The new transmembrane adaptor protein NTAL described in this paper (a product of the previously described gene WBSCR5;
) appears to be structurally closely related to the critical component of the TCR signaling pathway, LAT (4
). Moreover, the organization of the genes encoding LAT and NTAL, respectively, is also similar, indicating they probably have a common evolutionary origin. Interestingly, the expression pattern of NTAL in lymphocytes is largely complementary to that of LAT: while LAT is predominantly found in T but not B lymphocytes, the reverse is true for NTAL. Among the important features of the structure of NTAL is a potential palmitoylation site (CxxC) adjacent to the transmembrane domain that is presumably responsible for targeting the protein to membrane microdomains (rafts, GEMs). Furthermore, there are five potential Grb2 binding motifs (YxN). Our data using PTK inhibitors () and coexpression of NTAL and PTKs in 293T cells indicate that NTAL, again similarly to LAT, is phosphorylated by concerted action of Src- and Syk-family kinases (presumably the Src-family kinases are needed for activation of the Syk family kinases).
Induction of NTAL tyrosine phosphorylation after BCR cross-linking is reminiscent of the phosphorylation of LAT that is induced by TCR ligation (4
). Similar to LAT in activated T cells, NTAL immunoprecipitated from BCR-stimulated B lymphocytes is associated with the cytoplasmic linker protein Grb2 and the nucleotide exchange factor of the small G-protein Ras, Sos1 (27
). Surprisingly, and in clear contrast to LAT, we never observed an inducible association of NTAL with PLCγ or another cytoplasmic adaptor protein, SLP-76 (or its B cell analogue SLP-65/BLNK; references 28
), both of which are key components of the multicomponent complex that is organized by activated (tyrosine-phosphorylated) LAT (5
). This could suggest that the role of NTAL in BCR signaling differs from that of LAT in the TCR signaling; namely, that NTAL may be involved only in activation of the Grb2/Sos1-initiated pathway(s) but not in activation of the PLCγ-Ca2+
pathway. The finding of the adaptor protein Gab1 in the NTAL immunoprecipitates may further suggest that the NTAL-Grb2-Gab1 complex possibly regulates the activity of PI3-K in stimulated cells (30
), but this remains a speculation at this moment.
The lack of association of SLP-65 and PLCγ with phosphorylated NTAL may further indicate that SLP-65 does not require a LAT-like molecule in B cells for being targeted to the plasma membrane. Indeed, recent data suggested that SLP-65 binds directly with its Src-homology 2 (SH2)-domain to a highly conserved non-ITAM tyrosine motif within the cytoplasmic domain of CD79a (Igα; references 33
). Moreover in contrast to SLP-76, phosphorylated SLP-65 binds SH2 domains of PLCγ2 (35
). Thus, it is tempting to speculate that the multiple functions that LAT exerts in T cells are shared in B lymphocytes between NTAL and other molecules, for example, CD79a.
Similarly to the situation in B cells, NTAL becomes strongly tyrosine phosphorylated after cross-linking of FcγRI and Fc
RI and then associates with Grb2 and Sos1 (but again not with PLCγ and also not with SLP-76). Thus, also in these cells NTAL seems to be involved in linking the activated immunoreceptors to the Grb2/Sos pathway. In contrast to activated B cells, NTAL also interacts with c-Cbl in activated THP-1 cells (). LAT has been shown to be important for processing of the FcγRI and Fc
RI mediated signals; however, FcγRI and Fc
RI signaling in LAT−/−
cells is still partially functional (12
). It is therefore tempting to speculate that this residual Fc-receptor signaling capacity in myeloid cells is due to the presence of NTAL. It is important to note that FcγRI (CD64) is the only human Fc-receptor that binds soluble monomeric murine antibodies of the IgG2a isotype with sufficient affinity (22
). This indicates that under the used experimental conditions only this Fcγ-receptor became activated in our experiments with the THP-1 cells. Furthermore, an identical pattern of NTAL phosphorylation was observed in the THP-1 cells when FcγRI (CD64) was directly cross-linked by a CD64-specific monoclonal antibody; in contrast, no phosphorylation was observed after direct mAb-mediated cross-linking of FcγRII (CD32) or FcγRIII (CD16) in these cells (unpublished data).
Our experiments provide preliminary evidence for a functional LAT-like role of NTAL in immunoreceptor signaling: ectopic expression of this protein in LAT-deficient J.CaM2.5 Jurkat T cells partially rescues TCR/CD3-mediated signaling, namely activation of Erk1/2 (). The minimal calcium response accompanying the CD3-mediated stimulation may be in agreement with the observed lack of coprecipitation of PLCγ and SLP-76 with activated NTAL in these transfectants, as discussed above. The striking conservation of the exon-intron organization of the genes encoding LAT and NTAL, respectively (), suggests that they probably derive from a duplication of an ancestral gene. As reported for other gene families, in the course of evolution, the original position of the exon borders has been blurred by splice junction sliding (36
). It is important to note that the exon-intron organization and splice frame diagram of genes encoding other transmembrane adaptor proteins involved in immunoreceptor signaling, e.g., SIT (37
) or TRIM (38
) differ totally from the distinctive organization found in the genes enconing LAT and NTAL. A similar relationship of gene organization was previously noted for the functionally closely related signal transducing subunits of several immunoreceptors (39
). Thus, NTAL appears to be structurally, evolutionarily and probably also functionally related to the transmembrane adaptor protein LAT.