A weak Lck tail bite is necessary for Lck function in T cell antigen receptor signaling

Src family kinases are suppressed by a "tail bite" mechanism, in which the binding of a phosphorylated tyrosine in the C terminus of the protein to the Src homology (SH) 2 domain in the N-terminal half of the protein forces the catalytic domain into an inactive conformation stabilized by an additional SH3 interaction. In addition to this intramolecular suppressive function, the SH2 domain also mediates intermolecular interactions, which are crucial for T cell antigen receptor (TCR) signaling. To better understand the relative importance of these two opposite functions of the SH2 domain of the Src family kinase Lck in TCR signaling, we created three mutants of Lck in which the intramolecular binding of the C terminus to the SH2 domain was strengthened. The mutants differed from wild-type Lck only in one to three amino acid residues following the negative regulatory tyrosine 505, which was normally phosphorylated by Csk and dephosphorylated by CD45 in the mutants. In the Lck-negative JCaM1 cell line, the Lck mutants had a much reduced ability to transduce signals from the TCR in a manner that directly correlated with SH2-Tyr(P)(505) affinity. The mutant with the strongest tail bite was completely unable to support any ZAP-70 phosphorylation, mitogen-activated protein kinase activation, or downstream gene activation in response to TCR ligation, whereas other mutants had intermediate abilities. Lipid raft targeting was not affected. We conclude that Lck is regulated by a weak tail bite to allow for its activation and service in TCR signaling, perhaps through a competitive SH2 engagement mechanism.     

Subdomain X of the kinase domain of Lck binds CD45 and facilitates dephosphorylation

CD45 is a transmembrane, two-domain protein-tyrosine phosphatase expressed exclusively in nucleated hematopoietic cells. The Src family kinase, Lck, is a major CD45 substrate in T cells and CD45 dephosphorylation of Lck is important for both T cell development and activation. However, how the substrate specificity of phosphatases such as CD45 is achieved is not well understood. Analysis of the interaction between the cytoplasmic domain of CD45 and its substrate, Lck, revealed that the active, membrane-proximal phosphatase domain of CD45 (CD45-D1) bound to the phosphorylated Lck kinase domain, the SH2 domain, and the unique N-terminal region of Lck. The second, inactive phosphatase domain (CD45-D2) bound only to the kinase domain of Lck. CD45-D2 was unable to bind phosphotyrosine, and its interaction with the kinase domain of Lck was independent of tyrosine phosphorylation. The binding of CD45-D2 was localized to subdomain X (SD10) of Lck. CD45-D2 bound similarly to Src family kinases but bound Csk to a lesser extent and did not bind significantly to the less related kinase, Erk1. CD45 dephosphorylated Lck and Src at similar rates but dephosphorylated Csk and Erk1 at lower rates. Replacement of Erk1 SD10 with that of Lck resulted in the binding of CD45-D2 and the conversion of Erk1 to a more efficient CD45 substrate. This demonstrates a role for CD45-D2 in binding substrate and identifies the SD10 region in Lck as a novel site involved in substrate recognition.     

Identification of Grb2 as a novel binding partner of the signaling lymphocytic activation molecule-associated protein binding receptor CD229

Ag recognition by the TCR determines the subsequent fate of the T cell and is regulated by the involvement of other cell surface molecules, termed coreceptors. CD229 is a lymphocyte cell surface molecule that belongs to the CD150 family of receptors. Upon tyrosine phosphorylation, CD229 recruits various signaling molecules to the membrane. One of these molecules is the signaling lymphocytic activation molecule-associated protein, of which a deficiency leads to the X-linked lymphoproliferative syndrome. We report that CD229 interacts in a phosphorylation-dependent manner with Grb2. We mapped this interaction showing that the Src homology 2 domain of Grb2 and the tyrosine residue Y606 in CD229 are required for CD229-Grb2 complex formation. The Grb2 motif in the cytoplasmic tail of CD229 is distinct and independent from the two tyrosines required for efficient signaling lymphocytic activation molecule-associated protein recruitment. CD229, but not other members of the CD150 family, directly bound Grb2. We also demonstrate that CD229 precipitates with Grb2 in T lymphocytes after pervanadate treatment, as well as CD229 or TCR ligation. Interestingly, the CD229 mutant lacking the Grb2 binding site is not internalized after CD229 engagement with specific Abs. Moreover, a dominant negative form of Grb2 (containing only Src homology 2 domain) impaired CD229 endocytosis. Unexpectedly, Erk phosphorylation was partially inhibited after activation of CD229 plus CD3. Consistent with this, CD229 ligation partially inhibited TCR signaling in peripheral blood cells and CD229-Jurkat cells transfected with the 3XNFAT-luciferase reporter construct. Altogether, the data suggest a model whereby CD229 ligation attenuates TCR signaling and Grb2 recruitment to CD229 controls its rate of internalization.     

