were made, where each mutant SP/TP site was additively mutated from the N terminus to generate 1S-A, 2S-A, 3T/S-A etc. (Fig. 5a). Both qRT-PCR analysis and semi-quantitative analysis of ectopic neurogenesis as judged by in situ hybridisation show that sequential introduction of additional mutations creates a step-wise increase in neural-β-tubulin expression (Fig. 5b-d). Taken together, our data support the linear model where it is the number not the location of SP/TP sites that controls the activity of NeuroD4. Under this model, NeuroD4 may act as a “rheostat” to sense the level of proline-directed kinase activity and translate this into propensity to drive neuronal differentiation.Fig. 5


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

Hardwick LJ, Philpott A - Neural Dev (2015)

Cumulative mutation of phosphorylation sites creates step-wise increases in proneural activity (a-d). a Schematic representation of the phospho-mutant series of constructs demonstrating the SP and/or TP sites that are mutated to AP sites in each. The series consists of cumulative mutation of sites, working from N to C termini. 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 = 59-77]) 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

Fig5: Cumulative mutation of phosphorylation sites creates step-wise increases in proneural activity (a-d). a Schematic representation of the phospho-mutant series of constructs demonstrating the SP and/or TP sites that are mutated to AP sites in each. The series consists of cumulative mutation of sites, working from N to C termini. 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 = 59-77]) 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: Our data is therefore consistent with a model in which proneural activity varies with the number rather than location of sites phosphorylated. However, the data presented above do not distinguish between either a linear model, whereby mutation of each additional phosphorylation site additively increases the neurogenic activity of NeuroD4, or a threshold model, whereby mutation of a particular number of sites is enough to trigger a dramatic increase in activity. In order to investigate these two possibilities, a further panel of cumulative mutants were made, where each mutant SP/TP site was additively mutated from the N terminus to generate 1S-A, 2S-A, 3T/S-A etc. (Fig. 5a). Both qRT-PCR analysis and semi-quantitative analysis of ectopic neurogenesis as judged by in situ hybridisation show that sequential introduction of additional mutations creates a step-wise increase in neural-β-tubulin expression (Fig. 5b-d). Taken together, our data support the linear model where it is the number not the location of SP/TP sites that controls the activity of NeuroD4. Under this model, NeuroD4 may act as a “rheostat” to sense the level of proline-directed kinase activity and translate this into propensity to drive neuronal differentiation.Fig. 5

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.