Phosphorylation in intrinsically disordered regions regulates the activity of Neurogenin2.
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.
Department of Oncology, MRC/Hutchison Research Centre, University of Cambridge, Cambridge Biomedical Campus, Cambridge, CB2 0XZ, UK. Gary.McDowell@tufts.edu.|Current address: Center for Regenerative and Developmental Biology, Department of Biology, Tufts University, 200 Boston Avenue, Medford, MA, 02155, USA. Gary.McDowell@tufts.edu.
Gary S
GS
McDowell
Department of Oncology, MRC/Hutchison Research Centre, University of Cambridge, Cambridge Biomedical Campus, Cambridge, CB2 0XZ, UK. cjh87@cam.ac.uk.|Current address: The Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK. cjh87@cam.ac.uk.
Christopher J
CJ
Hindley
CNRS, Université de Lille 1, UMR 8576, Villeneuve d'Ascq, France. Guy.Lippens@univ-lille1.fr.
Guy
G
Lippens
CNRS, Université de Lille 1, UMR 8576, Villeneuve d'Ascq, France. Isabelle.Landrieu@univ-lille1.fr.
Isabelle
I
Landrieu
Department of Oncology, MRC/Hutchison Research Centre, University of Cambridge, Cambridge Biomedical Campus, Cambridge, CB2 0XZ, UK. ap113@cam.ac.uk.
Anna
A
Philpott
D000818:Animals; D064267:Intrinsically Disordered Proteins / Q000737:chemistry / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D055495:Neurogenesis; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D029867:Xenopus Proteins / Q000737:chemistry / Q000378:metabolism*; D014982:Xenopus laevis
D064267:Intrinsically Disordered Proteins; C553500:NEUROG2 protein, Xenopus; D009419:Nerve Tissue Proteins; D029867:Xenopus Proteins