Miller Patrick J, Dietz Kevin N, Hollenbach Andrew D
Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
Protein Sci. 2008 Nov;17(11):1979-86. doi: 10.1110/ps.035956.108. Epub 2008 Aug 15.
Pax3, a member of the paired class homeodomain family of transcription factors, is essential for early skeletal muscle development. Previously, others and we have shown that the stability of Pax3 is regulated on a post-translational level. Evidence in the literature and from our laboratory suggests that phosphorylation, a common form of regulation, may play a role. However, at present, the sites of Pax3 phosphorylation are not known. We demonstrate here the first evidence that Pax3 exists as a phosphoprotein in proliferating mouse primary myoblasts. Using an in vitro kinase assay, deletion, and point mutant analysis, we conclusively identify Ser205 as a site of phosphorylation. The phosphorylation of Ser205 on endogenously expressed Pax3 was confirmed in vivo using antibodies specific for phosphorylation at Ser205. Finally, we demonstrate for the first time that the phosphorylation status of endogenous Pax3 changes rapidly upon the induction of myogenic differentiation. The presence of phosphorylation in a region of Pax3 important for mediating protein-protein interactions, and the fact that phosphorylation is lost upon induction of differentiation, allow for speculation on the biological relevance of phosphorylation.
Pax3是配对类同源结构域转录因子家族的成员之一，对早期骨骼肌发育至关重要。此前，我们和其他研究人员已表明，Pax3的稳定性在翻译后水平受到调控。文献及我们实验室的证据表明，磷酸化这种常见的调控形式可能发挥了作用。然而，目前Pax3的磷酸化位点尚不清楚。我们在此首次证明，在增殖的小鼠原代成肌细胞中，Pax3以磷蛋白形式存在。通过体外激酶分析、缺失分析和点突变分析，我们最终确定Ser205为磷酸化位点。使用针对Ser205磷酸化的特异性抗体在体内证实了内源性表达的Pax3上Ser205的磷酸化。最后，我们首次证明，内源性Pax3的磷酸化状态在成肌分化诱导后迅速改变。Pax3中对介导蛋白质-蛋白质相互作用很重要的区域存在磷酸化，且在分化诱导后磷酸化消失，这使得我们能够推测磷酸化的生物学意义。
