טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBlecher-Gonen Ronnie
SubjectThe Role and Regulation of TCR-Induced Serine
Phosphorylation of SLP76 and SLP76-Associated
Proteins
DepartmentDepartment of Medicine
Supervisor Dr. Deborah Yablonski
Full Thesis textFull thesis text - English Version


Abstract

SH2-domain-containing leukocyte protein of 76kD (SLP76) is an adaptor protein, essential for T-cell development and activation. It nucleates a protein complex that links T-cell receptor (TCR)-induced tyrosine-kinases to the activation of PLCγ1, and to transcriptional activation of IL-2.

TCR-induced phosphorylation of three N-terminal tyrosine residues of SLP76 is essential for downstream signaling. SLP76 also undergoes serine-phosphorylation on residue 376, which then binds to the adaptor protein 14-3-3. Here, we explore the role and regulation of SLP76 serine-phosphorylation and its interaction with 14-3-3.

We identified that the kinase responsible for SLP76 phosphorylation on Ser376 is associated with the SH2-domain of SLP76, and is likely to be HPK-1. SLP76 serine-phosphorylation required the N-terminal phospho-tyrosine sites and the Gads binding site of SLP76. We suggest that signaling molecules recruited to the N-terminus of SLP76, together with the adaptor Gads, promote the activation of HPK-1, which in turn phosphorylates SLP76 at Ser376.

To explore the functional significance of SLP76 serine-phosphorylation, we compared cells expressing WT-SLP76 to those expressing an S376A-SLP76 mutant. Mutation of Ser376 did not affect TCR-induced tyrosine-phosphorylation of SLP76 or PLCγ1, CD69 upregulation or AP-1 activation, and somewhat decreased NFAT activation. In contrast, the S376A mutation dramatically increased RE/AP activation. RE/AP is part of the IL-2 promoter, and is activated by co-binding of NFκB, AP-1 and CREB transcription factors. This suggests that the S376A mutation may increase activation of NFκB and/or CREB.

14-3-3 proteins form dimers containing two binding sites for serine-phosphorylated proteins. Ser376 of SLP76 occupies one of the sites, and we hypothesized that the second site may be occupied by a SLP76-associated protein. A 40kD SLP76-associated protein was identified, which inducibly bound to 14-3-3 following TCR ligation. Its association with 14-3-3 depended on SLP76 tyrosine-phosphorylation, but not on SLP76 serine-phosphorylation or CD28 co-stimulation. Using 2-dimentional gel electrophoresis we identified this 40kD protein as the adaptor Gads. Gads amino-acid sequence contains one potential 14-3-3 binding site, located within its SH2-domain. We are currently investigating whether Gads is indeed serine-phosphorylated at this site, and if so, what are the implications for TCR signaling.

Together, our results imply that phosphorylation of SLP-76 at Ser376 is related to negative regulation of the TCR pathway. We provide evidence of cross-talk between molecules bound to different regions of SLP76, leading to activation of HPK-1. The previously unknown phosphorylation-dependent interaction of Gads with 14-3-3 may have far-reaching results for the regulation of TCR signaling.