Non-T cell activation linker (NTAL): a transmembrane adaptor protein involved in immunoreceptor signaling

Abstract

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non-T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcgamma- and Fc epsilon -receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non-T cells.

Figure 1.

NTAL isolation. (A) Proteins of myeloid cell (HL-60) vs. T cell (Jurkat) membrane microdomains (GEMs) phosphorylated under the conditions of the in vitro kinase assay; a pattern very similar to that of HL-60 was observed also in the case of the THP-1 cell preparation (unpublished data). (B) Proteins of THP-1 GEMs were mixed with the preparation obtained by in vitro kinase assay, separated by 2-dimensional gel electrophoresis and detected by silver staining or autoradiography. Arrows indicate the position of pp30.

Figure 2.

Predicted amino acid sequence of human NTAL (product of the WBSCR5 gene). The putative transmembrane region is boxed, the potential palmitoylation sequence, the tyrosine-x-asparagine motifs, and all other tyrosines are in bold and underlined. These sequence data are available from GenBank/EMBL/DDBJ under accession no. AAF74978.

Figure 3.

Comparison of the exon-intron organization and of the splice frame diagrams of the mouse genes encoding LAT and NTAL, respectively. Exons are shown by boxes; the positions of the initiation (Start) and termination (Stop) codons are indicated by vertical arrows. Based on splice frame junctions, three types of introns can be distinguished in a given gene: phase 0 intron interrupts the reading frame between two consecutive codons, whereas phase 1 and phase 2 introns interrupt the reading frame between the first and the second nucleotide of a codon or between the second and the third nucleotide of a codon, respectively (reference 40). According to that classification, the phase class of each intron is indicated by a solid circle on the diagram shown below each gene. For the sake of clarity, the length of introns is not drawn to scale. The structure of the mouse NTAL (WBSCR5) gene is reported in (reference 25) and that of LAT in this paper.

Figure 4.

Expression of NTAL. (A) cDNA encoding human NTAL was expressed in J.CaM2.5 cells and the protein product was visualized by Western blotting of the transfectants detergent lysate as compared with Ramos cells (expressing endogenous NTAL). (B) Western blotting of the indicated subpopulations of human peripheral blood cells (immunostaining for NTAL or Erk; the latter was used as a loading control).

Figure 5.

Tissue and subcellular localization of NTAL. (A) Paraffin section of lymphoid tissue immunoperoxidase stained for NTAL; the major positive structures are germinal centers. (B) Localization of NTAL in buoyant detergent-resistant microdomains (GEMs). THP-1 cells were solubilized in the presence of 3% nonionic detergent Brij-58 or 1% laurylmaltoside (LM; a detergent known to disrupt GEMs) and subjected to sucrose density gradient ultracentrifugation; the fractions (numbered from top to bottom) were analyzed by Western blotting. (C) Biosynthetic labeling of NTAL with [³H]palmitate; NTAL immunoprecipitate was analyzed by SDS-PAGE followed by fluorography of the gel. (D) Plasma membrane localization of NTAL (green) as determined by confocal microscopy in THP-1 cells and J.CaM2.5-NTAL transfectants; nuclei are shown in red.

Figure 6.

Induction of NTAL tyrosine phosphorylation and association with cytoplasmic signaling proteins. (A) THP-1 cells or purified human monocytes were stimulated via their FcγRI receptors, Ramos cells or murine B lymphocytes via BCR, and mouse BMMC via FcɛRI receptors. NTAL was immunoprecipitated from unstimulated (−) or stimulated (+) cells and analyzed by SDS-PAGE and Western blotting using anti-phosphotyrosine antibody to visualize tyrosine-phosphorylated NTAL (top panel). The bottom panel represents immunostaining of NTAL in the same samples and in the same position of the blot (around 30 kD). (B) The same NTAL immunoprecipitates as shown in part A were analyzed by Western blotting using antibodies to the indicated associated molecules. (C) Blots of NTAL immunoprecipitates from unstimulated (−) or anti-BCR-stimulated (+) Ramos cells were immunostained by antibodies to NTAL or ubiquitin (Ubq.). Only the relevant parts of the blots are shown in parts A and B, corresponding to the size of the relevant proteins.

Figure 7.

Kinases phosphorylating NTAL. (A) Inhibition of NTAL tyrosine phosphorylation in THP-1 cells stimulated via FcγRI by the indicated PTK inhibitors. NTAL immunoprecipitates prepared from the treated and control cells were analyzed by Western blotting to detect P-Tyr or NTAL, respectively. (B) Phosphorylation of NTAL coexpressed in 293T cells with various Src- and Syk-family kinases. Total cell lysates were analyzed by SDS-PAGE and Western blotting to detect the indicated molecules.

Figure 8.

Functional analysis of NTAL in LAT-defective J.CaM2.5 transfectants. (A) NTAL immunoprecipitates obtained from unstimulated (−) or anti-CD3 stimulated (+) J.CaM2.5 mutants and J.CaM2.5-NTAL transfectants were analyzed by Western blotting for the presence of the indicated molecules. The top panel corresponds to tyrosine-phosphorylated NTAL (30 kD). (B) Wild-type Jurkat, J.CaM2.5, and J.CaM2.5-NTAL transfectants were stimulated by anti-CD3 IgM mAb (added at time points indicated by arrows) and increase of cytoplasmic Ca²⁺ was measured. (C) Wild-type Jurkat, J.CaM2.5, and J.CaM2.5-NTAL transfectants were stimulated by optimally diluted anti-CD3 IgM mAb and after 5 min of activation Erk1/2 was detected in the cell lysates by Western blotting using anti-phospho Erk antibody; bottom panel represents control staining by anti-Erk. (D) J.CaM2.5 cells transiently transfected with the indicated FLAG-tagged constructs were stimulated for 2 min by anti-CD3 and anti-CD28 mAbs and activation of Erk1/2 was detected as in C (top panel); presence of equal amounts of Erk1/2 in all samples was ascertained (middle panel) and the level of expression of individual FLAG-tagged proteins was determined (bottom panel).