Targets of p56(lck) activity in immature thymoblasts: stimulation of the Ras/Raf/MAPK pathway.

Previous studies suggest that p56(lck) activity influences thymocyte development at a stage prior to TCR alphabeta expression. Transgenic mice that express high levels of p56(lck) activity during thymopoiesis develop thymic lymphomas consisting of cells with immature surface phenotypes. We have utilized cell lines derived from lck-induced thymic tumors to define biochemical pathways regulated by p56(lck) activity in immature thymocytes. Here we report that components of the Ras/Raf/MAPK pathway are constitutively activated in these lck-transformed immature thymoblasts. p56(lck) utilizes Shc and Grb2 adaptors to mediate activation of p21(ras) in the thymoblast lines by promoting tyrosine phosphorylation of the Shc protein and constitutive interaction between Shc and Grb2. The putative guanine nucleotide exchange factor p95(vav) is also maintained in constitutively tyrosine phosphorylated form as a result of elevated Lck activity. One target of activated Ras, the Raf-1 kinase, is hyperphosphorylated and downstream targets of activated Raf-1, Erk1 and Erk2, are hyperphosphorylated and activated in Lck-transformed thymocytes. Forskolin treatment reverses Raf-1 hyperphosphorylation in the cells and inhibits proliferation by blocking G1/S transition. In contrast, conventional protein tyrosine kinase inhibitors block proliferation by arresting Lck thymoblasts at G2/M. Lck-mediated stimulation of the Ras/Raf/MAPK pathway is also required to maintain cell viability by preventing programmed cell death. In summary, p56(lck) activity stimulates G1/S transition in immature thymoblasts and maintains cell viability via transduction of constitutive activation signals downstream to components of the Ras/Raf/MAPK pathway.