Serine 6 of Lck tyrosine kinase: a critical site for Lck myristoylation, membrane localization, and function in T lymphocytes

Lck is a member of the Src family kinases expressed predominantly in T cells, and plays a pivotal role in TCR-mediated signal transduction. Myristoylation of glysine 2 in the N-terminal Src homology 4 (SH4) domain of Lck is essential for membrane localization and function. In this study, we examined a site within the SH4 domain of Lck regulating myristoylation, membrane localization, and function of Lck. A Lck mutant in which serine 6 (Ser6) was substituted by an alanine was almost completely cytosolic in COS-7 cells, and this change of localization was associated with a drastic inhibition of myristoylation in this mutant. To assess the role of Ser6 of Lck in T cell function, we established stable transfectants expressing various Lck mutants using Lck-negative JCaM1 cells. The Lck mutant of Ser6 to alanine, most of which did not target to the plasma membrane, was not able to reconstitute TCR-mediated signaling events in JCaM1 cells, as analyzed by tyrosine phosphorylation of intracellular proteins and CD69 expression. These results demonstrate that Ser6 is a critical factor for Lck myristoylation, membrane localization, and function in T cells, presumably because the residue is important for N-myristoyl transferase recognition.     

Association of CD38 with nonmuscle myosin heavy chain IIA and Lck is essential for the internalization and activation of CD38

Activation of CD38 in lymphokine-activated killer (LAK) cells involves interleukin-8 (IL8)-mediated protein kinase G (PKG) activation and results in an increase in the sustained intracellular Ca(2+) concentration ([Ca(2+)](i)), cADP-ribose, and LAK cell migration. However, direct phosphorylation or activation of CD38 by PKG has not been observed in vitro. In this study, we examined the molecular mechanism of PKG-mediated activation of CD38. Nonmuscle myosin heavy chain IIA (MHCIIA) was identified as a CD38-associated protein upon IL8 stimulation. The IL8-induced association of MHCIIA with CD38 was dependent on PKG-mediated phosphorylation of MHCIIA. Supporting these observations, IL8- or cell-permeable cGMP analog-induced formation of cADP-ribose, increase in [Ca(2+)](i), and migration of LAK cells were inhibited by treatment with the MHCIIA inhibitor blebbistatin. Binding studies using purified proteins revealed that the association of MHCIIA with CD38 occurred through Lck, a tyrosine kinase. Moreover, these three molecules co-immunoprecipitated upon IL8 stimulation of LAK cells. IL8 treatment of LAK cells resulted in internalization of CD38, which co-localized with MHCIIA and Lck, and blebbistatin blocked internalization of CD38. These findings demonstrate that the association of phospho-MHCIIA with Lck and CD38 is a critical step in the internalization and activation of CD38.     

CD45 down-regulates Lck-mediated CD44 signaling and modulates actin rearrangement in T cells

The tyrosine phosphatase CD45 dephosphorylates the negative regulatory tyrosine of the Src family kinase Lck and plays a positive role in TCR signaling. In this study we demonstrate a negative regulatory role for CD45 in CD44 signaling leading to actin rearrangement and cell spreading in activated thymocytes and T cells. In BW5147 T cells, CD44 ligation led to CD44 and Lck clustering, which generated a reduced tyrosine phosphorylation signal in CD45(+) T cells and a more sustained, robust tyrosine phosphorylation signal in CD45(-) T cells. This signal resulted in F-actin ring formation and round spreading in the CD45(+) cells and polarized, elongated cell spreading in CD45(-) cells. The enhanced signal in the CD45(-) cells was consistent with enhanced Lck Y394 phosphorylation compared with the CD45(+) cells where CD45 was recruited to the CD44 clusters. This enhanced Src family kinase-dependent activity in the CD45(-) cells led to PI3K and phospholipase C activation, both of which were required for elongated cell spreading. We conclude that CD45 induces the dephosphorylation of Lck at Y394, thereby preventing sustained Lck activation and propose that the amplitude of the Src family kinase-dependent signal regulates the outcome of CD44-mediated signaling to the actin cytoskeleton and T cell spreading.     

