A study of the isolation, characterization, and function of chicken Janus kinase 3 in T lymphocyte signaling

The Janus kinases (JAKs) are a family of cytoplasmic protein tyrosine kinases (PTKs) that play critical roles in the signal transduction pathways mediated by various cytokines. The diversity and specificity of cellular responses to ligand binding are partly determined by the association of various receptors with distinct JAK combinations. The overlapping activation of JAKs by different ligands makes the transcriptional specificity of the individual ligands difficult to understand. Identification of new intracellular signaling targets provides insight into protein-protein interactions with JAK kinases.; The primary focus of this work was to isolate, characterize, and study the function of chicken JAK3 in T lymphocyte signaling. During the course of sequencing chicken JAK3, we identified a potential SH2 domain. Computational analysis showed this region contains the proper primary, secondary, and tertiary elements of an SH2 domain. Several proteins were shown to specifically bind to the SH2 domain of cJAK3 in a tyrosine-phosphorylated manner in a protein binding assay. In addition, by using the yeast-two-hybrid system, several interacting proteins were identified, including Hem-1, a potential interacting protein.; The effect of the SH2 domain on gene expression was evaluated using reporter gene assays. COS-7 cells transiently expressing full length human JAK3, murine STAT5A, and a STAT5-responsive luciferase plasmid results in an increase in luciferase activity indicating overexpression of these proteins leads to activation of the JAK-STAT pathway. When the SH2 domain of hJAK3 was included in the transfection, an inhibition of luciferase activity was seen, suggesting that overexpression of the SH2 domain blocks signaling through the JAK-STAT pathway. However, we also found that this SH2 construct can act to blunt expression of other luciferase constructs independent of JAK-STAT activation.; The role of cJAK3 in T lymphocyte signaling was evaluated using chicken DNA microarrays to profile gene expression. We identified several immune response genes that are differentially expressed in JAK3-inhibited lymphocytes, compared to normal, activated lymphocytes. For example, IFN-inducible protein and MHC class II invariant chain mRNA levels are reduced approximately 2-fold. The differences identified indicate that JAK3-controlled pathways in T lymphocyte signaling are important in activation and proliferation responses to Con A/IL2 treatment.