title: Identification of serines 201 and 209 as sites of Pax3 phosphorylation and the altered phosphorylation status of Pax3-FOXO1 during early myogenic differentiation.
abstract: Pax3, a member of the paired class homeodomain family of transcription factors, is essential for early skeletal muscle development and is key in the development of the childhood solid muscle tumor alveolar rhabdomyosarcoma (ARMS). ARMS is primarily characterized by a t(2;13)(q35;q14) chromosomal translocation, which fuses the 5'-coding sequences of Pax3 with the 3'-coding sequence of the forkhead transcription factor FOXO1 generating the oncogenic fusion protein Pax3-FOXO1. We previously demonstrated that Pax3 and Pax3-FOXO1 are phosphorylated by the protein kinase CK2 at serine 205 in proliferating primary myoblasts and that this phosphorylation event is rapidly lost from Pax3, but not Pax3-FOXO1 upon the induction of differentiation. However, reports suggested that additional sites of phosphorylation might be present on Pax3. In this report we use in vitro and in vivo analyses to identify serines 201 and 209 as additional sites of phosphorylation and along with serine 205 are the only sites of phosphorylation on Pax3. We provide solid evidence supporting the role of the protein kinase GSK3β as phosphorylating Pax3 at serine 201. Using phospho-specific antibodies we demonstrate a changing pattern of phosphorylation at serines 201, 205, and 209 throughout early myogenic differentiation and that this pattern of phosphorylation is different for Pax3-FOXO1 in primary myoblasts and in several ARMS cell lines. Taken together, our results allow us to propose a molecular model to describe the changing pattern of phosphorylation for Pax3 and the altered phosphorylation for Pax3-FOXO1 during early myogenic differentiation.
authors: Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
authors: Kevin N
authors: KN
authors: Dietz
authors: Patrick J
authors: PJ
authors: Miller
authors: Aditi S
authors: AS
authors: Iyengar
authors: Jacob M
authors: JM
authors: Loupe
authors: Andrew D
authors: AD
authors: Hollenbach
mesh_terms: D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000378:metabolism; D047390:Casein Kinase II / Q000378:metabolism; D045744:Cell Line, Tumor; D002648:Child; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D024510:Muscle Development; D019042:Muscle Neoplasms / Q000378:metabolism* / Q000473:pathology; D032446:Myoblasts / Q000378:metabolism* / Q000473:pathology; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D018232:Rhabdomyosarcoma, Alveolar / Q000378:metabolism* / Q000473:pathology; D012694:Serine / Q000235:genetics
chemical_list: D045163:Antibodies, Phospho-Specific; D015514:Oncogene Proteins, Fusion; D000072041:PAX3 Transcription Factor; C471563:PAX3-FOXO1A fusion protein, human; D051761:Paired Box Transcription Factors; C068686:Pax3 protein, mouse; D012694:Serine; D047390:Casein Kinase II; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3