TY - JOUR
T1 - The ALX Src homology 2 domain is both necessary and sufficient to inhibit T cell receptor/CD28-mediated up-regulation of RE/AP
AU - Shapiro, Michael J.
AU - Powell, Penda
AU - Ndubuizu, Adanma
AU - Nzerem, Chima
AU - Shapiro, Virginia Smith
PY - 2004/9/24
Y1 - 2004/9/24
N2 - Activation of naive T cells occurs when two signals are received. The first signal is received through the T cell antigen receptor (TCR), and a second costimulatory signal is primarily provided by CD28. We have recently identified a novel adaptor molecule, ALX, which is expressed exclusively in hematopoietic cells. ALX contains several sites for potential protein-protein interaction, including an Src homology 2 (SH2) domain, four PXXP polyproline sequences, and two likely sites of tyrosine phosphorylation. Overexpression of ALX inhibits the transcriptional activation of the interleukin 2 promoter during T cell activation, specifically affecting CD28-mediated activation of the RE/AP element of the interleukin 2 promoter. To understand how ALX functions downstream of CD28, we generated a panel of site-directed mutants as well as truncations in which potential protein-binding sites were mutated or absent. We found that the ALX SH2 domain is both necessary and sufficient to mediate inhibition of RE/AP activation. Mutation of the SH2 domain did not affect ALX expression, relative localization in the cytoplasm and nucleus, phosphorylation, or a mobility shift in response to TCR signaling alone. However, an activation-induced mobility shift triggered by CD28 was reduced in the ALX SH2 domain mutant. In addition, the isolated ALX SH2 domain was found to associate with a phosphoprotein from Jurkat T cells on TCR/CD28 stimulation. Therefore, the ALX SH2 domain plays a critical role in ALX function downstream of CD28.
AB - Activation of naive T cells occurs when two signals are received. The first signal is received through the T cell antigen receptor (TCR), and a second costimulatory signal is primarily provided by CD28. We have recently identified a novel adaptor molecule, ALX, which is expressed exclusively in hematopoietic cells. ALX contains several sites for potential protein-protein interaction, including an Src homology 2 (SH2) domain, four PXXP polyproline sequences, and two likely sites of tyrosine phosphorylation. Overexpression of ALX inhibits the transcriptional activation of the interleukin 2 promoter during T cell activation, specifically affecting CD28-mediated activation of the RE/AP element of the interleukin 2 promoter. To understand how ALX functions downstream of CD28, we generated a panel of site-directed mutants as well as truncations in which potential protein-binding sites were mutated or absent. We found that the ALX SH2 domain is both necessary and sufficient to mediate inhibition of RE/AP activation. Mutation of the SH2 domain did not affect ALX expression, relative localization in the cytoplasm and nucleus, phosphorylation, or a mobility shift in response to TCR signaling alone. However, an activation-induced mobility shift triggered by CD28 was reduced in the ALX SH2 domain mutant. In addition, the isolated ALX SH2 domain was found to associate with a phosphoprotein from Jurkat T cells on TCR/CD28 stimulation. Therefore, the ALX SH2 domain plays a critical role in ALX function downstream of CD28.
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U2 - 10.1074/jbc.M404198200
DO - 10.1074/jbc.M404198200
M3 - Article
C2 - 15284240
AN - SCOPUS:4644329608
SN - 0021-9258
VL - 279
SP - 40647
EP - 40652
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 39
ER -