Requirements for activation and RAFT localization of the T-lymphocyte kinase Rlk/Txk.
BACKGROUND
The Tec family kinases are implicated in signaling from lymphocyte antigen receptors and are activated following phosphorylation by Src kinases. For most Tec kinases, this activation requires an interaction between their pleckstrin homology (PH) domains and the products of phosphoinositide 3-Kinase, which localizes Tec kinases to membrane RAFTs. Rlk/Txk is a Tec related kinase expressed in T cells that lacks a pleckstrin homology domain, having instead a palmitoylated cysteine-string motif. To evaluate Rlk's function in T cell receptor signaling cascades, we examined the requirements for Rlk localization and activation by Src family kinases.


RESULTS
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.


CONCLUSIONS
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.
Chamorro
M
M
National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. chamorrm@mskcc.org
Czar
M J
MJ
Debnath
J
J
Cheng
G
G
Lenardo
M J
MJ
Varmus
H E
HE
Schwartzberg
P L
PL
D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D019169:Jurkat Cells; D021962:Membrane Microdomains / Q000201:enzymology*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology; D014443:Tyrosine / Q000378:metabolism
D011518:Proto-Oncogene Proteins; D014443:Tyrosine; C089848:Tec protein-tyrosine kinase; D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn