טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentLibman Evgeny
Subject14-3-3 Protein Binding to GADS and SLP76 as Negative-
Feedback in TCR-signaling
DepartmentDepartment of Medicine
Supervisor Dr. Deborah Yablonski
Full Thesis textFull thesis text - English Version


Abstract

T cell activation starts with activation of the T cell receptor (TCR) on the cell surface, and ends in the nucleus, with the ignition of transcription of the interleukin 2 gene. TCR activation initiates a series of tyrosine phosphorylation events, which trigger the assembly of a signaling complex, nucleated by three adaptor molecules, SH2-domain containing protein of 76kD (SLP-76), Grb2-related Adapter Downstream of Shc (GADS) and Linker for Activation of T-cells (LAT), which recruit a number of enzymes. This complex controls the progress of the signal.

Although TCR-induced signaling begins with tyrosine phosphorylation, the majority of phosphorylation events are actually on serine and threonine residues. One of the consequences of these phosphorylation events is binding of 14-3-3 proteins, a large family of dimeric proteins, with regulatory properties, which bind to phosphorylated serine or threonine residues in certain contexts. Possible effects of 14-3-3 binding will be considered here.

SLP-76 undergoes phosphorylation on S376 and this site serves as a docking site for 14-3-3 protein, an event which has down-regulatory properties. We explored the functional significance of SLP-76 serine-phosphorylation, by comparing cells expressing wild type SLP-76 to those expressing an S376A-SLP-76 mutant. The S376A mutation dramatically increased activation of a luciferase reporter gene driven by a multimerized RE/AP element. This element is part of the IL-2 promoter, and is activated by co-binding of NF-κB, AP-1 and CREB transcription factors. The S373A mutation did not affect NF-κB or AP1 activation, but RE/AP was hypersensitive to calcium flux, which implies CREB involvement. But we couldn’t show directly that CREB is affected by the S376A mutation on SLP-76.

In addition to SLP-76, GADS also binds 14-3-3 after TCR stimulation. Previously, our lab showed that GADS phosphorylation depends on tyrosine phosphorylation of SLP-76. We showed that GADS phosphorylation and binding to 14-3-3 protein did not depend on S376 of SLP-76 and but did depend on the SH2 domain of SLP-76. In addition the time course of 14-3-3 binding to SLP-76 and GADS was different. We identified the 14-3-3 binding site on GADS as T262.  In vitro both HPK-1 and PKA phosphorylated GADS at T262 as well as other sites on GADS.

We hypothesize that docking of 14-3-3 to GADS causes the SLP-76-GADS-LAT complex to be dismantled. Thus, we hypothesize that 14-3-3 binding to GADS inhibits TCR signaling, and may serve as a down-regulatory mechanisms for TCR signaling. This model of action is consistent with our results and other recently published results, but requires further investigation.