Abstract
c-myc oncogene activation is critical in the pathogenesis of a spectrum of human malignancies. The c-Myc NH2-terminal domain (MycNTD) is essential for cellular transformation, and mediates critical protein interactions that modulate c-Myc oncogenic properties. In medulloblastoma, the most common malignant pediatric brain tumor, deregulated c-myc expression is linked with poorer disease phenotypes and outcomes. The biological basis for these associations is, however, not well understood. To better understand mechanisms underlying Myc-mediated transformation of medulloblastoma, we sought to identify novel MycNTD protein interactors from a medulloblastoma cell line library using a unique two-hybrid system. We identified a novel MycNTD binding protein, JPO2, which shows nuclear colocalization with c-Myc, and interacts with c-Myc both in vitro and in mammalian cells. In Rat1a transformation assays, JPO2 potentiates c-Myc transforming activity, and can complement a transformation-defective Myc mutant. Immunohistochemical studies indicate tumor-specific JPO2 expression in human medulloblastoma, and an association of JPO2 expression with metastatic tumors. Significantly, JPO2 expression induces colony formation in UW228, a medulloblastoma cell line, whereas RNAi-mediated JPO2 knockdown impairs colony formation in UW228, and in Myc-transformed UW228 cells. These data provide evidence for biochemical and functional interaction between c-Myc and JPO2 in medulloblastoma transformation. JPO2 is closely related to JPO1, a Myc transcriptional target with transforming activity. As tumor-specific JPO1 expression in human and murine medulloblastoma has also been reported; these collective observations suggest important functional links between the novel JPO protein family and c-Myc in medulloblastoma transformation.
Highlights
Introduction c-myc oncogene activation is a critical transforming event in the pathogenesis of many human malignancies [1,2,3,4]; including medulloblastoma, the most common pediatric malignant brain tumor [5, 6]
In the repressed transactivator assay (Fig. 1A), the Myc NH2-terminal domain (MycNTD) bait is expressed as a Gal4-DNA binding domain fusion; prey proteins are fused to the repression domain of the yeast TUP1 protein and a Gal4-DNA-binding domain is located upstream of Ura3, a reporter gene that converts 5-fluoroorotic acid (FOA) to a toxic metabolite when expressed
When a bait/prey protein interaction occurs, the Ura3 reporter gene is turned off by the prey-associated TUP1 repression domain, and yeast cells containing bait/prey interactions grow on otherwise toxic FOAselective media
Summary
Introduction c-myc oncogene activation is a critical transforming event in the pathogenesis of many human malignancies [1,2,3,4]; including medulloblastoma, the most common pediatric malignant brain tumor [5, 6]. The NH2terminal domain of c-Myc (MycNTD) is not known to bind DNA but is indispensable for all known Myc functions including cellular transformation, cell cycle regulation, apoptosis, and transcriptional activation and repression [3, 23,24,25]. The MBII domain (amino acids 129-143), which is essential for Myc-mediated transformation, interacts with several different proteins which are components of large transcriptional complexes with histone acetylation and chromatin remodeling activities, including TRRAP [30, 31], BAF53 [32], and TIP60 [33]. We were interested in identifying MycNTD protein interactors with potential roles in Myc-mediated transformation of medulloblastoma cells. We show that JPO2 knockdown impairs anchorage-independent growth in UW228 and Myctransformed UW228 cells, pointing to an important role for this novel protein in Myc-mediated transformation of medulloblastoma
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