Reif, Karin Alma Frieda; (1997) The regulation and function of phosphatidylinositol 3-kinase. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase that is likely to play an important role in lymphocyte biology and immune function. PI 3-kinase is activated by the T cell antigen receptor (TCR) and the interleukin-2 (IL-2) receptor which govern T cell activation and growth, respectively. Moreover, accessory receptors such as CD28 and CTLA-4 or other cytokine receptors regulate PI 3-kinase activity in T cells. One aim of this thesis was to examine mechanism by which the TCR couples to PI 3-kinase. It was shown that the adaptor molecule Grb2 via its SH3 domains can bind to the p85 subunit of PI 3-kinase and provides a potential link between the TCR and PI 3-kinase. Importantly, these studies led to the first identification of a novel haematopoietic-lineage specific 75 kDa protein that associates with Grb2 SH3 domains. p75 is tyrosine phosphorylated in response to TCR engagement. In parallel studies by other investigators, a 76 kDa Grb2 associated protein was cloned, termed SLP-76. I show that p75 and SLP-76 are identical. p75/SLP-76 is involved in TCR signal transduction pathways leading to IL-2 gene transcription. In order to analyse the cellular functions of PI 3-kinase I generated a constitutively active form of the enzyme. The strategy I employed was to membrane localise its p110 catalytic subunit. P110 was fused to a truncated rat CD2 cell surface receptor giving rise to rCD2p110. Expression of the rCD2p110 chimera elevates the cellular levels of D-3 phosphorylated inositol lipids. Using rCD2p110 it was demonstrated that PI 3-kinase signals are sufficient to stimulate p70S6k but not the MAP kinases Erk or Sapk/Jnk in fibroblasts or T cells. However, in T cells, PI 3-kinase signals can contribute to Erk activation. Importantly, it was established that the protooncogene PKB/Akt is activated by IL-2 in a PI 3-kinase-dependent fashion. Active PI 3-kinase can substitute for IL-2 in stimulating PKB and an active form of PKB can substitute for rCD2p110 or IL-2 in activating p70S6k. Rac/Rho-dependent effector pathways play a role in cytoskeletal changes and regulation of gene transcription. PI 3-kinase is a putative upstream regulator of Rac and hence Rho. Here, I show that PI 3-kinase only regulates a subset of Rac/Rho-mediated cellular responses. PI 3-kinase signals are sufficient to induce Rac/Rho-controlled actin rearrangements but fail to trigger Rac/Rho-mediated effector pathways for activation of transcription factors. I propose that specific subcellular compartmentalisation mechanisms exist that localise different exchange factor/GTPase complexes to divergent downstream effector pathways. Finally, I describe a previously unrecognised function for PI 3-kinase during T cell activation. PI 3-kinase can act as a selective negative regulator of TCR-mediated induction of the transcription factor NF-AT. This involves a novel uncharacterised effector of PI 3-kinase. The significance of these findings for immune homeostasis will be discussed.

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