Triggering the T cell receptor (TCR)/CD3 complex generates signals that traverse the cell to the nucleus, leading to gene transcription, activation and proliferation. Elucidation of signaling events is crucial to understand T cell behavior in physiological and pathological (autoimmune, malignant) responses. Our previous studies focused on a phospholipase C (PLC) - mediated, second messenger - generating pathway that activates protein kinase C (PKC) and mobilizes Ca2+, respectively. It is becoming clear, however, that this pathway is regulated in a critical, yet poorly understood, manner by tyrosine protein kinases (TPKs). The proposed studies address the roles of, and interaction between, the inositol phospholipid and TPK signaling pathways in T cells. First, polymerase chain reaction (PCR) and gene cloning techniques will be used to clone and characterize cDNAs encoding T cell-expressed PKC isoforms, including a putative new isoform revealed by preliminary studies. A multiprobe RNAse protection assay will be used to analyze expression patterns of PKC genes in thymic and peripheral T cell subsets and during human T cell activation. Second, the following approaches will be undertaken to elucidate the roles of two T cell-expressed TPKs, p59fyn and p56lck, that are associated with the TCR/CD3 complex or the CD4/CD8 coreceptors, respectively, in T cell signaling, in particular the mode by which they may regulate PLC activation: 1) The activation status of these two TPKs will be sequentially analyzed in TCR/CD3-triggered T cells by following the phosphorylation levels of two conserved tyrosine residues involved in positive or negative regulation of the TPK's activity, respectively. 2) T cells will be stably transfected with expression vectors designed to selectively inhibit the expression of the lck or fyn genes, using antisense RNA and targeted ribozyme approaches as well as expression of mutated, inactive TPKs. The effects of these experimental manipulations on signal transduction events (PLC activation, gene induction and proliferation) will be analyzed and correlated with p56lck or p59fyn levels. 3) Establish "maps" of TPK substrates phosphorylated upon TCR/CD3 triggering using anti- phosphotyrosine antibodies, attempt to identify them, and determine whether inositol phospholipid hydrolysis is coupled to TPK activation via tyrosine phosphorylation of PLC or other substrates associated with receptor TPKs. 4) Various TPK inhibitors will be analyzed in parallel for their effects on p56lck or p59fyn activity, TCR/CD3-induced tyrosine phosphorylation and later signaling events. 5) Activation-defective CD4-CD8- lpr T cells or their representative T cell hybridomas, that display increased constitutive tyrosine phosphorylation (a result of overexpressed p59fyn) and phosphoinositide hydrolysis, will be studied as a possible natural model manifesting a causal relationship between tyrosine phosphorylation and PLC activation. Understanding the rol...