Cloning and Characterization of ALX, an Adaptor Downstream of CD28

Abstract

T cell activation requires two signals: specific recognition of antigen through the T cell receptor (TCR) and a costimulatory signal provided primarily by CD28 in naïve T cells. We cloned a novel gene with considerable homology to RIBP/TSAd/Lad, an adaptor involved in T cell activation and interleukin-2 (IL-2) promoter activation. Expression of this gene is limited to the spleen and thymus. We have named this gene ALX, adaptor in lymphocytes of unknown function X. Because the related adaptor RIBP is involved in IL-2 regulation, we investigated whether ALX had a similar function. ALX overexpression in Jurkat T cells results in inhibition of IL-2 promoter activation after stimulation with superantigen. The IL-2 promoter contains several binding sites for transcription factors including the composite element RE/AP, which is the primary site of CD28 transcriptional activation. ALX overexpression had the greatest effect on the activation of a RE/AP reporter as opposed to an AP-1 reporter. Interestingly, ALX overexpression strongly inhibited RE/AP activation in response to anti-CD28/phorbol 12-myristate 13-acetate (PMA) stimulation but had minimal effect when anti-TCR/PMA was used. Therefore, it appears that ALX may function downstream of CD28 costimulation during T cell activation. In addition, the mobility of ALX shifts upon TCR/CD28 costimulation to a greater extent than what is observed with either stimulus alone demonstrating that ALX is a target of both TCR and CD28 costimulatory signaling pathways.The activation of T cells is critical to the generation of an immune response. Minimally, two signals are required to activate resting T cells into effector T cells: an antigen-specific signal through the T cell receptor (TCR) 1 and a second antigenindependent "costimulatory" signal, which is primarily pro- 2). The biochemical events downstream of CD28 and how they synergize with TCR signals to result in T cell activation are much less understood. A critical step in T cell activation is the induction of the IL-2 gene, which occurs through both a transcriptional up-regulation of its promoter and increased stability of its mRNA (3, 4).One molecule involved in TCR signaling is the adaptor molecule TSAd (also designated Lad, and for the mouse ortholog RIBP) (5). This adaptor was identified in separate two-hybrid screens for proteins that associated with the Tec family kinases Rlk and Itk (6), the Src family kinase Lck (7), and the mitogenactivated protein kinase kinase kinase MEKK2 (8). Although the mechanism of TSAd action is not well characterized, a role for TSAd in the regulation of IL-2 was demonstrated in the RIBP knock-out mouse (6). These mice have no gross abnormalities in T cell development, but mature T cells show a moderate defect (ϳ70% decrease) in proliferation and IL-2 production upon TCR or TCR/CD28 stimulation. TSAd has been reported to localize to the cytoplasm in T cells and to translocate to the immunological synapse during T cell activation (8), although it also has been reported to be primarily nucle...

The C terminus of T cell‐specific adapter protein (TSAd) is necessary for TSAd‐mediated inhibition of Lck activity

“…Recently the adapter protein ALX or HSH2 was found to have substantial homology to TSAd [30]. Both ALX and TSAd were found to inhibit the activity of IL-2 promoter constructs [9,30]; however, TSAd has also been found to promote IL-2 promoter activity [10], and activated T cells from TSAd knockout mice express less IL-2 [6].…”

“…TSAd cDNA was cloned into the expression vectors pEF/HA and pEF6/Myc-His B [3,9,30]. The Lck SH3 domain was subcloned into the bacterial expression vector pGEX-6P (Amersham Biosciences, Uppsala, Sweden).…”

“…Jurkat TAg T cells (20Â10 6 cells in RPMI 1640) were transfected with 5-20 lg plasmid DNA using a BTX electroporator (Genetronix, San Diego, CA) at 200 V, 70 ms. Stable transfectants have been described [9,30]. Transient transfectants were cultured for 16-48 h. 293T cells (2Â10 6 -3Â10 6 ) were transfected for 5-8 h with 0.5-2 lg DNA in 25 ll lipofectin (GIBCO-BRL) and further propagated for 16-24 h.…”

The Adaptor Protein HSH2 Attenuates Apoptosis in Response to Ligation of the B Cell Antigen Receptor Complex on the B Lymphoma Cell Line, WEHI-231

“…Based on a search of expressed sequence tag databases using the BLAST algorithm, we identified the mouse and rat homologues of human HSH2 consisting of 334 and 335 amino acids, respectively. Recently, another group has cloned the mouse homologue of HSH2 referred to as ALX (29). Primary amino acid sequence analysis of mouse HSH2 revealed an N-terminal SH2 domain and three conserved PXXP motifs that are likely to facilitate protein-protein interactions; however, no domains associated with catalytic activity were detected.…”

“…HSH2 is expressed predominantly in cells of the lymphoid lineage but can be detected in cells of the myelomonocytic lineage as well (28,29). HSH2 contains a single SH2 domain, three conserved proline-rich regions and two tyrosine residues that are potential sites of phosphorylation.…”