The precise control of cell differentiation and proliferation is mediated by the interaction of extracellular growth factors with discrete membrane receptors. Transduction of the surface signal to the various cellular compartments, including the nucleus, is mediated by a phosphorylation cascade of intracellular substrates, culminating in the activation of specific transcription factors. Because p21ras functions as the critical switch which converts a receptor tyrosine kinase signal initiated at the membrane into a serine/threonine kinase cascade, p21ras expression vectors have been used to analyze growth factor-induced responses. Typically, p21ras-induced protein phosphorylation has been assayed. However, the functional consequence of such modifications, and whether these modification are common to all cell types, irrespective of their developmental history, remains ill-defined. Therefore, defining the precise molecular mechanisms by which growth factor/ras-induced signaling pathways common to all cell types result in cell-specific responses, remains a fundamental problem in current biology. To this end, we have developed a transient co-transfection approach in cultured GH4 rat pituitary cells to test the effects of oncogenic V-12 ras on the promoter activity of a cellular gene whose expression is highly restricted to the pituitary cell type: rat prolactin (rPRL). Using this assay, we have shown that V-12 ras selectively and significantly activities the rPRL promoter, and we have identified and ordered the functional components of the ras signaling cascade, from the membrane to the nucleus, as: ras raf kinase MAP kinase ets-1/GHF-1. Further studies have shown that a ras-dependent functional interaction of the ubiquitous nuclear proto- oncogene transcription factor, ets-1, with the pituitary-specific transcription factor, GHF-1/Pit-1, is required for the synergistic enhancement of the oncogene-mediated activation of the rPRL promoter; whereas neither factor alone is sufficient to mediate this response. Moreover, we have mapped the ras responsive element (RRE) of the rPRL promoter to a DNA region which contains both a putative ets site and an adjacent GHF-1/Pit-1 site. Thus, we have discovered that a ras- dependent, functional interaction of these two factors confers a pituitary-specific response to the general ras signaling pathway. Based on these preliminary results, the long-term goal of the current proposal is to show that the molecular mechanism by which the ras signaling cascade governs pituitary-specific gene expression is via a signal- activated and phosphorylation-dependent interaction of ets-1 with GHF-1, resulting in a more stable ets-1/GHF-1 transcription factor complex on the rPRL RRE.
The specific aims to accomplish this goal are: (1) to fine-map the precise DNA sequences of the rPRL ras responsive element; (2) to determine whether ets-1 and GHF-1 physically interact; (3) to map the ras-mediated phosphorylation site(s) on ets-1 and/or GHF-1; and, (4) to map the ras-dependent, functionally-interactive domains of ets-1 and GHF-1. Our discovery not only has important scientific implications regarding the molecular mechanisms by which generic signal are harnessed for the control of cell-specific processes, but also provides novel insights into the molecular mechanisms by which pituitary tumors might arise.
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