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.
From the Cell Signalling Section, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom.
Hebin
H
Liu
Youg Raj
YR
Thaker
Loren
L
Stagg
Helga
H
Schneider
John E
JE
Ladbury
Christopher E
CE
Rudd
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D017252:CD3 Complex / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D002942:Circular Dichroism; D006801:Humans; D007376:Interleukin-2 / Q000276:immunology / Q000378:metabolism; D019169:Jurkat Cells; D008027:Light; D008213:Lymphocyte Activation / Q000276:immunology*; D018613:Microscopy, Confocal; D008958:Models, Molecular; D009154:Mutation; D050778:NFATC Transcription Factors / Q000235:genetics; D010750:Phosphoproteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011948:Receptors, Antigen, T-Cell / Q000276:immunology* / Q000378:metabolism; D012542:Scattering, Radiation; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D014158:Transcription, Genetic
D048868:Adaptor Proteins, Signal Transducing; D017252:CD3 Complex; D007376:Interleukin-2; D050778:NFATC Transcription Factors; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; C092947:SLP-76 signal Transducing adaptor proteins
Adaptor Proteins; Biophysics; Immunology; Protein Complexes; SAM Domain; SLP-76; Signal Transduction; T-cell