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BACKGROUND: Neuronal differentiation is largely under the control of basic Helix-Loop-Helix (bHLH) proneural transcription factors that play key roles during development of the embryonic nervous system. In addition to well-characterised regulation of their expression, increasing evidence is emerging for additional post-translational regulation of proneural protein activity. Of particular interest is the bHLH proneural factor Neurogenin2 (Ngn2), which orchestrates progression from neural progenitor to differentiated neuron in several regions of the central nervous system. Previous studies have demonstrated a key role for cell cycle-dependent multi-site phosphorylation of Ngn2 protein at Serine-Proline (SP) sites for regulation of its neuronal differentiation activity, although the potential structural and functional consequences of phosphorylation at different regions of the protein are unclear.
RESULTS: Here we characterise the role of phosphorylation of specific regions of Ngn2 on the stability of Ngn2 protein and on its neuronal differentiation activity in vivo in the developing embryo, demonstrating clearly that the location of SP sites is less important than the number of SP sites available for control of Ngn2 activity in vivo. We also provide structural evidence that Ngn2 contains large, intrinsically disordered regions that undergo phosphorylation by cyclin-dependent kinases (cdks).
CONCLUSIONS: Phosphorylation of Ngn2 occurs in both the N- and C-terminal regions, either side of the conserved basic Helix-Loop-Helix domain. While these phosphorylation events do not change the intrinsic stability of Ngn2, phosphorylation on multiple sites acts to limit its ability to drive neuronal differentiation in vivo. Phosphorylated regions of Ngn2 are predicted to be intrinsically disordered and cdk-dependent phosphorylation of these intrinsically disordered regions contributes to Ngn2 regulation.
Figure 1. Phosphorylation at SP sites in both the N- and C-terminal regions regulates xNgn2 activity. (A)35S-labelled IVT xNgn2, or the indicated Serine-Proline to Alanine-Proline xNgn2 mutants, full phosphomutant (9S-AxNgn2), N-terminal region (NT-S-AxNgn2) or C-terminal region (CT-S-AxNgn2), were added to Xenopus laevis mitotic egg extracts and incubated at 21°C. Samples were taken at 0, 15, 30, 45, 60, 75, 90 and 120 mins and separated on 15% SDS-PAGE gels. Gels were analyzed by quantitative phosphorimaging analysis, calculating the average stabilization relative to wild-type xNgn2 within mitotic extract. Half-lives were calculated using first-order rate kinetics, and errors calculated using the Standard Error of the Mean (SEM). (B) Embryos were injected into 1 cell of 2 cells with the indicated amount of mRNA encoding GFP, xNgn2, CT-S-AxNgn2 or 9S-AxNgn2, injected side to the left. Embryos were fixed at stage 15 and subjected to in situ hybridization for neural ß-tubulin expression before being scored for increased neurogenesis on the injected side compared to the uninjected side on a scale of 0¿3 [38]. The experiment was performed in duplicate (n?=?17-37). (C) 1 cell-stage embryos were injected with 20 pg of mRNA encoding GFP, xNgn2, NT-S-AxNgn2, CT-S-AxNgn2 or 9S-AxNgn2, harvested at stage 15 and expression of xNeuroD analysed by qPCR (5 embryos per sample, n?=?3).
Figure 2. SP site availability semi-quantitatively controls neuronal differentiation activity of xNgn2in vivo. (A) Embryos were injected into 1 cell of 2 cells with 20 pg of mRNA encoding GFP, xNgn2, or the indicated mutant version of xNgn2 from the cumulative mutant series (see Additional file 2: Figure S2), injected side to the left. Embryos were fixed at stage 15 and subject to in situ hybridization for neural ß-tubulin expression. (B) Scoring of ectopic neurogenesis on the injected side of embryos from (A) on a scale of ?1 - +3 [38]. The experiment was performed in triplicate (n?=?47-87).
Figure 3. The terminal regions of Ngn2 are predicted to be intrinsically disordered. PONDR-FIT disorder predictions of (A) xNgn2 and (B) mNgn2. (C) DISOPRED2 and (D) FoldIndex disorder predictions of mNgn2.
Figure 4. Annotated1H,15N 2D spectra of phosphorylated15N-mNgn2 showing altered structural positions of phosphorylated residues. (A) Detail of overlayed 2D [1H, 15N] HSQC spectra of 15N mNgn2 phosphorylated with cycA/CDK2. Resonances corresponding to phosphorylated Ser and Thr residues are labelled. (B) Secondary Structure Propensity (SSP, [46]) along the mNgn2 sequence calculated based on the available CA and CB chemical shifts (Additional file 4: Table S1). SSP scores correspond to a calculated percentage of occupancy: the 0.1/-0.1 thresholds, here represented as red lines, being empirically proposed for significance. Regions with positive values are associated with a preferential ?-helical conformation, here presented as cylinders. Regions with negative values are associated with a preferential extended conformation, here presented as arrows.
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