It has become increasingly clear that non-receptor tyrosine kinases are critical components of the signaling cascade following lymphocyte activation. One subclass of these kinases belong to the src-family, a group of genes originally identified by their oncogenic potential. It has been postulated that these kinases normally control cell growth when activated. These studies are aimed at understanding how one Src-related kinase, Fyn, transduces signals during T cell activation. Mice lacking fyn show anomalies in T cell activation. T cells fail to flux calcium following crosslinking of the T cell receptor (TCR) complex and are impaired in IL-2 synthesis, probably because of the defective Ca2+ response. In addition, alternative activation pathways through the GPI- linked proteins Thy-1 and Ly-6 are compromised. To study the defects in better detail with the overall goal of determining how Fyn regulates signaling pathways, an ovalbumin responsive T cell clone was generated from the fyn mutant mice. The T cell clone will be transfected with a series of mutant Fyn molecules in order to identify structural motifs and residues which confer unique signaling properties of Fyn. The transfected clones will be analyzed for restoration of Ca2+ fluxes following TCR stimulation and ability to promote proliferation following cross-linking of Thy-1. Specifically, chimeric proteins between Fyn and the related kinase Lck will be used to determine the regions required for Fyn-specific signaling. Fyn is modified by the addition of the fatty acids, palmitate and myristate. The role of fatty acids in modulating Fyn funcTIon will be addressed by introducing mutants which can only be modified by one type of fatty acids then scored for effects on Thy-1 proliferation or Ca2+ fluxes. T cells express an alternatively spliced form of Fyn (FynT) which contains an altered and unusual tyrosine kinase domain. As it is unclear if Fyn T transduces different signals than Fyn, the T cell clone will be transfected with either Fyn or Fyn T and Ca+2+ fluxes measured. In addition proliferation will be monitored following cross-linking of Thy-1. A partial cDNA has been isolated which encodes a putative Ca2+ channel that interacts with Fyn. A full-length cDNA will be isolated and transfected into T cell lines to determine if it can modulate Ca2+ dependent responses associated with TCR activation. Together, these experiments will help clarify the mechanisms by which Fyn facilitates T cell activation. Such studies will provide insights into the etiology of inappropriate immune responses such as autoimmunity, and will provide a better understanding of the oncogenic activation of Src kinases in general.
