Bioinformatic analysis of OLIG2 using online services NetPhos 2.0 Server ( Blom et al., 1999) and NetPhosK 1.0 Server ( Blom et al., 2004) identified one potential PKA and one potential protein kinase C (PKC) phosphorylation site ( Table S1). Alignment of OLIG2 protein sequences from different species revealed that the predicted PKA site (R[R/K]X[S/T]) (X, any amino acid)—at S147 in the bHLH Helix-2 (H2)

domain—is conserved during evolution ( Figure 1B). This site is also conserved in OLIG1 but not in NGN1–3 or other bHLH proteins examined. We mutated S147 to p53 inhibitor Alanine (S147A), transfected the mutant (Olig2S147A) and wild-type (Olig2WT) constructs into Cos-7 cells, and visualized OLIG2 proteins by 2D PAGE and WB as before. This revealed an altered phosphorylation pattern of OLIG2S147A ( Figure 1D), indicating that S147 is a bone fide phosphate acceptor. Moreover, cotransfection of Olig2WT with a plasmid encoding a dominant-negative form of PKA (dnPKA) strongly reduced the phosphoprotein signal in the low pH range of GSK-3 beta phosphorylation the 2D gel (the region affected by S147A mutation), implying that OLIG2 can be phosphorylated by PKA on several sites, including S147 ( Figure 1E). To examine OLIG2-S147 phosphorylation status directly, we raised an antiserum in rabbits against a custom

phosphopeptide and purified an antibody fraction that specifically recognizes the S147-phosphorylated form of OLIG2 (anti-OLIG2 Cell press ph-S147; Figure S1). We prepared nuclear extracts of embryonic day 9.5 (E9.5), E11.5, and E13.5 mouse spinal cord tissue, immunoprecipitated endogenous OLIG2 protein with a goat anti-OLIG2 antibody, and visualized the precipitates by PAGE and WB with either rabbit anti-OLIG2 or rabbit anti-OLIG2 ph-S147. OLIG2 was expressed more or less equally at all stages examined (Figure 1G). The specific phosphorylated form OLIG2 ph-S147 was present at E9.5 and to a lesser extent at E11.5 (∼3.2-fold decrease; see Experimental Procedures) but was not detected at E13.5 (Figure 1H). These results demonstrate

that endogenous OLIG2 is phosphorylated on S147 during MN production (∼E9–12) but is later dephosphorylated, coinciding with the switch from MN to OLP production that occurs around E12.5 in mice (Pringle et al., 1996 and Richardson et al., 2006). Given the location of the S147 phosphorylation site in the bHLH domain, it seemed likely that the phosphorylation status of this site might affect the interactions of OLIG2 with other proteins or with DNA. OLIG2 can form strong homodimers with itself as well as heterodimers with OLIG1 but forms weak heterodimers with other bHLH proteins, such as E12 or NGN2 (Lee et al., 2005 and Li et al., 2007). It also interacts physically with other non-bHLH transcription factors, including SOX10, NKX2.