Characterization of Gads N Terminal SH3 Domain Role on Modulating TCR+ CD28 Co-Stimulation Responsiveness

GRB2-related adaptor downstream of Shc (GADS) is a member of the Grb2-family adaptor

proteins, composed of a central SH2 domain and a proline-rich linker flanked by two SH3

domains. Via its different domains, Gads can mediate diverse interactions with key signaling

proteins. These interactions may amplify or attenuate the intensity of T cell receptor signals,

or change the balance of downstream pathways, leading to different cellular responses.

Early studies defined the role of Gads SH2 and C terminal SH3 domains, which interact with

two adaptor proteins LAT and SLP76 respectively, thereby bridging these two proteins to

create a large complex that activates the enzyme-PLCγ and contributes to T cell responses.

The N terminal SH3 domain (N-SH3) of Gads has no known function or binding partners,

yet it is extremely conserved.

We uncovered a newly phosphorylation site pY45 in the Gads N terminal SH3 domain. Our data show that

this site is phosphorylated by the tec family kinase ITK in vitro and in intact cells .

Functional comparison of a Gads-deficient T cell line (dG32), reconstituted with wild type Gads,

or Gads muted to Y45F on its N-SH3 domain, revealed that the Y45F mutation abrogates activation

of the RE/AP composite element which is dependent on CD28 signaling, while the CD69 activation marker

and the calcium flux were not affected.

Based on these preliminary results, Gads Y45 phosphorylation may be involved in uncovered pathway

leading to the activation of the RE/AP transcriptional element via a co-stimulation dependent manner.

In conclousion , we aim to explore the Y45 phosphorylation mechanism using in-vitro and in-vivo models

to facilitate a better understanding of the TCR+ CD28 Co-stimulation signaling responsiveness.