Analysis of in vivo expression and function of Src-family protein tyrosine kinases during lymphocyte development

Abstract

Src-family protein tyrosine kinases (PTKs) are critical components of T and B lymphocyte signaling cascades. To examine the relationship between Src-PTK expression and lymphocyte development, we defined quantitative and qualitative expression patterns of Src-PTKs during lymphocyte ontogeny. A quantitative RT-PCR assay was designed to distinguish transcripts derived from the lck proximal (Type I) vs distal (Type II) promoter elements, and fynT vs fynB isoforms. These assays were then used to measure lck and fyn expression in normal fetal and adult tissue. Changes in Type I and Type II promoter utilization correlate with pre-TCR and alphabetaTCR expression suggesting that as receptor complexes develop they couple to different intracellular signaling cascades that differentially regulate lck expression. The fynT isoform, not fynB, is detected in both lineages, including pre-T and pre-B cells, suggesting a role for Fyn in lymphocyte development before TCRalphabeta or IgM expression. In spite of much lower levels of fynT and lck transcripts in the B cell lineage, compared to lck transcripts in thymocytes; accumulation of lck and fynT correlates temporally with acquisition of immune receptor expression in both lineages. In addition to immune receptors, Src family PTKs are activated by receptors regulating growth and differentiation in thymic stroma which is required for lymphocyte development. To examine the impact of Src-PTK function in thymic stroma on lymphocyte development, a transgenic model system was developed in which the expression of polyoma virus middle T antigen (PymT), a molecule that activates Src-PTKs, was directed to thymic subcapsular epithelial cells using a mutant Class II promoter element. PymT expression constitutively activated endogenous Src-PTKs which phosphorylated PymT, inducing a PymT association with p85, the regulatory subunit of PI 3-kinase. Src-PTK activation was associated with changes in tyrosine phosphorylation of cellular proteins. Transgene expression in subcapsular stroma resulted in upregulation of epidermal growth factor (EGF) receptor and 4 to 5-fold thymic lymphoid hyperplasia. However, normal thymic architecture, proliferation and apoptotic responses, thymocyte differentiation, repertoire selection, and thymic involution were maintained. These findings suggest that the level and/or persistence of signals generated via Src-PTKs in thymic subcapsular epithelium serve as major defining factors influencing homeostatic control of thymic organ size and cellularity.