Cofactor-activated phosphorylation is required for inhibition of cortical neuron differentiation by Groucho/TLE1.
BACKGROUND
Transcriptional co-repressors of the Groucho/transducin-like Enhancer of split (Gro/TLE) family regulate the expression of a variety of genes and are involved in numerous developmental processes in both invertebrate and vertebrate species. More specifically, Gro/TLE1 participates in mechanisms that inhibit/delay the differentiation of cerebral cortex neural progenitor cells into neurons during mammalian forebrain development. The anti-neurogenic function of Gro/TLE1 depends on the formation of protein complexes with specific DNA-binding transcription factors that engage Gro/TLE1 through WRP(W/Y) sequences. Interaction with those transcription partners results in Gro/TLE1 recruitment to selected DNA sites and causes increased Gro/TLE1 phosphorylation. The physiological significance of the latter event, termed "cofactor-activated phosphorylation," had not been determined. Therefore, this study aimed at clarifying the role of cofactor-activated phosphorylation in the anti-neurogenic function of Gro/TLE1.


RESULTS
A combination of site-directed mutagenesis, mass spectrometry, biochemistry, primary cell culture, and immunocytochemical assays was utilized to characterize point mutations of Ser-286, a residue that is phosphorylated in vivo and is located within the serine/proline-rich (SP) domain of Gro/TLE1. Mutation of Ser-286 to alanine or glutamic acid does not perturb the interaction of Gro/TLE1 with DNA-binding partners, including the basic helix-loop-helix transcription factor Hes1, a prototypical anti-neurogenic WRP(W/Y) motif protein. Ser-286 mutations do not prevent the recruitment of Gro/TLE1 to DNA, but they impair cofactor-activated phosphorylation and weaken the interaction of Gro/TLE1 with chromatin. These effects are correlated with an impairment of the anti-neurogenic activity of Gro/TLE1. Similar results were obtained when mutations of Ser-289 and Ser-298, which are also located within the SP domain of Gro/TLE1, were analyzed.


CONCLUSIONS
Based on the positive correlation between Gro/TLE1 cofactor-activated phosphorylation and ability to inhibit cortical neuron differentiation, we propose that hyperphosphorylation induced by cofactor binding plays a positive role in the regulation of Gro/TLE1 anti-neurogenic activity.
Buscarlet
Manuel
M
Centre for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Canada.
Hermann
Robert
R
Lo
Rita
R
Tang
Yeman
Y
Joachim
Kerline
K
Stifani
Stefano
S
D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002540:Cerebral Cortex / Q000166:cytology*; D002843:Chromatin / Q000378:metabolism; D056970:Co-Repressor Proteins; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D055495:Neurogenesis; D009474:Neurons / Q000166:cytology*; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D017354:Point Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
D002843:Chromatin; D056970:Co-Repressor Proteins; D010455:Peptides; D012097:Repressor Proteins; C499043:TLE1 protein, human; D014157:Transcription Factors; D012694:Serine