ZAP-70-independent Ca2+ mobilization and Erk activation in Jurkat T cells in response to T-Cell antigen receptor ligation

The tyrosine kinase ZAP-70 has been implicated as a critical intermediary b etween T-cell antigen receptor (TCR) stimulation and Erk activation on the basis of the ability of dominant negative ZAP-70 to inhibit TCR-stimulated Erk activation, and the reported inability of anti-CD3 antibodies to activa te Erk in ZAP-70-negative Jurkat cells. However, Erk is activated in T cell s receiving a partial agonist signal, despite failing to activate ZAP-70. T his discrepancy led us to reanalyze the ZAP-70-negative Jurkat T-cell line P116 for its ability to support Erk activation in response to TCR/CD3 stimu lation. Erk was activated by CD3 cross-linking in P116 cells. However, this response required a higher concentration of anti-CD3 antibody and was dela yed and transient compared to that in Jurkat T cells. Activation of Raf-1 a nd MEK-1 was coincident with Erk activation. Remarkably, the time course of Ras activation was comparable in the two cell lines, despite proceeding in the absence of LAT tyrosine phosphorylation in the P116 cells. CD3 stimula tion of P116 cells also induced tyrosine phosphorylation of phospholipase C -gamma1 (PLC gamma1) and increased the intracellular Ca2+ concentration. Pr otein kinase C (PKC) inhibitors blocked CD3-stimulated Erk activation in P1 16 cells, while parental Jurkat cells were refractory to PKC inhibition. Th e physiologic relevance of these signaling events is further supported by t he finding of PLC gamma1 tyrosine phosphorylation, Erk activation, and CD69 upregulation in P116 cells on stimulation with superantigen and antigen-pr esenting cells. These results demonstrate the existence of two pathways lea ding to TCR-stimulated Erk activation in Jurkat T cells: a ZAP-70-independe nt pathway requiring PKC and a ZAP-70-dependent pathway that is PKC indepen dent.