ZAP70 (zeta-chain (TCR) associated protein kinase 70kDa)

	Figure 1. Schematic structure of inactive ZAP70 (adopted from Wang et al 2010). The ZAP70 domains indicated are the amino-terminal SH2 domain (N-SH2), interdomain A (I-A), carboxy-terminal SH2 domain (C-SH2), interdomain B (I-B) and the kinase domain.
Description	ZAP70 is composed of two SH2 domains and a carboxy-terminal kinase domain (Fig. 1). The crystal structure of the tandem SH2 domains of the human PTK ZAP70 in complex with a peptide derived from the zeta-subunit of the T-cell receptor (TCR) was revealed by Hatada et al (Hatada 1995). A coiled coil of alpha-helices connects the two SH2 domains, producing an interface that constitutes one of the two critical phosphotyrosine binding sites, providing the molecular basis for highly selective association of ZAP70 with the T-cell receptor. The crystal structure of autoinhibited ZAP70 revealed a new mechanism for maintaining an inactive kinase domain conformation (Deindl 2007), which also have been shown in cell-based experiments (Brdicka 2005; Deindl 2009). The two tandem SH2 domains that are separated by a linker region, termed interdomain A. Upon activation, conformational changes in ZAP70 promote disassembly of the interface mediating the autoinhibited conformation, and exposure of tyrosines Y292, Y315 and Y319 in interdomain B, leading to their phosphorylation that further destabilizes the interface (Wang 2010).
Expression	ZAP70 protein is expressed in all major thymocyte populations, with the level of expression being comparable to that found in both CD4+ and CD8+ peripheral T cells (Chan 1991;Irving 1993). Stimulation of the TCR results in tyrosine phosphorylation of a number of cellular substrates. One of these is the TCR zeta chain, which can mediate the transduction of extracellular stimuli into cellular effector functions. When ZAP70 was discovered, it represented a novel PTK and was found to be expressed in T cells and natural killer (NK) cells. The tyrosine phosphorylation and association of ZAP70 with TCR zeta was shown to require the presence of src family PTKs and provide a potential mechanism by which the src family PTKs and ZAP70 may interact to mediate TCR signal transduction (Chan 1991;Irving 1993). The first indication that ZAP70 may be expressed in the B cell lineage came from studies of B-cell chronic lymphocytic leukemia (CLL) (Rosenwald 2001). ZAP70 was thereafter also reported to be expressed throughout B cell development at different maturational stages and that it plays a role in the transition of pro-B to pre-B cells in the bone marrow, a checkpoint controlled by signals from the pre-B cell receptor (pre-BCR), which monitors for successful rearrangement of immunoglobulin heavy chain genes (Schweighoffer 2003; Nolz 2005; Crespo 2006; Scielzo 2006).
Localisation	In the cytoplasmic compartment of the T-cell, once recruited to tyrosines in the immunoreceptor tyrosine-based activation motif (ITAM) of the TCR-subunit, ZAP70 is activated by phosphorylation, at Tyr-493 in its activation loop, by Lck (Chan 1995; Wange 1995). Upon phosphorylation, these bound ZAP70 molecules autophosphorylate to create docking sites for SH2-domain-containing signalling proteins (Neumeister 1995; Katzav 1994; Couture 1994; Duplay 1994). Docking of ZAP70 at the plasma membrane is required for its activation (reviewed in Fischer 2010).