Expression of CD45 lacking the catalytic protein tyrosine phosphatase domain modulates Lck phosphorylation and T cell activation

The function of the second protein tyrosine phosphatase domain (D2) in two-domain protein tyrosine phosphatases (PTP) is not well understood. In CD45, D2 can interact with the catalytic domain (D1) and stabilize its activity. Although D2 itself has no detectable catalytic activity, it can bind substrate and may influence the substrate specificity of CD45. To further explore the function of D2 in T cells, a full-length construct of CD45 lacking the D1 catalytic domain (CD45RABC-D2) was expressed in CD45+ and CD45- Jurkat T cells. In CD45- Jurkat T cells, CD45RABC-D2 associated with Lck but, unlike its active counterpart CD45RABC, did not restore the induction of tyrosine phosphorylation or CD69 expression upon T cell receptor (TCR) stimulation. Expression of CD45RABC-D2 in CD45+ Jurkat T cells resulted in its association with Lck, increased the phosphorylation state of Lck, and reduced T cell activation. TCR-induced tyrosine phosphorylation was delayed, and although MAPK phosphorylation and CD69 expression were not significantly affected, the calcium signal and IL2 production were severely reduced. This indicates that the non-catalytic domains of CD45 can interact with Lck in T cells. CD45RABC-D2 acts as a dominant negative resulting in an increase in Lck phosphorylation and a preferential loss of the calcium signaling pathway, but not the MAPK pathway, upon TCR signaling. This finding suggests that, in addition to their established roles in the initiation of TCR signaling, CD45 and Lck may also influence the type of TCR signal generated.     

CD38 signaling in T cells is initiated within a subset of membrane rafts containing Lck and the CD3-zeta subunit of the T cell antigen receptor

In this study we present data supporting that most CD38 is pre-assembled in a subset of Brij 98-resistant raft vesicles, which were stable at 37 degrees C, and have relatively high levels of Lck and the CD3-zeta subunit of T cell antigen receptor-CD3 complex in contrast with a Brij 98-soluble pool, where CD38 is associated with CD3-zeta, and Lck is not detected. Our data further indicate that following CD38 engagement, LAT and Lck are tyrosine phosphorylated exclusively in Brij 98-resistant rafts, and some key signaling components translocate into rafts (i.e. Sos and p85-phosphatidylinositol 3-kinase). Moreover, N-Ras results activated within rafts immediately upon CD38 ligation, whereas activated Erk was mainly found in soluble fractions with delayed kinetics respective to Ras activation. Furthermore, full phosphorylation of CD3-zeta and CD3-epsilon only occurs in rafts, whereas partial CD3-zeta tyrosine phosphorylation occurs exclusively in the soluble pool, which correlated with increased levels of c-Cbl tyrosine phosphorylation in the non-raft fractions. Taken together, these results suggest that, unlike the non-raft pool, CD38 in rafts is able to initiate and propagate several activating signaling pathways, possibly by facilitating critical associations within other raft subsets, for example, LAT rafts via its capacity to interact with Lck and CD3-zeta. Overall, these findings provide the first evidence that CD38 operates in two functionally distinct microdomains of the plasma membrane.     

Evidence that Llck-mediated phosphorylation of p56dok and p62dok may play a role in CD2 signaling

The Lck tyrosine kinase is involved in signaling by T cell surface receptors such as TCR/CD3, CD2, and CD28. As other downstream protein-tyrosine kinases are activated upon stimulation of these receptors, it is difficult to assign which tyrosine-phosphorylated proteins represent bona fide Lck substrates and which are phosphorylated by other tyrosine kinases. We have developed a system in which Lck can be activated independently of TCR/CD3. We have shown that activation of an epidermal growth factor receptor/Lck chimera leads to the specific phosphorylation of Ras GTPase-activating protein (RasGAP) and two RasGAP-associated proteins, p56(dok) and p62(dok). Activation of the chimeric protein correlates with an increase in cellular Ca(2+) in the absence of ZAP-70 and phospholipase Cgamma1 phosphorylation. Furthermore, we have found that p62(dok) co-immunoprecipitates with the activated epidermal growth factor receptor/LckF505 and that phosphorylated Dok proteins bind to the Src homology 2 domain of Lck in vitro. In addition, we have shown that activation via the CD2 but not the TCR/CD3 receptor leads to the phosphorylation of p56(dok) and p62(dok). Using JCaM1.6 cells, we have demonstrated that Lck is required for CD2-mediated phosphorylation of Dok proteins. We propose that phosphorylation and Src homology 2-mediated association of p56(dok) and p62(dok) with Lck play a selective function in accessory receptor signal transduction mechanisms.     

