Search

The ARF tumor suppressor signals through p53 and other poorly defined anti-proliferative pathways to block carcinogenesis. In a search for new regulators of ARF signaling, we discovered a novel nuclear protein that we named NIAM (nuclear interactor of ARF and MDM2) for its ability to bind both ARF and the p53 antagonist MDM2. NIAM protein is normally expressed at low to undetectable levels in cells because of, at least in part, MDM2-mediated ubiquitination and proteasomal degradation. When reintroduced into cells, NIAM activated p53, caused a G1 phase cell cycle arrest, and collaborated with ARF in an additive fashion to suppress proliferation. Notably, NIAM retains growth inhibitory activity in cells lacking ARF and/or p53, and knockdown experiments revealed that it is not essential for ARF-mediated growth inhibition. Thus, NIAM and ARF act in separate anti-proliferative pathways that intersect mechanistically and suppress growth more effectively when jointly activated. Intriguingly, silencing of NIAM accelerated chromosomal instability, and microarray analyses showed reduced NIAM mRNA expression in numerous primary human tumors. This study identifies a novel protein with tumor suppressor-like behaviors and functional links to ARF-MDM2-p53 signaling.

The development of erythroid progenitor cells depends upon exposure to the glycoprotein hormone, erythropoietin (EPO). Binding of EPO to its transmembrane receptor leads to the rapid tyrosine phosphorylation of several cellular targets including Shc, Raf-1, Gap120, the cloned EPO receptor (EPOR), pp100/97, and a M(r) 130,000 EPO-activated receptor-associated Janus protein tyrosine kinase, Jak2. A membrane-proximal cytosolic region of the EPOR recently has been shown to be essential for the activation of Jak2 and sufficient for EPO-induced mitogenesis. This cytosolic region includes 8-12 amino acid box 1 and box 2 subdomains, which are conserved in certain class I receptors as well as a more distal 10-40 amino acid subdomain (extended box 2 subdomain, ExBx2), which likewise is implicated in mitogenic signaling. Through the expression of EPOR carboxyl-terminal truncation mutants in FDC-P1 cells, we presently show that an EPOR form truncated within the ExBx2 domain efficiently activates Jak2, yet is deficient in mitogenesis. Efficient expression of this mutant receptor at the cell surface and its ability to activate Jak2 indicate that poor mitogenic activity does not result from aberrant transport or folding. Rather, failure of this mutant to support proliferation above nominal rates underlines an apparent role for the EPOR ExBx2 subdomain in the activation of a distinct primary mitogenic effector.

Receptors for the hematopoietic growth factors erythropoietin, interleukin 3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) are members of a structurally related receptor superfamily. Interestingly, while none of these receptors encode tyrosine kinase activities, induced tyrosine phosphorylation has been observed in various responsive cells stimulated with each factor. Toward defining possible common transduction pathways which are activated by these three cytokines, we have studied induced protein phosphorylation in murine myeloid FDC-P1 cells stably transfected with an erythropoietin receptor cDNA (FDC-ER cells). FDC-ER cells proliferate in response to erythropoietin (Quelle, D. E., and Wojchowski, D. M. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4801-4805), and presently are shown to rapidly phosphorylate a M(r) 100,000 cytosolic protein (pp100) at tyrosine residues in response to this factor. Phosphorylation of pp100 also is induced in FDC-P1 and FDC-ER cells in response to IL-3 or GM-CSF. Importantly, quantitative analyses showed identical concentration dependencies for factor-induced pp100 phosphorylation and induced cell proliferation. Moreover, a selective loss of proliferative responsiveness to GM-CSF in FDC-ER cells was associated with a reduced capacity of GM-CSF to induce pp100 phosphorylation. Finally, limited differences in tryptic phosphopeptide maps of pp100 as isolated following exposure to erythropoietin, IL-3, or GM-CSF were observed, suggesting that these factors also may preferentially induce phosphorylation of pp100 at distinct sites. These findings are consistent with a role for pp100 as a common cytosolic transducer in the apparently convergent pathways of erythropoietin-, IL-3-, and GM-CSF-induced proliferation of myeloid progenitor cells.