Function	Indeed, PTKs play an integral role in T cell activation. TCR signal transduction and the events leading to activation of downstream signaling pathways involve many molecules, including the PTKs Src, Syk, Csk and Tec families, as well as adaptor proteins and effector enzymes in tyrosine phosphorylation cascades (review in Mustelin, 2003). Both SH2-domains of ZAP70 are crucial for high avidity binding and for function in signal transduction (Bu 1995; review by Au-Yeung 2009 and Wang 2010). ZAP70 is phosphorylated on tyrosine residues upon TCR stimulation, and functions in the initial step of TCR-mediated signal transduction in combination with the clustering of coreceptor (CD4 or CD8)-associated LCK with T cell receptors allows Lck to phosphorylate tyrosine residues in ITAMs in the intracellular tails of zeta chains of the TCR-complex (Yan 2013). Doubly phosphorylated ITAMs in the stimulated TCR complex recruit ZAP-70 to the plasma membrane. Activated ZAP70 subsequently phosphorylates at least two critically important adaptor proteins, linker for the activation of T cells (LAT) and SH2-domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76 ) (Bubeck Wardenburg 1996; Zhang 1998; Au-Yeung 2009). Interdomain B in the ZAP70 molecule, between the C-terminal SH2-domain and the kinase domain, is also a critical region because of the three tyrosine residues Y292, Y315 and Y319 that are phosphorylated by Lck upon TCR-triggering (Fischer 2010). The tyrosine Y292 binds the ubiquitin ligase c-Cbl and control both zeta (of the TCR-CD3-zeta complex) ubiquitination and TCR downmodulation, whereas Y315 interacts with the CT10 regulator of kinase II (CrkII ) adapter protein (Lupher 1997; Gelkop 1999; Fischer 2010). The tyrosine Y319 is also a binding site for PLC-gamma , resulting in a positive ZAP70 kinase-mediated regulation through PLC-gamma phosphorylation and Ca2+ mobilization (Di Bartolo 1999; Williams 1999). The kinase domain itself also contains two tyrosine residues that are phosphorylated. Phosphorylation of Y493 by Src-family PTKs upregulate ZAP70 activity, whereas phosphorylation of Y492 seems to do the opposite, by negatively regulate ZAP70 kinase activity (Chan 1995; Wange 1995). Moreover, the low molecular weight protein-tyrosine phosphatase (LMPTP) specifically dephosphorylates the negative regulatory Tyr-292 of ZAP-70, thereby counteracting inactivation of ZAP70 (Bottini 2002). The proteins phosphorylated by ZAP70 are not that well investigated. VHR , a Vaccinia virus VH-1 related dual-specific protein phosphatase that inactivates the mitogen-activated kinases Erk1 and Jnk, is phosphorylated at Y138 by ZAP70 (Alonso 2002). The role of ZAP70 in B cells have been investigated, but is poorly understood possibly due to the functional redundancy between Syk and ZAP70 (Fallah-Arani 2008). Studies in CLL reveal that the ability of ZAP70 to enhance BCR signalling was independent of its kinase activity as both WT ZAP70 and a catalytically inactive ZAP70 mutant induced similar increases in intracellular free Ca2+ concentration upon BCR triggering (Chen 2008).
Homology	Together with Syk, ZAP70 defines the Syk family of PTKs, with structural homology composed of non-myristylated cytoplasmic peptides of two N-terminal Src-homology 2 (SH2) domains and a C-terminal catalytic domain (Taniguchi 1991; Chan 1992). Although Syk protein is also present in all thymocyte subsets, expression of Syk protein is down-regulated three- to fourfold in peripheral T cells. In contrast to ZAP70, expression of Syk is 12- to 15-fold higher in peripheral B cells when compared with peripheral T cells (Chan 1994a). Both ZAP-70 and Syk are dependent upon a Src-family protein tyrosine kinase for association with the phosphorylated zeta-chain. Thus, the differential expression of these kinases suggests the possibility of different roles for ZAP70 and Syk in TCR signaling and thymic development (Palacios 2007). Another study showed functional homology in antigen receptor signaling by demonstrating that expression of ZAP70 in Syk- B cells reconstitutes BCR function (Kong 1995). Reconstitution required the presence of functional Src homology 2 (SH2) and catalytic domains of ZAP70. In addition, they demonstrated that both ZAP70 and Syk can bind directly to the phosphorylated Ig alpha and Ig beta subunits with affinities comparable to their binding to the TCR CD3 epsilon subunit (Kong 1995). Another feature that distinguish ZAP70 from Syk is its greater dependency on Src kinases for activation and its ability to phosphorylate and promote the auto-activation of the downstream mitogen-activated protein kinase (MAPK) p38 (reviewed in Au-Yeung 2009).

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