The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling.

The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs. However, there is a CD45-deficient cell line that can signal through its TCR. We have studied this cell line to identify a TCR signaling pathway that is independent of CD45 regulation. In the course of these experiments, we found that the Syk PTK, but not the ZAP-70 PTK, is able to mediate TCR signaling independently of CD45 and of Lck. For this function, Syk requires functional kinase and SH2 domains, as well as intact phosphorylation sites in the regulatory loop of its kinase domain. Thus, differential expression of Syk is likely to explain the paradoxical phenotypes of different CD45-deficient T cells. Finally, these results suggest differences in activation requirements between two closely related PTK family members, Syk and ZAP-70. The differential activities of these two kinases suggest that they may play distinct, rather than completely redundant, roles in lymphocyte signaling.     

Inefficient phospholipase C activation and reduced Lck expression characterize the signaling defect of umbilical cord T lymphocytes.

Adult and neonatal immunocompetent cells exhibit important functional distinctions, including differences in cytokine production and susceptibility to tolerance induction. We have investigated the molecular features that characterize the immune response of cord blood-derived T lymphocytes compared with that of adult T lymphocytes. Our findings demonstrate that phospholipase C (PLC) isozymes, which play a pivotal role in the control of protein kinase C activation and Ca2+ mobilization, are differently expressed in cord and adult T lymphocytes. PLCbeta1 and delta1 are expressed at higher levels in cord T cells, while PLCbeta2 and gamma1 expression is higher in adult T lymphocytes. PLCdelta2 and gamma2 appear to be equally expressed in both cell types. In addition, a functional defect in PLC activation via CD3 ligation or pervanadate treatment, stimuli that activate tyrosine kinases, was observed in cord blood T cells, whereas treatment with aluminum tetrafluoride (AlF4-), a G protein activator, demonstrated a similar degree of PLC activation in cord and adult T cells. The impaired PLC activation of cord blood-derived T cells was associated with a a very low expression of the Src kinase, Lck, along with a reduced level of ZAP70. No mitogenic response to CD3 ligation was observed in cord T cells. However, no signaling defect was apparent downstream of PLC activation, as demonstrated by the mitogenic response of cord T cells to the pharmacologic activation of protein kinase C and Ca2+ by treatment with PMA and ionomycin. Thus, neonatal cord blood-derived T cells show a signaling immaturity associated with inadequate PLCgamma activation and decreased Lck expression.     

The inhibitory function of CTLA-4 does not require its tyrosine phosphorylation

CTLA-4 is a negative regulator of T cell responses. Sequence analysis of this molecule reveals the presence of two cytoplasmic tyrosine residues at positions 165 and 182 that are potential Src homology (SH)-2 domain binding sites. The role of phosphorylation of these residues in CTLA-4-mediated signaling is unknown. Here, we show that sole TCR ligation induces zeta-associated protein (ZAP)-70-dependent tyrosine phosphorylation of CTLA-4 that is important for cell surface retention of this molecule. However, CTLA-4 tyrosine phosphorylation is not required for down-regulation of T cell activation following CD3-CTLA-4 coengagement. Specifically, inhibition of extracellular signal-regulated kinase (ERK) activation and of IL-2 production by CTLA-4-mediated signaling occurs in T cells expressing mutant CTLA-4 molecules lacking the cytoplasmic tyrosine residues, and in lck-deficient or ZAP-70-deficient T cells. Therefore, CTLA-4 function involves interplay between two different levels of regulation: phosphotyrosine-dependent cell surface retention and phosphotyrosine-independent association with signaling molecules.     

Functional requirements for interactions between CD84 and Src homology 2 domain-containing proteins and their contribution to human T cell activation

