TCR, BCR, Soluble Tyrosine Kinases and NFAT
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The high-affinity receptor for IgE (IgE-R). Present on mast cells and blood-borne basophils,4 this plays an important role in hypersensitivity and in the initiation of acute inflammatory responses.
Erythropoietin receptors. The cytokine erythropoietin (EPO) plays an important role in the final stage of maturation of erythroid progenitor cells into mature red blood cells. The receptor is also present on other cell types suggesting that erythropoietin may have effects on other tissues. EPO has been abused by athletes aiming to boost their performance in endurance sports such as cycling.
Prolactin receptors. Primarily involved in the regulation of lactation. In addition, prolactin has been implicated in modulation of immune
responses. For instance, it regulates the level of NK-mediated cytotoxicity. It has also gained attention as a potential male contraceptive.
Other non-receptor PTKs are discussed in Chapter 13, where they are shown to play pivotal roles in cell survival and proliferation.
T-cell receptor signalling
T lymphocytes are central role to cell-mediated immunity. When activated, they proliferate and differentiate, becoming cytotoxic T cells, helper T cells, or long-lasting memory cells. Cytotoxic T cells kill specific targets, most commonly
virus-infected cells; helper T cells assist other cells of the immune system, mainly B lymphocytes. Effective T cell activation requires the coordinated attentions of several different ligands with their receptors. This is not the only unusual aspect. The primary ligands that activate T cells are not classical first messengers (i.e. blood-borne soluble molecules), but proteins presented on the membranes
of neighbouring cells. For example, the stimulation of T lymphocytes that are naive to any previous form of activation involves the T cell receptor (Figure 17.2a). This detects foreign proteins (antigens) in the form of short peptides that are proffered on the surface of the target cells. An infected cell, expressing viral proteins, presents fragments fixed in a groove on its MHC class I molecules. These are expressed on virtually all nucleated cells and are characteristic of the host so that they are recognized by the immune system as ‘self’. The task of the T cell is to kill the virus-infected cell. Circulating antigens are processed by
specialized cells such as dendritic cells, B lymphocytes, and macrophages which then present protein fragments on their surfaces, again attached to an antigenpresenting molecule, this time MHC class II (Figure 17.2b). Here, the main function of the T cell is to help the target cell, the B lymphocyte, to make antibodies.
More than one lymphocyte receptor must be engaged to ensure activation
The cell–cell interaction necessary for T cell activation requires intimate contact, calling on specialized adhesion molecules, as well as three quite
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Fig 17.2 Clonal expansion of naive T lymphocytes.
(a) Clonal expansion is activated through binding of an antigen, presented in the groove of MHC II on an antigen-presenting dendritic cell (above), to the receptor (TCR) on the naive T cell (below). There are also essential costimulatory interactions involving CD4, B7, and CD28. The result is the production of IL-2 together with its receptor on the T cell. This induces a proliferation signal that is responsible for the expansion of a clone of T cells that exclusively recognize this particular antigen. (b) The TCR is a disulfide-linked heterodimer of and chains. These have hypervariable regions that detect the antigen, presented as a short peptide in the groove of the MHC molecule. This heterodimer forms a complex with six polypeptides, two chains and the , , and chains of the CD3 molecule. A CD4 or CD8 molecule is also associated with the TCR in helper or cytotoxic T cells respectively. Importantly, attachment of CD4 (or CD8) to the MHC chain brings the non-receptor PTK Lck, into the vicinity of the T cell receptor.
distinct ligand–receptor interactions (Figure 17.2a). It is the combination of antigen and MHC that initiates the T cell response, but there are two possible outcomes. Only in the presence of a second, ‘costimulatory’ signal do the cells become fully activated. The full response comprises transcription of early response genes, followed by synthesis and release of the cytokine IL-2, entry into the cell cycle, and differentiation into an effector or memory cell. In the absence of a costimulatory signal, the T cell becomes unresponsive or anergic.
Initial cell–cell contact involves low-specificity interaction between B7 (on the antigen-presenting cell) and CD28 (on the T cell). The and chains of the
T cell receptor (TCR) may then bind to the peptide presented by the antigenpresenting cell. The TCR is associated with CD3 as components of a complex of six polypeptides, all of which span the membrane (Figure 17.2b). The TCR/CD3 bound to MHCII assemble as microclusters, comprising 11–150 copies of each that converge to form the so-called immunological synapse.5 By the recruitment of non-receptor PTKs, components of the TCR/CD3 complex
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are rapidly phosphorylated. In addition, there is activation of PLC- 1 with production of IP3 and diacylglycerol and elevation of cytosol Ca2 . Thus, the consequences of receptor ligation are not dissimilar from those induced by the receptors for EGF or PDGF.
