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

Brdicka T, Imrich M, Angelisová P, Brdicková N, Horváth O, Spicka J, Hilgert I, Lusková P, Dráber P, Novák P, Engels N, Wienands J, Simeoni L, Osterreicher J, Aguado E, Malissen M, Schraven B, Horejsí V - J. Exp. Med. (2002)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4, Czech Republic.

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.

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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.
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fig3: 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.

Mentions: In vitro kinase assays performed on GEMs immunoprecipitated from myeloid cell lines HL-60 and THP-1 revealed the presence of an unidentified 30 kD phospho-protein (pp30) which was not detectable under similar conditions in T cells (Fig. 1 A). To further characterize this protein, the in vitro labeled proteins were mixed with GEMs prepared from 5 × 108 THP-1 cells as described previously (18). This mixture was then subjected to two-dimensional gel electrophoresis and a doublet of acidic protein spots of 29–30 kD visualized by silver staining was found to colocalize with radiolabeled pp30 (Fig. 1 B). The spots were excised, digested in-gel with trypsin, and resulting peptides were analyzed by mass spectrometry (MALDI-TOF). Database searching revealed that six of the peptides (Table I) fit precisely to those predicted for a so far uncharacterized protein encoded by a previously cloned full-length cDNA corresponding to a broadly expressed human gene termed WBSCR5 which is located on human chromosome 7 (7q11.23; references 24 and 25). The WBSCR5 cDNA codes for a polypeptide of 243 amino acid residues (Fig. 2) and a predicted molecular weight of 26.550 daltons while the predicted mouse homologue is shorter by 40 amino acid residues and is encoded by a gene residing on chromosome 5 (25). The protein resembles in its general organization the GEM-associated transmembrane adaptor proteins PAG/Cbp (18, 26) and LAT (4). Thus, it consists of a very short NH2-terminal extracellular peptide (6aa), a single putative hydrophobic transmembrane domain which is followed by a potential palmitoylation site (a CxxC motif). The predicted cytoplasmic domain contains a total of 10 tyrosines but no other clearly recognizable motifs. Importantly, the mouse LAT and WBSCR5 genes show a strikingly similar organization. Both of them are composed of 11 exons that split the respective coding sequence in a very similar manner (Fig. 3) . The two genes (residing on different chromosomes) also display the same splice frame diagrams (Fig. 3) further suggesting that they are closely related and likely are derived from a common ancestor (see Discussion). Because of the structural similarity to LAT and its broad expression we termed to the novel protein NTAL.


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

Brdicka T, Imrich M, Angelisová P, Brdicková N, Horváth O, Spicka J, Hilgert I, Lusková P, Dráber P, Novák P, Engels N, Wienands J, Simeoni L, Osterreicher J, Aguado E, Malissen M, Schraven B, Horejsí V - J. Exp. Med. (2002)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196071&req=5

fig3: 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.
Mentions: In vitro kinase assays performed on GEMs immunoprecipitated from myeloid cell lines HL-60 and THP-1 revealed the presence of an unidentified 30 kD phospho-protein (pp30) which was not detectable under similar conditions in T cells (Fig. 1 A). To further characterize this protein, the in vitro labeled proteins were mixed with GEMs prepared from 5 × 108 THP-1 cells as described previously (18). This mixture was then subjected to two-dimensional gel electrophoresis and a doublet of acidic protein spots of 29–30 kD visualized by silver staining was found to colocalize with radiolabeled pp30 (Fig. 1 B). The spots were excised, digested in-gel with trypsin, and resulting peptides were analyzed by mass spectrometry (MALDI-TOF). Database searching revealed that six of the peptides (Table I) fit precisely to those predicted for a so far uncharacterized protein encoded by a previously cloned full-length cDNA corresponding to a broadly expressed human gene termed WBSCR5 which is located on human chromosome 7 (7q11.23; references 24 and 25). The WBSCR5 cDNA codes for a polypeptide of 243 amino acid residues (Fig. 2) and a predicted molecular weight of 26.550 daltons while the predicted mouse homologue is shorter by 40 amino acid residues and is encoded by a gene residing on chromosome 5 (25). The protein resembles in its general organization the GEM-associated transmembrane adaptor proteins PAG/Cbp (18, 26) and LAT (4). Thus, it consists of a very short NH2-terminal extracellular peptide (6aa), a single putative hydrophobic transmembrane domain which is followed by a potential palmitoylation site (a CxxC motif). The predicted cytoplasmic domain contains a total of 10 tyrosines but no other clearly recognizable motifs. Importantly, the mouse LAT and WBSCR5 genes show a strikingly similar organization. Both of them are composed of 11 exons that split the respective coding sequence in a very similar manner (Fig. 3) . The two genes (residing on different chromosomes) also display the same splice frame diagrams (Fig. 3) further suggesting that they are closely related and likely are derived from a common ancestor (see Discussion). Because of the structural similarity to LAT and its broad expression we termed to the novel protein NTAL.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4, Czech Republic.

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.

Show MeSH