The ALX Src homology 2 domain is both necessary and sufficient to inhibit T cell receptor/CD28-mediated up-regulation of RE/AP.
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.
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Michael J
MJ
Shapiro
Penda
P
Powell
Adanma
A
Ndubuizu
Chima
C
Nzerem
Virginia Smith
VS
Shapiro
D048868:Adaptor Proteins, Signal Transducing; D001665:Binding Sites; D015153:Blotting, Western; D018106:CD28 Antigens / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004789:Enzyme Activation; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008156:Luciferases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism; D013997:Time Factors; D015533:Transcriptional Activation; D014162:Transfection; D015854:Up-Regulation; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D018106:CD28 Antigens; D002352:Carrier Proteins; C442142:HSH2D protein, human; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D005982:Glutathione Transferase