ZAP-70 and SLP-76 regulate protein kinase C-theta and NF-kappa B activation in response to engagement of CD3 and CD28

The transcription factor NF-KB is a critical regulator of T cell function t hat becomes strongly activated in response to coengagement of TCR and CD28. Although events immediately proximal to NF-kappaB activation are well unde rstood, uncertainty remains over which upstream signaling pathways engaged by TCR and CD28 lead to NF-kappaB activation. By using Jurkat T cell lines that are deficient or replete for either the protein tyrosine kinase ZAP-70 or the cytosolic adapter molecule SLP-76, the role of these proteins in mo dulating NF-kappaB activation was examined. NF-kappaB was not activated in response to coengagement of TCR and CD28 in either the ZAP-70- or SLP-76-ne gative cells, whereas stimuli that bypass these receptors (PMA plus A23187, or TNF-alpha) activated NF-kappaB normally. Protein kinase C (PKC) theta a ctivation, which is required for NF-kappaB activation, also was defective i n these cells. Reexpression of ZAP-70 restored PKC theta and NF-kappaB acti vation in response to TCR and CD28 coengagement. p95(vav) (Vav)-1 tyrosine phosphorylation was largely unperturbed in the ZAP-70-negative cells; howev er, receptor-stimulated SLP-76/Vav-1 coassociation was greatly reduced. Wil d-type SLP-76 fully restored PKC theta and NF-kappaB activation in the SLP- 76-negative cells, whereas 3YF-SLP-76, which lacks the sites of tyrosine ph osphorylation required for Vav-1 binding, only partially rescued signaling. These data illustrate the importance of the ZAP-70/SLP-76 signaling pathwa y in CD3/CD28-stimulated activation of PKC theta and NF-kappaB, and suggest that Vav-1 association with SLP-76 may be important in this pathway.