SLP-76 sterile α motif (SAM) and individual H5 α helix mediate oligomer formation for microclusters and T-cell activation.
Despite the importance of the immune adaptor SLP-76 in T-cell immunity, it has been unclear whether SLP-76 directly self-associates to form higher order oligomers for T-cell activation. In this study, we show that SLP-76 self-associates in response to T-cell receptor ligation as mediated by the N-terminal sterile α motif (SAM) domain. SLP-76 co-precipitated alternately tagged SLP-76 in response to anti-CD3 ligation. Dynamic light scattering and fluorescent microscale thermophoresis of the isolated SAM domain (residues 1-78) revealed evidence of dimers and tetramers. Consistently, deletion of the SAM region eliminated SLP-76 co-precipitation of itself, concurrent with a loss of microcluster formation, nuclear factor of activated T-cells (NFAT) transcription, and interleukin-2 production in Jurkat or primary T-cells. Furthermore, the H5 α helix within the SAM domain contributed to self-association. Retention of H5 in the absence of H1-4 sufficed to support SLP-76 self-association with smaller microclusters that nevertheless enhanced anti-CD3-driven AP1/NFAT transcription and IL-2 production. By contrast, deletion of the H5 α helix impaired self-association and anti-CD3 induced AP1/NFAT transcription. Our data identified for the first time a role for the SAM domain in mediating SLP-76 self-association for T-cell function.
['Liu|Hebin|H|', 'Thaker|Youg Raj|YR|', 'Stagg|Loren|L|', 'Schneider|Helga|H|', 'Ladbury|John E|JE|', 'Rudd|Christopher E|CE|']
D048868:Adaptor Proteins, Signal Transducing D020816:Amino Acid Motifs D000595:Amino Acid Sequence D001665:Binding Sites D017252:CD3 Complex D002478:Cells, Cultured D002942:Circular Dichroism D006801:Humans D007376:Interleukin-2 D019169:Jurkat Cells D008027:Light D008213:Lymphocyte Activation D018613:Microscopy, Confocal D008958:Models, Molecular D009154:Mutation D050778:NFATC Transcription Factors D010750:Phosphoproteins D011485:Protein Binding D055503:Protein Multimerization D017434:Protein Structure, Tertiary D011948:Receptors, Antigen, T-Cell D012542:Scattering, Radiation D013601:T-Lymphocytes D014158:Transcription, Genetic
SLP-76 T-cell SAM Domain Adaptor Proteins Biophysics Immunology Protein Complexes Signal Transduction