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.
METHODS AND PRINCIPAL FINDINGS: 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.
CONCLUSION: 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.