casein-kinase-ii and Rhabdomyosarcoma--Alveolar

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.

Topics: Animals; Antibodies, Phospho-Specific; Casein Kinase II; Cell Line, Tumor; Child; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Mice; Mice, Inbred C57BL; Muscle Development; Muscle Neoplasms; Myoblasts; Oncogene Proteins, Fusion; Paired Box Transcription Factors; PAX3 Transcription Factor; Phosphorylation; Rhabdomyosarcoma, Alveolar; Serine

The myogenic transcription factor Pax3 plays an essential role in early skeletal muscle development and is a key component in alveolar rhabdomyosarcoma (ARMS), a childhood solid muscle tumor. ARMS is characterized by a t(2;13) chromosomal translocation resulting in the fusion of the 5' Pax3 sequences to the 3' FOXO1 sequences to encode the oncogenic fusion protein, Pax3-FOXO1. Posttranslational modifications such as phosphorylation are common mechanisms by which transcription factors are regulated. Consistent with this fact, we demonstrated in a previous report that Pax3 is phosphorylated on Ser205 in proliferating, but not differentiated, primary myoblasts. However, the kinase that mediates this phosphorylation event has yet to be identified. In addition, it is not known whether Pax3-FOXO1 is phosphorylated at this site or how the phosphorylation of the fusion protein changes during early myogenic differentiation. In this report we identify CK2 (formerly termed "casein kinase II") as the kinase responsible for phosphorylating Pax3 and Pax3-FOXO1 at Ser205 in proliferating mouse primary myoblasts. Furthermore, we demonstrate that, in contrast to wild-type Pax3, phosphorylation at Ser205 persists on Pax3-FOXO1 throughout early myogenic differentiation. Finally, we show that Pax3-FOXO1 is phosphorylated at Ser205 in a variety of translocation-containing ARMS cell lines. The results presented in this report not only suggest a possible mechanism by which the disregulation of Pax3-FOXO1 may contribute to tumorigenesis but also identify a novel target for the development of therapies for the treatment of ARMS.

Topics: Animals; Casein Kinase II; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Humans; Mice; Mice, Inbred C57BL; Muscle Development; Myoblasts; Oncogene Proteins, Fusion; Paired Box Transcription Factors; PAX3 Transcription Factor; Phosphorylation; Rhabdomyosarcoma, Alveolar; Serine; Translocation, Genetic