The Dok proteins share a common structure, shown in Figure 1.5, that bears some similarity to the insulin receptor substrate (IRS) family of proteins, which act downstream of the insulin and IGF-1 receptors mediating signal transduction to the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways (102).
A.
B.
Figure 1.5 Protein structure of DOKs. A. Function domains of DOK protein family members 1-5. PH (grey) and PTB (black) domains are shown and positions of tyrosine residues (Y) and PXXP motifs (P) are indicated. Illustration taken from Grimm et al (104) B. Phylogenetic tree of human DOK proteins. The phylogenetic analysis was performed using the ClustalW (http://www.ebi.ac.uk/clustalw/), and the dendrogram tree was plotted using the Treeview (Version 1.6.6, http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).
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The MAP kinase pathway is a signalling cascade initiated by the binding of a ligand, such as epidermal growth factor (EGF) to the extracellular binding site of a receptor tyrosine kinase (RTK). Binding of the ligand results in the dimerization of two subunits of the RTK. This in turn catalyses the phosphorylation of the intracellular portion of each RTK subunit and enables growth factor receptor bound protein 2 (GRB2) to bind to the phosphorylated RTK, which in turn binds the protein SOS. SOS can then bind onto the membrane-bound protein RAS, which in its inactive form is bound to GDP. Binding of SOS to Ras catalyses the conversion of GDP into GTP and the activation of the Ras protein. Once activated, Ras is able to bind to several effector proteins, including the kinase B-Raf. Active B-Raf phosphorylates and activates kinases MEK1 and MEK2, which in turn, phosphorylate and activate ERK1 and ERK2. Ultimately this cascade of phosphorylation and activation results in the activation of activator protein 1 (AP-1) transcription factors, including jun and fos. These transcription factors then form a heterodimer in the cell nucleus and bind to AP-1 motifs in the DNA resulting in the expression of genes involved in cell proliferation.
The Dok family proteins, like other docking/scaffolding proteins, characteristically contain protein-protein interaction motifs such as multiple tyrosine residues (111). When
phosphorylated, these tyrosine residues act as binding sites for Src homology 2 (SH2) domain containing signalling proteins, such as growth factor receptor-bound protein 2 (Grb2), PI3K and the SHP-2 tyrosine phosphatase (111).
Initial work investigating the structure of Dok-1 revealed prominent tyrosine residues and nearby SH2 binding sites, suggesting its likely role as a signalling molecule (94, 95). Since then, the structure of the Dok family proteins has been elucidated and is characterized by three distinct regions: an amino (N-) terminal pleckstrin homology (PH) domain, a central phosphotyrosine-binding (PTB) domain and a carboxyl (C-) terminal, which contains multiple sites for tyrosine phosphorylation and proline-rich (PxxP) motifs which may enable the docking of SH2 and SH3 containing proteins (89, 101-103, 112).
The N-terminal PH domain is a region containing around 120 amino acids. This domain is found in many proteins involved in cell signalling and is thought to regulate signal
transduction by mediating the association of the PH domain containing protein with the cell membrane (113). Studies of Dok-1 isoforms suggest that the N-terminal plays an important role in determining the cellular localization of Dok-1 throughout the cytoplasm, and in Dok-2, the PH domain acts to stabilise Dok-2-EGFR binding and tyrosine phosphorylation of Dok-2 (93, 114). Phosphorylation of Dok-4 by receptor tyrosine kinase RET or cytosolic tyrosine kinase Fyn requires both its PH and PTB domains and results in the inhibition of Elk-1 activation (115). This effect, as well as the negative regulation of T cell activation, requires the PH domain of Dok-4 (116). The PH domain has also been shown to be important for the constitutive localization of Dok-4 and Dok-6 at the cell membrane (117). Itoh et al found that the mitochondrial location of Dok-4, and c-Src with which it associates, is dependent on the N-terminal and PTB domains and identified a putative mitochondrial targeting sequence in the N terminal of Dok-4 and Dok-5 (118). Expression of Dok-4 in endothelial mitochondria enhances TNF-α-induced reactive oxygen species (ROS) production and NF-κB production (118). In Dok-7, the N-terminal PH domain enables the nuclear import of Dok-7 (119).
