No abstract

Erythropoietin (EPO)1 is an essential growth factor that promotes survival, proliferation, and differentiation of erythroid progenitor cells (1). These effects are conveyed through engagement of the EPO receptor (EPO-R) (2), a member of the superfamily of cytokine receptors. Erythropoietin induces dimerization of its receptor, resulting in trans-phosphorylation of receptor-associated Janus Kinase 2 (JAK2) molecules (3, 4). Activated JAK2 phosphorylates some or all of the eight tyrosine residues in the intracellular domain of the EPO-R. Phosphorylation of these intracellular tyrosines in turn recruits other intracellular proteins that bind to the EPO-R via their Src homology 2 (SH2) domains (reviewed in Ref. 5). The critical importance of EPO (6), , and JAK2 (9, 10) for efficient erythopoiesis have been demonstrated by targeted deletion in mice.The EPO-R recruits several distinct SH2-containing proteins, including signal transducer and activator of transcription-1 (STAT1) (11, 12), STAT3 (12), and STAT5 proteins (13-18). The EPO-R also associates with a diverse array of SH2-domain containing proteins, including Ship1 inositol phosphatase (19), Shp1 (20), Shp2 (21), phospholipase C␥1/C␥2 (22, 23), phosphatidylinositol 3Ј-kinase (24), as well as many non-catalytic adapter molecules (reviewed in Ref. 5). Binding of some of these molecules leads to their JAK2-dependent tyrosine phosphorylation.STAT proteins are an important class of signaling molecules that are recruited to the erythropoietin receptor. These latent transcription factors are activated at the receptor and transduce signals to the nucleus. While seven STAT molecules have been identified, only STAT5A and STAT5B are consistently activated by EPO in all cell types (13-18), whereas STAT1 and STAT3 are activated in erythroid cell types or by Friend virus transformation (11,12,25). STAT5A/B knockout mice have defects in prolactin (26), growth hormone (26), and interleukin-2 signaling (27), display anemia during fetal development (28, 29) and decreased formation of CD71 lo Ter119 hi cells in STAT5A/B Ϫ/Ϫ adult mice (30). No erythroid abnormalities were been described in the original reports of STAT1 Ϫ/Ϫ mice (31, 32), however, a more recent report suggested differences in clonogenic potential of burst forming unit-erythroid cells (33). STAT3 Ϫ/Ϫ mice are embryonic lethal, precluding analysis of their role in erythropoiesis by conventional gene targeting approaches (34).While tyrosine phosphorylation is required for cytokine-induced STAT dimerization, nuclear translocation, and DNA binding, full transcriptional activity of the homodimer is manifested only when a serine residue in the transcription activation domain is also phosphorylated (35). While the identity of the serine kinases is unclear, these putative proline-directed phosphorylation sites may be targetted by the mitogen-activated protein kinases (MAPKs).The MAPKs comprise a family of serine/threonine kinases that are activated by extracellular signals. There are at least three distinct types of MAPKs:...

Mass cytometry (MC) offers unparalleled potential for the development of highly parameterized assays for characterization of single cells within heterogeneous populations. Current reagents compatible with MC analysis employ antibody-metal-chelating polymer conjugates to report on the presence of biomarkers. Here, we expand the utility of MC by developing the first activity-based probe designed specifically for use with the technology. A compact MC-detectable telluroether is linked to a bioreductively sensitive 2-nitroimidazole scaffold, thereby generating a probe sensitive to cellular hypoxia. The probe exhibits low toxicity and is able to selectively label O2-deprived cells. A proof-of-concept experiment employing metal-bound DNA intercalators demonstrates that a heterogeneous mixture of cells with differential exposure to O2 can be effectively discriminated by the quantity of tellurium-labeling. The organotellurium reagents described herein provide a general approach to the development of a large toolkit of MC-compatible probes for activity-based profiling of single cells.