Cell surface receptors belonging to the CD2 subset of the Ig superfamily of molecules include CD2, CD48, CD58, 2B4, signaling lymphocytic activation molecule (SLAM), Ly9, CD84, and the recently identified molecules NTB-A/Ly108/SLAM family (SF) 2000, CD84H-1/SF2001, B lymphocyte activator macrophage expressed (BLAME), and CRACC (CD2-like receptor-activating cytotoxic cells)/CS-1. Some of these receptors, such as CD2, SLAM, 2B4, CRACC, and NTB-A, contribute to the activation and effector function of T cells and NK cells. Signaling pathways elicited via some of these receptors are believed to involve the Src homology 2 (SH2) domain-containing cytoplasmic adaptor protein SLAM-associated protein (SAP), as it is recruited to SLAM, 2B4, CD84, NTB-A, and Ly-9. Importantly, mutations in SAP cause the inherited human immunodeficiency X-linked lymphoproliferative syndrome (XLP), suggesting that XLP may result from perturbed signaling via one or more of these SAP-associating receptors. We have now studied the requirements for SAP recruitment to CD84 and lymphocyte activation elicited following ligation of CD84 on primary and transformed human T cells. CD84 was found to be rapidly tyrosine phosphorylated following receptor ligation on activated T cells, an event that involved the Src kinase Lck. Phosphorylation of CD84 was indispensable for the recruitment of SAP, which was mediated by Y(262) within the cytoplasmic domain of CD84 and by R(32) within the SH2 domain of SAP. Furthermore, ligating CD84 enhanced the proliferation of anti-CD3 mAb-stimulated human T cells. Strikingly, this effect was also apparent in SAP-deficient T cells obtained from patients with XLP. These results reveal a novel function of CD84 on human lymphocytes and suggest that CD84 can activate human T cells via a SAP-independent mechanism.     

SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation

To study the cis- and trans-acting factors that mediate programmed death 1 (PD-1) signaling in primary human CD4 T cells, we constructed a chimeric molecule consisting of the murine CD28 extracellular domain and human PD-1 cytoplasmic tail. When introduced into CD4 T cells, this construct mimics the activity of endogenous PD-1 in terms of its ability to suppress T cell expansion and cytokine production. The cytoplasmic tail of PD-1 contains two structural motifs, an ITIM and an immunoreceptor tyrosine-based switch motif (ITSM). Mutation of the ITIM had little effect on PD-1 signaling or functional activity. In contrast, mutation of the ITSM abrogated the ability of PD-1 to block cytokine synthesis and to limit T cell expansion. Further biochemical analyses revealed that the ability of PD-1 to block T cell activation correlated with recruitment of Src homology region 2 domain-containing phosphatase-1 (SHP-1) and SHP-2, and not the adaptor Src homology 2 domain-containing molecule 1A, to the ITSM domain. In TCR-stimulated T cells, SHP-2 associated with PD-1, even in the absence of PD-1 engagement. Despite this interaction, the ability of PD-1 to block T cell activation required receptor ligation, suggesting that colocalization of PD-1 with CD3 and/or CD28 may be necessary for inhibition of T cell activation.     

The kinase, SH3, and SH2 domains of Lck play critical roles in T-cell activation after ZAP-70 membrane localization.

Antigenic stimulation of the T-cell antigen receptor initiates signal transduction through the immunoreceptor tyrosine-based activation motifs (ITAMs). When its two tyrosines are phosphorylated, ITAM forms a binding site for ZAP-70, one of the cytoplasmic protein tyrosine kinases essential for T-cell activation. The signaling process that follows ZAP-70 binding to ITAM has been analyzed by the construction of fusion proteins that localize ZAP-70 to the plasma membrane. We found that membrane-localized forms of ZAP-70 induce late signaling events such as activation of nuclear factor of activated T cells without any stimulation. This activity was observed only when Lck was expressed and functional. In addition, each mutation that affects the function of Lck in the kinase, Src homology 2 (SH2), and SH3 domains greatly impaired the signaling ability of the chimeric protein. Therefore, Lck functions in multiple manners in T-cell activation for the steps following ZAP-70 binding to ITAM.     

Growth transformation of human T cells by herpesvirus saimiri requires multiple Tip-Lck interaction motifs

Lymphoma induction and T-cell transformation by herpesvirus saimiri strain C488 depends on two viral oncoproteins, StpC and Tip. The major interaction partner of Tip is the protein tyrosine kinase Lck, a key regulator of T-cell activation. The Lck binding domain (LBD) of Tip comprises two interaction motifs, a proline-rich SH3 domain-binding sequence (SH3B) and a region with homology to the C terminus of Src family kinase domains (CSKH). In addition, biophysical binding analyses with purified Lck-SH2 domain suggest the phosphorylated tyrosine residue 127 of Tip (pY127) as a potential third Lck interaction site. Here, we addressed the relevance of the individual binding motifs, SH3B, CSKH, and pY127, for Tip-Lck interaction and for human T-cell transformation. Both motifs within the LBD displayed Lck binding activities and cooperated to achieve a highly efficient interaction, while pY127, the major tyrosine phosphorylation site of Tip, did not enhance Lck binding in T cells. Herpesvirus saimiri strain C488 recombinants lacking one or both LBD motifs of Tip lost their transforming potential on human cord blood lymphocytes. Recombinant virus expressing Tip with a mutation at position Y127 was still able to transform human T lymphocytes but, in contrast to wild-type virus, was strictly dependent on exogenous interleukin-2. Thus, the strong Lck binding mediated by cooperation of both LBD motifs was essential for the transformation of human T cells by herpesvirus saimiri C488. The major tyrosine phosphorylation site Y127 of Tip was particularly required for transformation in the absence of exogenous interleukin-2, suggesting its involvement in cytokine signaling pathways.     

