Role of Two Adaptor Molecules SLP-76 and LAT in the PI3K Signaling Pathway in Activated T Cells

Abstract

Previously, we identified p85, a subunit of PI3K, as one of the molecules that interacts with the N-terminal region of Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76). We also demonstrated that tyrosine phosphorylation either at the 113 and/or 128 position is sufficient for the association of SLP-76 with the Src homology 2 domain near the N terminus of p85. The present study further examines the role of the association of these two molecules on the activation of PI3K signaling cascade. Experiments were done to determine the role of SLP-76, either wild-type, tyrosine mutants, or membrane-targeted forms of various SLP-76 constructs, on the membrane localization and phosphorylation of Akt, which is an event downstream of PI3K activation. Reconstitution studies with these various SLP-76 constructs in a Jurkat variant cell line that lacks SLP-76 or linker for activation of T cells (LAT) show that the activation of PI3K pathway following TCR ligation requires both SLP-76 and LAT adaptor proteins. The results suggest that SLP-76 associates with p85 after T cell activation and that LAT recruits this complex to the membrane, leading to Akt activation.

Annular PIP3accumulation controls actin architecture and modulates cytotoxicity at the immunological synapse

“…The TCR signaling complex contains several phosphoproteins that could potentially engage PI3K, including LAT and Slp76, two scaffolding molecules known to interact with p85 in a pTyr-dependent manner (Zhang et al, 1998;Shim et al, 2004Shim et al, , 2011. Hence, we favor a model in which early TCR signaling drives pTyr-mediated recruitment and allosteric activation of class IA PI3K in centripetally motile microclusters.…”

“…It remains controversial, however, precisely which isoforms contribute to this process (Alcázar et al, 2007;Garçon et al, 2008;Sauer et al, 2008), and it is also unclear how these proteins might be recruited and activated by TCR signaling. Previous studies in T cells have focused on the role of phosphotyrosine (pTyr)-containing signaling motifs that can bind and allosterically activate certain PI3K isoforms (Carpenter et al, 1993;Holt et al, 1994;Pagès et al, 1994;Zhang et al, 1998;Shim et al, 2004Shim et al, , 2011. However, class I PI3Ks also interact with the small GTPase Ras (Rodriguez-Viciana et al, 1994, 1996, which functions synergistically with pTyr peptides to induce full PI3K activity (Jimenez et al, 2002).…”

ARAP, a Novel Adaptor Protein, Is Required for TCR Signaling and Integrin-Mediated Adhesion

“…All studies were approved by the Institutional Human Ethics Review Board of Ewha Womans University Medical Center. The isolation of mononuclear cells and T cell fraction using Ficoll-Paque Plus density centrifugation followed by depletion of non-T cells with superparamagnetic microbeads was described previously (29). Isolated T cells were resuspended in RPMI 1640 medium and rested at 37˚C.…”

“…Conjugation of Jurkat T cells and Raji B cells as APCs, followed by immunocytochemistry, was described previously (29). Jurkat T cells were transfected with expression plasmids encoding GFP-ARAP constructs, and Raji B cells were stained with CellTracker Orange CMTMR.…”

“…The Brij-insoluble rafts fractions were separated from the cell lysates by sucrose density gradient ultracentrifugation as described previously (18,29). Jurkat T cells (1 3 10 8 ) were stimulated with either control Ig or with anti-CD3 Ab (2 mg/ml) for 5 min at 37˚C.…”