1999). Acknowledgments We thank Dr. rapidly upon the induction of myogenic differentiation. The presence of phosphorylation in a region of Pax3 important for mediating proteinCprotein interactions, and the fact that phosphorylation is lost upon induction of differentiation, allow for speculation on the biological relevance of phosphorylation. mice are embryonic lethal due to defects in skeletal muscle (Xia and Barr 2005), and human patients AZD3514 with haploinsufficiency display limb muscle hypoplasia (Epstein et al. 1996). It has been demonstrated that the ectopic expression of Pax3 is capable of activating the myogenic program in mesoderm tissue by activating the expression of the myogenic regulatory factors MyoD, Myf-5, and myogenin (Maroto et al. 1997). Because of the importance of Pax3 in early muscle development and in the expression of early myogenic genes, it is critical that the expression and activity of Pax3 be tightly regulated throughout differentiation. Recently, it was determined that the stability of Pax3 is regulated on a post-translational level during myogenic differentiation. We have shown that Pax3 protein levels decrease significantly in the first 24 h of myogenic differentiation and that this change in protein levels are regulated post-translationally since changes in mRNA levels and protein translation for Pax3 do not correlate with the decrease in Pax3 protein levels (Miller and Hollenbach 2007). Furthermore, it has been shown Rabbit Polyclonal to MOBKL2A/B that Pax3 stability is regulated, in part, via the ubiquitin-proteasome system (Boutet et al. 2007). In addition to the ubiquitin-proteasome system, it has been speculated that phosphorylation may also be important in the regulation of Pax3 biological activities (Boutet et al. 2007; Miller and Hollenbach 2007). Phosphorylation has been widely studied due to its various roles in transcription factor regulation (Hunter and Karin 1992). However, at present it is not known whether Pax3 is phosphorylated in a physiologically relevant cell type, nor have the sites of phosphorylation been identified. Therefore, in the present study, we demonstrate that Pax3 is indeed phosphorylated in proliferating mouse primary myoblasts. Furthermore, we use both in vitro and in vivo mapping techniques along with a phosphospecific antibody to identify Ser205 as a site of phosphorylation on Pax3 in proliferating mouse primary myoblasts. Finally, we demonstrate that Pax3 is only phosphorylated at Ser205 in proliferating mouse primary myoblasts and that the phosphorylation status of Pax3 changes rapidly upon the induction of myogenic differentiation. The fact that a site of phosphorylation occurs in a region of Pax3 required for mediating proteinCprotein interactions and that phosphorylation of Pax3 changes upon induction of differentiation allows for speculation into the role of this phosphorylation event in the regulation of AZD3514 Pax3 activities. Results Pax3 is phosphorylated in vivo In order to demonstrate that Pax3 is phosphorylated in a physiologically relevant cell type, we stably transduced proliferating mouse primary myoblasts AZD3514 with a retroviral construct containing a FLAG epitope tagged Pax3 (FLAG-Pax3) construct, metabolically labeled the cells with [32P]-orthophosphate or [35S]-Methionine, immunoprecipitated FLAG-Pax3 with a FLAG-specific antibody, and examined incorporated radiolabel by SDS-PAGE analysis. We observed the specific incorporation of both radiolabels into Pax3 (Fig. 1), demonstrating that Pax3 is both expressed and AZD3514 phosphorylated in proliferating primary myoblasts. This result provides the first evidence that Pax3 exists as a phosphoprotein in a physiologically relevant cell type. In AZD3514 order to further characterize the in vivo phosphorylation of Pax3, we generated a two-dimensional phosphopeptide map of FLAG-Pax3 that had been metabolically labeled with [32P]-orthophosphate. The phosphopeptide analysis demonstrates the presence of five distinct radiolabeled peptides, suggesting multiple sites of phosphorylation may be present (Fig. 2A). Open in a separate window Figure 1. Pax3 is phosphorylated in proliferating mouse primary myoblasts. Proliferating mouse primary myoblasts stably transduced with an amino-terminal FLAG epitope tagged Pax3 were metabolically labeled with either [35S]-Methionine or [32P]-orthophosphate, and FLAG-Pax3 was immunoprecipitated from total cell extracts using an anti-FLAG antibody, as described in Materials and Methods. The resulting immunoprecipitates were separated by 12% SDS-PAGE, and the radiolabeled species were detected by autoradiography. Open in a separate window Figure 2. Development of an in vitro kinase assay to facilitate the identification of phosphorylation sites on Pax3. (panel) Coomassie staining to demonstrate similar amounts of protein; (panel) independent radiolabeling to confirm the phosphorylation status; (panel) Western blot analysis using the anti-Pax3(p205) antibody. The arrow indicates the mobility of wild-type Pax3, while the asterisk indicates the.