Lck和Fyn对T细胞发育过程的影响

胸腺中T细胞的发育及次级淋巴组织中成熟T细胞的活化均需要细胞能够对环境信号分子做出适应性的反应。在共刺激分子及细胞因子受体介导的信号参与下通过TCR(T cell receptor)及其辅助受体CD4和CD8与MHC/抗原肽复合物相互作用,可以诱导TCR信号通路激活并最终导致T细胞免疫反应的发生。Src家族激酶Lck(Lymphocyte-specific protein tyrosine kinase)和Fyn(Proto-oncogene tyrosine-protein kinase)的激活是启动TCR信号通路的关键因素。在T细胞的发育、阳性选择、初始T细胞的外周存活及由淋巴细胞缺失诱导的细胞增殖中都起着关键性的作用。研究显示,虽然这两种信号分子紧密相关,但在某些条件下Lck发挥着比Fyn更重要的作用,并且Fyn仅可以补充Lck的部分功能。文章针对这两个激酶在T细胞发育的整个过程中的作用机制进行了论述。     

Lck和Fyn对T细胞发育过程的影响

胸腺中T细胞的发育及次级淋巴组织中成熟T细胞的活化均需要细胞能够对环境信号分子做出适应性的反应。在共刺激分子及细胞因子受体介导的信号参与下通过TCR(T cell receptor )及其辅助受体CD4和CD8与MHC/抗原肽复合物相互作用, 可以诱导TCR信号通路激活并最终导致T细胞免疫反应的发生。Src家族激酶Lck(Lymphocyte-specific protein tyrosine kinase)和Fyn (Proto-oncogene tyrosine-protein kinase)的激活是启动TCR信号通路的关键因素。在T细胞的发育、阳性选择、初始T细胞的外周存活及由淋巴细胞缺失诱导的细胞增殖中都起着关键性的作用。研究显示, 虽然这两种信号分子紧密相关, 但在某些条件下Lck发挥着比Fyn更重要的作用, 并且Fyn仅可以补充Lck的部分功能。文章针对这两个激酶在T细胞发育的整个过程中的作用机制进行了论述。     

Ig alpha and Ig beta are functionally homologous to the signaling proteins of the T-cell receptor.

Signal transduction by antigen receptors and some Fc receptors requires the activation of a family of receptor-associated transmembrane accessory proteins. One common feature of the cytoplasmic domains of these accessory molecules is the presence is at least two YXXA repeats that are potential sites for interaction with Src homology 2 domain-containing proteins. However, the degree of similarity between the different receptor-associated proteins varies from that of T-cell receptor (TCR) zeta and Fc receptor RIIIA gamma chains, which are homologous, to the distantly related Ig alpha and Ig beta proteins of the B-cell antigen receptor. To determine whether T- and B-cell antigen receptors are in fact functionally homologous, we have studied signal transduction by chimeric immunoglobulins bearing the Ig alpha or Ig beta cytoplasmic domain. We found that Ig alpha and Ig beta cytoplasmic domains were able to activate Ca2+ flux, interleukin-2 secretion, and phosphorylation of the same group of cellular substrates as the TCR in transfected T cells. Chimeric proteins were then used to examine the minimal requirements for activation of the Fyn, Lck, and ZAP kinases in T cells. Both Ig alpha and Ig beta were able to trigger Fyn, Lck, and ZAP directly without involvement of TCR components. Cytoplasmic tyrosine residues in Ig beta were required for recruitment and activation of ZAP-70, but these amino acids were not essential for the activation of Fyn and Lck. We conclude that Fyn and Lck are able to recognize a clustered nonphosphorylated immune recognition receptor, but activation of these kinases is not sufficient to induce cellular responses such as Ca2+ flux and interleukin-2 secretion. In addition, the molecular structures involved in antigen receptor signaling pathways are conserved between T and B cells.