MAP KINASE PHOSPHATASE-1 GENE EXPRESSION AND REGULATION IN NEUROENDOCRINE CELLS

Abstract

ABSTRACTLong-term cellular processes like proliferation, differentiation, and adaptive responses (e.g. neuronal plasticity) are initiated by the synthesis of immediate early gene (IEG) products which control the expression of late response genes. Immediate early genes encode for transcription factors, structural proteins, cytokines, and other regulatory proteins. One of the latter category of IEG products is the mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1), a dual specificity tyrosine phosphatase which inactivates the MAP kinase ERK in the nucleus. In GH4C1 neuroendocrine cells, MKP-1 is rapidly synthesised and translocated to the nucleus in response thyrotropin-releasing hormone (TRH) or epidermal growth factor (EGF). Regulation of MKP-1 gene expression in this cell line is controlled at the transcriptional level via a strong block to elongation in the exon 1 of the gene. After stimulation with TRH the block to elongation is released and gene transcription is completed. Nuclear run-on is traditionally used to identify blocks to elongation and to determine endogeneous levels of transcriptional activities, but this method has severe technical limitations. An alternative approach to nuclear run-on is presented here for the MKP-1 gene, which involves the purification and analysis of nascent and free nuclear RNA fractions. This method may be helpful to study in more detail the mechanisms of transcriptional elongation in mammalian cells.