Biochemical and genetic analyses of the Tec kinases Itk and Rlk/Txk

The Tee kinases have been implicated as important components of signalling pathways downstream of lymphocyte antigen receptors. Activation of these ki nases requires two steps: (i) phosphorylation by Src family kinases and (ii ) plasma membrane localization, which is mediated by interaction between th e pleckstrin homology (PH) domains of Tee kinases and the products of phosp hoinositide-3 kinase (PI-3K). Itk and Rlk/Txk are Tee kinases expressed in T-lymphocytes. Despite similarity to other Tee kinases, Rlk/Txk lacks a PH domain and instead possesses a palmitoylated cysteine-string motif. We have found that both Rlk/Txk and Itk are phosphorylated in response to T-cell r eceptor stimulation and can be activated by phosphorylation by Sire family kinases. However, consistent with its lack of PH domain, Rlk/Txk is phospho rylated independent of PI-3K activity. Furthermore, we demonstrated that li ke Itk, Rlk/Txk is associated with lipid RAFTs (detergent-insoluble, choles terol-rich regions of the membrane), but unlike Itk, Rlk/Txk's RAFT associa tion is independent of PI-3K activity. Despite these differences, Rlk/Txk p artially compensates for loss of Itk in gene-targeted animals, suggesting o verlapping functions for these kinases.