Woodrow, Melissa A.; (1994) The role of the guanine nucleotide binding proteins p21ras in T cell activation and growth. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Both the T cell receptor for antigen, the TCR, which mediates T cell activation, and the receptor for T cell growth factor, the IL2R, which drives T cell proliferation, are able to activate the guanine nucleotide binding proteins p21ras. The aim of this study was therefore to examine the role of the p21ras proteins in T cell activation and proliferation. Transient transfection of IL2 gene reporter constructs and mutant Ras genes was used to assess the effect of dominant positive or dominant inhibitory Ras mutants on T cell activation. The reporter constructs used to monitor T cell activation were comprised of either the IL2 enhancer, or multiple copies of NFAT(the nuclear factor of activated T cells), which restricts IL2 expression to activated T cells, driving the reporter gene CAT. Dominant positive oncogenic p21ras was shown to replace some TCR derived signals to activate IL2 and NFAT CAT, but in itself was not sufficient to drive IL2 reporter gene transcription. When the dominat inhibitory N17ras was used, the data showed that p21ras is crucial to the function of PKC and the TCR in the regulation of IL2 and NFAT CAT. In addition, these data highlight that p21ras imparts a third, essential signal from the TCR for IL2 gene regulation, that is independent of calcium and PKC pathways. The serine/threonine specific phosphatase calcineurin, involved in calcium regulation of NFAT, was also used in transient transfection assays with oncogenic Ras. Calcineurin is a major in vivo target for the immunosupressants cyclosporin A and FK506, which block T cell activation. When co-expressed, constitutively active calcineurin and oncogenic p21ras can substitute for TCR stimulation of NFAT induction. These data identify p21ras and calcineurin as cooperating partners for NFAT and IL2 gene regulation. For T cell proliferation studies, co-transfection of a cell surface marker and mutant Ras proteins was used to allow purification of transfected cells out of a mixed population via co-expression of the surface marker. This would in theory allow the examination of the effect of expression of mutant Ras proteins in a homogeneously transfected population. However, technical difficulties prevented a conclusive study of this issue. Ras proteins have been implicated as the initiators of a kinase cascade that involves Raf-1, MEK, and ERK, and which can regulate the activity of transcription factors as well as ribosomal S6 kinase p90, or RSK. RSK is regulated by the TCR but not the IL2R. The IL2R does however, regulate the p70 S6 kinase, and it is this kinase which is a target for the immunosupressant rapamycin. The final part of the thesis describes the development of a system for examining the signalling pathways that couple the IL2R to p70 S6 kinase.

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