Phosphotyrosine-binding (PTB) domains are found in a range of signalling and cytoskeletal proteins (120). Previous authors have shown that Dok-1 and Dok-2 associate with activated EGFR via their PTB domains and that the inhibition of Src tyrosine kinase-induced cellular transformation by Dok-1 is dependent on the Dok-1 PTB domain as well as the presence of C-terminal tyrosine phosphorylation sites (especially residues 336-363) (93, 112, 121). Upon tyrosine phosphorylation, Dok-1 and Dok-2 interact homotypically and heterotypically to form oligomers via the PTB domain and this oligomerisation is essential for Dok
phosphorylation and function (122). For example, the PTB domain and the amino terminal tyrosine 146 (Tyr (146)) residue enable Dok-1 to form homodimers, which are essential for the inhibition of Src transformation (112).
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The PTB domain of the Dok proteins has also been shown to play a key role in the formation of Dok-1 and Dok-5 homomeric and heteromeric associations, the interactions of Dok-1, Dok-2 and Dok-3 with SH2-containing inositol 5’-Phosphatase (SHIP)-1, the interaction of Dok-2 with receptor tyrosine kinases such as erbB-2 (HER2/neu) and Tek (Angiopoietin-1 receptor), and for the recruitment and phosphorylation of Dok-2 by activated EGFR, an interaction that is stabilized by the PH domain (93, 103, 123-126).
Dok-3 binds to, and is phosphorylated by, Abl tyrosine kinase via its PTB domain (102). Overexpression of Dok-3 reduces v-Abl-induced MAPK activation in a PTB dependent fashion but has no effect on constitutively activated Ras- and EGF-induced MAPK activation (102).
Unlike Dok-1, the PTB domain of Dok-4 and Dok-5 does not bind tyrosine phosphorylation sites in EGFR and when compared to Dok-1, the PTB domain of Dok-4 binds poorly to Ret with additional C-terminal residues being required for its binding (121, 125). However, the PTB domain of Dok-4 does bind SHIP-1, via phosphorylated NPXY motifs and SHIP-1 facilitates coupling of Abl to Dok-4 (125). In Dok-5, the PTB domain interacts with
Tropomyosin receptor kinase (Trk) B/C resulting in the activation of the MAPK pathway and in Dok-6, the PTB domain binds to the NPQY motif of TrkC (127, 128). TrkC is involved in nervous system development and this interaction with Dok-6 promotes neurotrophin 3 (NT-3) mediated neurite outgrowth in cortical neurons (105, 128). Dok-7 interacts with the
juxtamembrane region of MuSK via its PTB domain and both PH and PTB domains are required for MuSK activation (106, 119, 129).
The C-terminal of Dok-1 and Dok-2 (but not Dok-3) contains repeated YxxP motifs, which are involved in the interaction of the Dok proteins with SH domain-containing molecules, as well as providing multiple sites for tyrosine phosphorylation (102, 130). It is here that Dok-1 is tyrosine phosphorylated by p210 (bcr-abl) and specifically, Tyr (362) and Tyr (398) are
major sites for phosphorylation and for interaction of Dok-1 with GAP (131, 132). Tyr (362) is also involved in the interaction between Dok-1 and non-catalytic region of tyrosine kinase adaptor protein 1 (Nck) (132), whilst Tyr (336) and Tyr (340) are necessary for the negative regulation of Ras-Erk signalling and cellular transformation, though they are not required for p210rasGAP (RasGAP) binding (133). Dok-2 interacts with c-Abl in an interaction that is mediated by SH3 and a PMMP motif in the proline-rich tail of Dok-2 and which is enhanced by the SH2 domain (134). Through this interaction, the tyrosine phosphorylation and kinase activity of c-Abl is increased, with a corresponding increase in activity relating to cytoskeletal reorganization (134).
Hamuro et al found that the C-terminal region of Dok-7 contains a chromosome region maintenance 1-dependent nuclear export signal (NES) and that the NES-mediated
cytoplasmic location of Dok-7 is necessary for regulating its interaction with MuSK (119). The C-terminal of Dok-7 contains SH2 target motifs required for MuSK activation, and phosphorylation of tyrosine residues in this region by agrin results in the recruitment of the adapter proteins Crk and Crk-L which are required for normal NMJ formation (119, 129). Crk also contains N-terminal SH2 and C-terminal SH3 domains implicated in the positive and negative regulation of cellular transformation respectively (135). Overall, the PH, PTB, SH2 motifs and C-terminal NES are all important for Dok-7/MuSK signalling and disruption in these elements may give rise to NMJ synaptopathy (119).