In vivo disruption of T cell development by expression of a dominant‐negative polypeptide designed to abolish the SLP‐76/Gads interaction

Abstract

Multi-molecular complexes nucleated by adaptor proteins play a central role in signal transduction. In T cells, one central axis consists of the assembly of several signaling proteins linked together by the adaptors linker of activated T cells (LAT), Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), and Grb2-related adaptor downstream of Shc (Gads). Each of these adaptors has been shown to be important for normal T cell development, and their proper sub-cellular localization is critical for optimal function in cell lines. We previously demonstrated in Jurkat T cells and a rat basophilic leukemic cell line that expression of a 50-amino acid polypeptide identical to the site on SLP-76 that binds to Gads blocks proper localization of SLP-76 and SLP-76-dependent signaling events. Here we extend these studies to investigate the ability of this polypeptide to inhibit TCR-induced integrin activity in Jurkat cells and to inhibit in vivo thymocyte development and primary T cell function. These data provide evidence for the in vivo function of a dominant-negative peptide based upon the biology of SLP-76 action and suggest the possibility of therapeutic potential of targeting the SLP-76/Gads interaction. IntroductionStimulation of T cells through the T cell receptor (TCR) leads to recruitment and localization of several critical signaling proteins, which results in the generation of a macro-molecular signaling complex. Key components of this complex, or signalosome, include the adaptors Src homology 2 (SH2) domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), and Grb2-related adaptor downstream of Shc (Gads), which serves to link SLP-76 and its associated molecules to the transmembrane adaptor linker of activated T cells (LAT) [1].As an adaptor protein, SLP-76 possesses no enzymatic activity but functions via interaction with multiple proteins by way of three structural domains [2][3][4]. The N terminus harbors three tyrosines that become phos- phorylated upon TCR engagement [5,6] and have been reported to serve as docking sites for the guanine nucleotide exchange factor Vav, the adaptor protein Nck, the Tec family kinase Itk, and the p85 subunit of PI3K [7][8][9][10][11][12][13][14][15][16]. A single SH2 domain resides in the C-terminal portion of SLP-76 and links it to the adhesion and degranulation-promoting adaptor protein (ADAP) and to the hematopoietic progenitor kinase 1 [17][18][19]. Hematopoietic progenitor kinase 1, in turn, has been shown to phosphorylate SLP-76, creating a binding site for 14-3-3, a negative regulator of TCR signaling [20,21]. The central proline-rich region binds phospholipase Cc- Targeted deletion of SLP-76 in mice results in an inability of thymocytes to progress past the CD4 -CD8 -double-negative (DN) stage of development and thus a complete absence of mature T cells [28,29]. Since T cells do not develop in the absence of SLP-76, much of our knowledge of the role of SLP-76 in mature TCR signaling has been derived from the study of J14 ...

Src Homology 2-Domain Containing Leukocyte-Specific Phosphoprotein of 76 kDa Is Mandatory for TCR-Mediated Inside-Out Signaling, but Dispensable for CXCR4-Mediated LFA-1 Activation, Adhesion, and Migration of T Cells

“…Our previous data suggested that the signaling pathways controlled by the Gads/SLP76 complex also include those regulating adhesion processes (22). In this study, we confirm this hypothesis by showing that suppression of SLP76 expression by RNAi in human T cells or the Lysates were analyzed by Western blotting using the indicated Abs.…”

“…In line with this idea is the observation that disruption of the interaction between SLP76 and Gads blocks TCR-mediated adhesion to ICAM-1 (22). Furthermore, it was shown that mutation of those tyrosine residues within ADAP, which are believed to mediate the interaction between ADAP and SLP76, blocks TCR-mediated activation of LFA-1 (23).…”

“…Indeed, it was shown recently that SLP76 regulates CXCR4-induced Ca 2ϩ flux and ERK1/2 phosphorylation (31). Conversely, we had demonstrated that disruption of the SLP76/ Gads association, albeit impairing TCR-mediated signaling processes, does not affect CXCR4-induced Ca 2ϩ flux and chemotaxis (22). Thus, it is unclear whether SLP76 is also important for CXCR4-mediated activation of integrins and chemotaxis.…”

“…By using a small peptide that disrupts the constitutive interaction between SLP76 and Gads, we recently provided evidence that formation of the LAT/Gads/SLP76 signaling platform at the plasma membrane is required for TCR-mediated integrin activation (22). However, we formally could not exclude the possibility that the functional effects exerted by the Gads-binding fragment were not due to targeting the SLP76/Gads complex, but rather to disruption of a distinct signaling pathway that regulates intergrin activation in response to TCR stimulation.…”

SKAP-55, SKAP-55-related and ADAP adaptors modulate integrin-mediated immune-cell adhesion

“…Disruption of upstream components such as p56lck, ZAP-70, LAT, SLP-76 or GADS-SLP-76 interaction results in a generic impairment of LFA-1-mediated adhesion [17][18][19] (Figure 2). A recent study has shown that a polypeptide identical to the site on SLP-76 that binds to GADS can also inhibit TCR-induced integrin adhesion and thymocyte development [20].Despite the importance of these general upstream events, the quest over the past few years has been to identify the key downstream effector adaptors of the 'inside-out' adhesion pathway. A major advance in this area has recently come with the discovery of immune-cell adaptors ADAP [previously known as Fyn T-binding protein ( …”