Limits...
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.


6T/S-A NeuroD4 shows enhanced protein stability and enhanced chromatin binding relative to WT NeuroD4 (a-d). Embryos were injected at the one cell stage with 200 pg mRNA encoding HA-tagged versions of either WT or 6T/S-A NeuroD4. a Western blot analysis on extracts prepared from stage 13 embryos, with tubulin as a loading control. b The density of the protein band for WT or 6T/S-A NeuroD4 was quantified and expressed relative to the tubulin loading control of each sample. Mean values are shown from independent duplicate samples with the standard error of the mean. c At stage 14, cross-linking and chromatin isolation were performed prior to western blot analysis as before. No HA-tagged NeuroD4 protein was detected in the uninjected embryos confirming antibody specificity, and no tubulin protein was detected in the chromatin fraction. d NeuroD4 protein bands were again quantified relative to tubulin or histone H3 bands for cytoplasmic and chromatin fractions respectively. Mean values are shown from independent duplicate samples with the standard error of the mean
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: 6T/S-A NeuroD4 shows enhanced protein stability and enhanced chromatin binding relative to WT NeuroD4 (a-d). Embryos were injected at the one cell stage with 200 pg mRNA encoding HA-tagged versions of either WT or 6T/S-A NeuroD4. a Western blot analysis on extracts prepared from stage 13 embryos, with tubulin as a loading control. b The density of the protein band for WT or 6T/S-A NeuroD4 was quantified and expressed relative to the tubulin loading control of each sample. Mean values are shown from independent duplicate samples with the standard error of the mean. c At stage 14, cross-linking and chromatin isolation were performed prior to western blot analysis as before. No HA-tagged NeuroD4 protein was detected in the uninjected embryos confirming antibody specificity, and no tubulin protein was detected in the chromatin fraction. d NeuroD4 protein bands were again quantified relative to tubulin or histone H3 bands for cytoplasmic and chromatin fractions respectively. Mean values are shown from independent duplicate samples with the standard error of the mean

Mentions: In order to compare protein levels in vivo, HA-tagged WT or 6T/S-A NeuroD4 mRNA was injected into one cell embryos and extracts were prepared from stage 13 embryos for western blot analysis (Fig. 7a-b). WT NeuroD4 protein migrates as a broad band whereas 6T/S-A NeuroD4 migrates as a single band, demonstrating that these six SP/TP sites that are mutated in the phospho-mutant account for the phosphorylation seen in the WT protein (as discussed for Fig. 1c). The density of the NeuroD4 protein bands were calculated relative to the respective tubulin loading control: Injection of the same amount of WT or 6T/S-A NeuroD4 mRNA results in a 1.5 fold increase in 6T/S-A NeuroD4 band compared to WT NeuroD4.Fig. 7


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

Hardwick LJ, Philpott A - Neural Dev (2015)

6T/S-A NeuroD4 shows enhanced protein stability and enhanced chromatin binding relative to WT NeuroD4 (a-d). Embryos were injected at the one cell stage with 200 pg mRNA encoding HA-tagged versions of either WT or 6T/S-A NeuroD4. a Western blot analysis on extracts prepared from stage 13 embryos, with tubulin as a loading control. b The density of the protein band for WT or 6T/S-A NeuroD4 was quantified and expressed relative to the tubulin loading control of each sample. Mean values are shown from independent duplicate samples with the standard error of the mean. c At stage 14, cross-linking and chromatin isolation were performed prior to western blot analysis as before. No HA-tagged NeuroD4 protein was detected in the uninjected embryos confirming antibody specificity, and no tubulin protein was detected in the chromatin fraction. d NeuroD4 protein bands were again quantified relative to tubulin or histone H3 bands for cytoplasmic and chromatin fractions respectively. Mean values are shown from independent duplicate samples with the standard error of the mean
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: 6T/S-A NeuroD4 shows enhanced protein stability and enhanced chromatin binding relative to WT NeuroD4 (a-d). Embryos were injected at the one cell stage with 200 pg mRNA encoding HA-tagged versions of either WT or 6T/S-A NeuroD4. a Western blot analysis on extracts prepared from stage 13 embryos, with tubulin as a loading control. b The density of the protein band for WT or 6T/S-A NeuroD4 was quantified and expressed relative to the tubulin loading control of each sample. Mean values are shown from independent duplicate samples with the standard error of the mean. c At stage 14, cross-linking and chromatin isolation were performed prior to western blot analysis as before. No HA-tagged NeuroD4 protein was detected in the uninjected embryos confirming antibody specificity, and no tubulin protein was detected in the chromatin fraction. d NeuroD4 protein bands were again quantified relative to tubulin or histone H3 bands for cytoplasmic and chromatin fractions respectively. Mean values are shown from independent duplicate samples with the standard error of the mean
Mentions: In order to compare protein levels in vivo, HA-tagged WT or 6T/S-A NeuroD4 mRNA was injected into one cell embryos and extracts were prepared from stage 13 embryos for western blot analysis (Fig. 7a-b). WT NeuroD4 protein migrates as a broad band whereas 6T/S-A NeuroD4 migrates as a single band, demonstrating that these six SP/TP sites that are mutated in the phospho-mutant account for the phosphorylation seen in the WT protein (as discussed for Fig. 1c). The density of the NeuroD4 protein bands were calculated relative to the respective tubulin loading control: Injection of the same amount of WT or 6T/S-A NeuroD4 mRNA results in a 1.5 fold increase in 6T/S-A NeuroD4 band compared to WT NeuroD4.Fig. 7

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.