Suppression of microphthalmia transcriptional activity by its association with protein kinase C-interacting protein 1 in mast cells

Microphthalmia (mi) is a transcription factor that plays a major role in th e regulation of growth and function in mast cells and melanocytes. Associat ion of mi with other proteins is a critical step in the regulation of mi-me diated transcriptional activation We found protein kinase C-interacting pro tein 1 (PKCI) specifically associated with mi in yeast two-hybrid screening . Immunoprecipitation of mi 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 Fc epsilon RI of mast cells or engagement of the Kit receptor on melanocytes. Hence, cell activation caused disengagement of mi from PKCI. Microphthalmi a was previously shown to activate the mouse mast cell protease 6 (mMCP-6) promoter. Cotransfection of mi with PKCI in NIH 3T3 fibroblasts containing an mMCP-6 promoter-luciferase reporter demonstrated an up to 94% inhibition of mi-mediated transcriptional activation PKCI by itself, although localiz ed in the cytosol and nucleus of the cells, has no known physiological func tion and did not demonstrate transcriptional activity. Its ability to suppr es mi transcriptional activity in the transient transfected fibroblast syst em suggests that it can function in vivo as a negative regulator of mi-indu ced transcriptional activation.