Requirements for activation and RAFT localization of the T-lymphocyte kinase Rlk/Txk

We demonstrate that Rlk is also associated with RAFTs, despite its lack of a pleckstrin homology domain. Rlk RAFT association requires the cysteine-string motif and is independent of PI3 Kinase activity. We further demonstrate that Rlk can be phosphorylated and activated by Src kinases, leading to a decrease in its half-life. A specific tyrosine in the activation loop of Rlk, Y420, is required for phosphorylation and activation, as well as for decreased stability, but is not required for lipid RAFT association. Mutation of this tyrosine also prevents increased tyrosine phosphorylation of Rlk after stimulation of the T cell receptor, suggesting that Rlk is phosphorylated by Src family kinases in response to T cell receptor engagement.Like the other related Tec kinases, Rlk is associated with lipid RAFTs and can be phosphorylated and activated by Src family kinases, supporting a role for Rlk in signaling downstream of Src kinases in T cell activation.Engagement of antigen receptors on lymphocytes leads to the rapid sequential activation of non-receptor tyrosine kinases, including well-studied members of the Src and the ZAP-70/Syk families [1,2]. Recently, the Tec kinases have also been implicated as important components of signaling from antigen and other lymphocyte cell surface receptors (reviewed in [3,4,5].) This subfamily includes six members: Btk, Tec, Itk/Tsk/Emt, Bmx/Emt, Dsrc29, and Rlk/Txk [3,6,7]. The importance of the Tec kinases in antigen receptor signaling was first demonstrated by the observation that mutations in BTK result in the human disease X-linked agammaglobulinemia (XLA) and the murine counterpart X-linked immunodeficiency (xid). These severe B cell immunodeficiencies are characterized by diminished numbers of mature B cells and reduced immunoglobulin levels associated with impaired PLC-γ activation and Ca++ mobilization in response to surface IgM stimulation (sIgM) [8,9,10,11]. Btk kinase activity and tyrosine phosphorylation increase upon stim