An early candidate explaining the induction of tyrosine kinase activity emerged with the discovery of Lck (p56lck). This T cell-specific kinase of the Src family6,7 (see Figure 17.1 and Table 17.4) is associated with the membrane and with the cytosolic tail of CD4 (in helper T cells) or CD8 (in cytotoxic T cells)8 (Figure 17.2b). The extracellular domains of CD4 and CD8 attach to the MHC, strengthening the rather weak interaction established between the TCR and antigen. They also bring CD4/CD8 into the vicinity of the TCR complex, leading Lck to its targets on their and other CD3 chains.9
T cell receptor signal-complex formation
In resting cells, Lck exists in a primed state. This has an open conformation, accessible to substrate, but not phosphorylated at the activating residue (Y394) in its activation segment. The open state of Lck associated with CD4/CD8 is maintained by CD45, a transmembrane phosphatase that targets pY505 in the C-terminal tail. Its function is described more fully in Chapter 21. As with c-Src, Csk is thought to be the kinase that drives Lck into its inactive state10 (see Figure 17.13).
Activation of primed Lck is by transphosphorylation by another Lck molecule present in the TCR microcluster. It can then phosphorylate the , , and chains of CD3.11 The target tyrosines are confined to so-called ITAM motifs. Phosphorylation of ITAMs provides multiple docking sites for SH2 domainbearing molecules, amongst them ZAP-70, another kinase which binds to the chain of CD3.12 (Figure 17.3). ZAP-70 is now phosphorylated by Lck and
thereby activated, causing further phosphorylation of multiple substrates. The sequence of events then follows a pattern in which phosphotyrosines bind SH2 or PTB domain-containing proteins that may themselves be PTKs and that can phosphorylate and recruit yet more proteins in succession. At each stage there is the opportunity for the pathways to diverge, involving a range of effectors.9
One important branch point is offered by the integral membrane protein LAT, which presents no fewer than nine substrate tyrosine residues.13 When
phosphorylated, these recruit a broad range of signalling molecules that include Grb2, SLP76, PLC 1, PI 3-kinase (through its p85 regulatory subunit) and the guanine nucleotide exchange factors Dbl and Vav (see inset in Figure 17.3).
The formation of a signalling complex around the TCR and the branching pathways that emanate from it resemble the mechanisms used by the growth factors. However, not all the destinations of these pathways are clear. We outline two, one involving calcineurin, the other PKC (Figure 17.4). An important starting point is the production of diacylglycerol and IP3 by PLC 1.
TCR activation and Fyn. The ITAM motifs (immunoreceptor tyrosine-based activation motif ) in the , , , andchains of CD3 are also targets of Fyn (p59fyn) another member of the large Src kinase family. Fyn is also activated by phosphorylation but its mode of recruitment to the TCR microclusters is
not yet understood. Fyn is required for efficient TCR signalling during antigen presentation but it plays a more important role in the survival of naïve
T cells.9
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Fig 17.3 Signalling complex formation at the TCR complex.
The antigen/MHC-bound TCR activates Lck that phosphorylates the ITAM motifs present in the chains of CD3 (1). The resulting pY residues form a docking site for the SH2 domains of ZAP70 (2), another cytosolic PTK. ZAP70 is phosphorylated by Lck in the linker region between the SH2 domains and the catalytic domain (3). The activated ZAP70 then phosphorylates several (maximally nine) tyrosine residues on the transmembrane adaptor LAT (4). Various proteins attach. These include PLC (5), which upon attachment becomes phosphorylated and activated by Lck (6). Other molecules include the guanine nucleotide exchange factor Vav, the adaptors Grb2 and SLP76, and the p85 regulatory subunit of P I3-kinase. All play important roles in the activation
of the IL-2 gene. Generation of diacylglycerol and IP3 (7) by PLC is a starting point for the two signalling pathways described in this section. The main components of the TCR are indicated in Figure 17.2.
Fig 17.4 Two signalling pathways downstream of PLC .
