טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBlecher-Gonen Ronnie
SubjectThe Role of The P-1 Region in the Function on SLP-76
DepartmentDepartment of Medicine
Supervisor Dr. Deborah Yablonski


Abstract

SLP-76 is a central adaptor protein in the signal transduction pathways of ITAM-containing receptors. It is necessary for the T-cell receptor (TCR)-induced activation of phospholipase C-g1 (PLC-g1), which result in the activation of both the Ras and the calcium influx pathways. The primary structure of SLP-76 includes three domains, which mediate protein-protein interactions: an NH2-terminal acidic domain containing three tyrosine phosphorylation sites, a central proline-rich domain, and a COOH-terminal SH 2 domain . Recently, a new functional region within the SLP-76 proline-rich domain was identified, named the P-1 region. The P-1 region is capable of binding to the SH3 domain of PLC-g1, and the activation of PLC-g1 is abolished upon deletion of the P-1 region. These two findings are consistent with the concept that the P-1 region may directly regulate PLC-g1, either by stabilizing its recruitment to the lipid rafts, or by inducing a conformational change in PLC-g1.

To test the first hypothesis, we introduced a raft-targeted form of PLC-g1 into mutant cells, lacking the P-1 domain of SLP-76. This construct failed to rescue TCR-induced NFAT activation. Thus, recruitment of PLC-g1 to the lipid rafts cannot be the sole function of the P-1 domain.

We precisely mapped the PLCg1-binding sequence within the P-1 domain, and created a mutant form of SLP-76 that fails to bind to the SH3 domain of PLC-g1. Surprisingly, this mutant was fully functional, mediating TCR-induced calcium flux and NFAT activation.  Another SLP-76 mutant, in which the P-1 region was scrambled to abolish any sequence-dependent protein-binding motifs, also retained functionality.

Our results suggest that the P-1 domain of SLP-76 has primarily a structural role, and that SLP-76 need not interact with PLC-g1 in order to activate it.