Microphthalmia (mi) is a transcription factor that plays a major role in the regulation of growth and function in mast cells and melanocytes. Association ofmi with other proteins is a critical step in the regulation of mi-mediated transcriptional activation. We found protein kinase C-interacting protein 1 (PKCI) specifically associated withmi in yeast two-hybrid screening. Immunoprecipitation ofmi from quiescent rat basophilic leukemic cells or mouse melanocytes resulted in the specific co-immunoprecipitation of PKCI. This association was significantly reduced on engagement of the surface FcεRI of mast cells or engagement of the Kit receptor on melanocytes. Hence, cell activation caused disengagement of mi from PKCI. Microphthalmia was previously shown to activate the mouse mast cell protease 6 (mMCP-6) promoter. Cotransfection of miwith PKCI in NIH 3T3 fibroblasts containing an mMCP-6 promoter-luciferase reporter demonstrated an up to 94% inhibition ofmi-mediated transcriptional activation. PKCI by itself, although localized in the cytosol and nucleus of the cells, has no known physiological function and did not demonstrate transcriptional activity. Its ability to suppres mi transcriptional activity in the transient transfected fibroblast system suggests that it can function in vivo as a negative regulator ofmi-induced transcriptional activation.

The antigen-mediated activation of mast cells by means of IgE antibodies bound to the cell surface leads to direct interactions between FcɛRI receptor cytoplasmic domains and various intracellular proteins. These interactions initiate diverse signal-transduction pathways, and the activation of these pathways results in the immediate release of proinflammatory agents. A delayed response also occurs and includes the release of various cytokines. It is clear that the activation of kinases is a requirement for the exocytosis observed in mast cells. In addition to the tyrosine phosphorylation of the affected system by soluble tyrosine kinases, activity of protein kinase C (PKC) results in serine or threonine phosphorylation of multiple protein substrates. In this study, we found that mast cells derived from PKCβ-deficient mice produce less interleukin 6 in response to IgE-Ag. The inhibition of exocytosis in the PKCβ-deficient mast cells occurred whether the stimuli were due to the aggregation of the mast cell surface FcɛRI or to the calcium ionophore, ionomycin. However, no significant changes were observed in the proliferative response of the mast cells to interleukin 3 (IL-3) or in their apoptotic rate after IL-3 depletion.

Reports of the biological multifunctional activity of various aminoacyl tRNA synthetases have recently accumulated in the literature. The primary function of these critical enzymes is to charge various tRNAs with their appropriate amino acids, thus producing the building blocks of protein synthesis. We have previously shown that lysyl tRNA synthetase (LysRS) associates with microphthalmia transcription factor (MITF) and regulates its activity by synthesis of Ap₄A in mast cells. Here, we show for the first time that LysRS associates with another transcription factor, USF2, which unlike MITF, is ubiquitously expressed in eukaryotic cells. Using mast cells, we have found that USF2 is negatively regulated by Hint and Ap₄A acts as a positive regulator of USF2 by a molecular mechanism similar to that described for MITF. Since USF2 plays a significant role in a variety of cellular functions, our finding suggests that LysRS and Ap₄A may be involved in general regulation of gene transcription.

Microphthalmia transcription factor (MITF) and STAT3 are two transcription factors that play a major role in the regulation of growth and function in mast cells and melanocytes. In the present study, we explored the MITF-PIAS3-STAT3 network of interactions, how these interactions regulate gene expression, and how cytokine-mediated phosphorylation of MITF and STAT3 is involved in the in vivo interplay between these three proteins. In NIH 3T3 cells stimulated via gp130 receptor, transfected MITF was found to be phosphorylated at S409. Such phosphorylation of MITF leads to PIAS3 dissociation from MITF and its association with STAT3. Activation of mouse melanoma and mast cells through gp130 or c-Kit receptors induced the mobilization of PIAS3 from MITF to STAT3. In mast cells derived from MITF^di/di mice, whose MITF lacks the Zip domain (PIAS3-binding domain), we found downregulation in mRNA levels of genes regulated by either MITF or STAT3. This regulatory mechanism is of considerable importance since it is likely to advance the deciphering of a role for MITF and STAT3 in mast cells and melanocytes.