Anti-ETV3 monoclonal antibody

The mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 2 (ERK2) is ubiquitously expressed in mammalian tissues and is involved in a wide range of biological processes. Although MAPKs have been intensely studied, identification of their substrates remains challenging. We have optimized a chemical genetic system using analog-sensitive ERK2, a form of ERK2 engineered to use an analog of adenosine 5'-triphosphate (ATP), to tag and isolate ERK2 substrates in vitro. This approach identified 80 proteins phosphorylated by ERK2, 13 of which are known ERK2 substrates. The 80 substrates are associated with diverse cellular processes, including regulation of transcription and translation, mRNA processing, and regulation of the activity of the Rho family guanosine triphosphatases. We found that one of the newly identified substrates, ETV3 (a member of the E twenty-six family of transcriptional regulators), was extensively phosphorylated on sites within canonical and noncanonical ERK motifs. Phosphorylation of ETV3 regulated transcription by preventing its binding to DNA at promoters for several thousand genes, including some involved in negative feedback regulation of itself and of upstream signals.

Mast cell development is an important component of atopic and chronic inflammatory diseases such as asthma, multiple sclerosis, rheumatoid arthritis, and atherosclerosis. In this study, we found that IL-4 and IL-10 were produced constitutively in cultures of developing mast cells, correlating with mast cell purity. Deletion of either gene increased mast cell numbers and Fc epsilon RI expression during culture in IL-3 + stem cell factor (SCF). By adding exogenous IL-4 and IL-10 to bone marrow (BM) cultures containing IL-3 + SCF, we found that IL-4 + IL-10 suppressed mast cell development through mechanisms not used by either cytokine alone. IL-4 + IL-10 elicited a rapid cell death coincidental with reduced Kit receptor expression and signaling and enhanced mitochondrial damage and caspase activation. IL-4 or IL-10 costimulation, unlike either cytokine alone, altered mast cell ontogeny to yield predominantly macrophages in cultures that typically produce mast cells. This effect was observed consistently with unseparated BM cells, purified mouse BM stem cells, and erythrocyte-depleted human umbilical cord blood cells. These experiments demonstrated a major role for Stat6 and Stat3, but not the Stat3-induced transcriptional repressor Ets variant gene 3. Genetic background was also a critical factor, as BALB/c-derived BM cells were completely resistant to IL-10-mediated killing and expressed lower levels of IL-10R. Collectively, these results support the theory that IL-4 and IL-10 function as endogenous regulators of mast cell progenitor development, consistent with a role in immune homeostasis. Loss of this homeostasis, perhaps via genetic polymorphism, could contribute to the etiology of mast cell-associated disease. J. Leukoc. Biol. 85: 826-836; 2009.