Itk/Emt/Tsk activation in response to CD3 cross-linking in Jurkat T cells requires ZAP-70 and Lat and is independent of membrane recruitment

The Tec family tyrosine kinase, Itk has been implicated in T cell antigen r eceptor (TCR) signaling, yet little is known about Itk regulation. Here, we investigate the role of the tyrosine kinase ZAP-70 in regulating Itk. Wher eas Itk was activated in Jurkat T cells in response to CD3 cross-linking, I tk activation was defective in the ZAP-70-deficient P116 Jurkat T cell line . Itk responsiveness to TCR engagement was restored in Pile cells stably tr ansfected with ZAP-70 cDNA, ZAP-70 itself could not directly phosphorylate the Itk kinase domain, indicating an indirect regulation of Itk activity. N o role was found for ZAP-70 in regulating Itk recruitment to the plasma mem brane, an event that has been suggested to be rate-limiting for the activat ion of Tec family kinases. Indeed, Itk was found to be constitutively targe ted to the membrane fraction in both Jurkat and P116 cells. Lat, a prominen t in vivo substrate of ZAP-70 that mediates assembly of multimolecular sign aling complexes at the plasma membrane of T cells was also found to be requ ired for TCR-stimulated Itk activation. Itk could not be activated by CD3 c ross-linking in a Lat-negative cell line, unless Lat expression was restore d. Lat and Itk were observed to co-associate in response to CD3 crosslinkin g in Jurkat T cells, but not in pile T cells. The Lat-Itk association corre lated with Lat tyrosine phosphorylation, which was deficient in the P116 T cells. These data suggest that ZAP-70 and Lat play important, probably sequ ential, roles in regulating the activation of Itk following TCR engagement.