The ALX Src homology 2 domain is both necessary and sufficient to inhibit T cell receptor/CD28-mediated up-regulation of RE/AP

Michael J. Shapiro, Penda Powell, Adanma Ndubuizu, Chima Nzerem, Virginia M Shapiro

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)40647-40652
Number of pages6
JournalJournal of Biological Chemistry
Volume279
Issue number39
DOIs
StatePublished - Sep 24 2004
Externally publishedYes

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src Homology Domains
T-Cell Antigen Receptor
Up-Regulation
Chemical activation
T-cells
Phosphorylation
T-Lymphocytes
Interleukin-2
Jurkat Cells
Phosphoproteins
Protein Binding
Transcriptional Activation
Tyrosine
Cytoplasm
Proteins
Binding Sites
Mutation
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

The ALX Src homology 2 domain is both necessary and sufficient to inhibit T cell receptor/CD28-mediated up-regulation of RE/AP. / Shapiro, Michael J.; Powell, Penda; Ndubuizu, Adanma; Nzerem, Chima; Shapiro, Virginia M.

In: Journal of Biological Chemistry, Vol. 279, No. 39, 24.09.2004, p. 40647-40652.

Research output: Contribution to journalArticle

Shapiro, Michael J. ; Powell, Penda ; Ndubuizu, Adanma ; Nzerem, Chima ; Shapiro, Virginia M. / The ALX Src homology 2 domain is both necessary and sufficient to inhibit T cell receptor/CD28-mediated up-regulation of RE/AP. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 39. pp. 40647-40652.
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