The second messengers diacylglycerol and IP3 activate two signalling pathways. One involves IP3-released Ca2 , which results in the activation of the serine/ threonine phosphatase calcineurin and leads to activation of NFAT. The other involves the diacylglycerol-mediated activation of PKC which phosphorylates the adaptor CARMA1. The unfolded protein then acts as a docking site for the assembly of a TRAF6-ubiquitin ligase complex that causes activation of IKK and then nuclear translocation of NF- B. The genes regulated by NFAT and NF- B are involved in various aspects of the immune response.
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Fig 17.5 Calcineurin-mediated activation of NFAT.
Ca2 binds to calmodulin (CaM) and calcineurin B (CnB), leading to the activation of the serine/threonine phosphatase calcineurin. Dephosphorylation of NFAT1 ensues, exposing its nuclear localization sequence (NLS) and masking the nuclear export sequence (NES). NFAT enters the nucleus and associates with c-Jun and c-Fos (AP-1 complex) to drive gene expression.
PLC 1 to NFAT
Phosphorylated LAT recruits PLC 1 to the TCR/CD3 complex.14 This is facilitated by the concerted actions of an array of other proteins such as Vav1, SLP-76, Itk, and c-Cbl. In the assembled complex, PLC- 1 is activated through phosphorylation by Lck in the linker region between SH2 and SH3 (see Figure 17.3). The consequent production of DAG and IP3 and elevation of Ca2 leads to activation of calcineurin (PP2B, see page 682). This (serine/
threonine) phosphatase utilizes two Ca2 sensors, calmodulin and calcineurin B (Figure 17.5). Calcineurin dephosphorylates multiple pS residues in the N-terminal segment of the transcription factor NFAT, thereby exposing a nuclear localization sequence (NLS).15
Multiple protein kinases and multiple phosphorylation sites on NFAT.
The N-terminal segment of NFAT contains numerous serine phosphorylation sites (about 14) which reside in short conserved motifs, serine-rich regions (SRR), and serine/proline motifs (SP1, SP2, and SP3) (see figure 17.6). These serine residues surround the NLS. Phosphorylation occurs in the nucleus. DYRKA initially phosphorylates the SP3 site and this opens the way to further phosphorylations by casein kinase and glycogen synthase kinase
. Phosphorylation leads to the masking of the NLS and unmasking of the nuclear export signal NES.17 (see Figure 17.5). NFAT1 is actively transported out of the nucleus by exportin-1/RanGTP.
Two calcium sources.
An initial IP3-induced release of Ca2 from intracellular stores is augmented by cADPRinduced release, and then by influx of extracellular calcium ions through plasma membrane channels (CRAC; see page 210). This source of Ca2 is required for expression of NFAT-controlled genes. Loss of function of CRAC causes immune deficiency.16
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Fig 17.6 Domain architecture of proteins involved in TCR-mediated activation of NFAT.
A variety of proteins associates with the TCR. They are brought together through a cascade of phosphotyrosine-SH2 interactions. An important signal is transmitted via phospholipase C which, through activation of calcineurin, results in the nuclear translocation of NFAT2. The domain organization of PLC is illustrated in Figure 5.11, page 151.
Nuclear translocation of NFAT is essential for clonal expansion of T cells because of its pivotal role in the induction of IL-2 and IFN- (for Th1 cells) and IL-4 (for Th2 cells). Full transcriptional activity of NFAT only occurs after phosphorylation by ERK1/2 or p38.18 In order to drive full expression of IL-2, NFAT must associate with the AP-1 complex and with NF- B (see below). The domain architecture of proteins involved in this pathway is illustrated in Figure 17.6.
Calcineurin, NFAT, and islet development in the pancreas. Ciclosporin, a cyclic peptide of 11 residues, of fungal origin, has been applied following
transplantation surgery to suppress tissue rejection. However, it was found that it can raise the concentration of blood glucose, effectively causing diabetes mellitus, due to the loss of the insulin-producing -cells in the pancreatic islets of Langerhans.19 This raised the possibility that calcineurin (PP2B), being the target of ciclosporin, might play a normal role in adaptive islet responses. Mice deficient in the calcineurin regulatory subunit (CnB1) develop age-related diabetes due to loss of -cells. Conditional expression of active NFAT1 in these -cells rescues the defect and prevents diabetes. NFAT1 regulates transcription of genes critical for-cell endocrine function and which are linked to type 2 (late onset) diabetes.20
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