The hepatocyte growth factor/scatter factor receptor (cMet) is a target in oncology particularly due to its role in the development of invasive and metastatic tumor phenotypes. It is an a-P heterodimer with an extracellular a-chain linked by a disulphide bond to a membrane-spanning P-chain. Phosphorylation of tyrosines 1234 and 1235 in the A-loop activate the kinase, which then leads to autophosphorylation of tyrosines 1349 and 1356 in the C-terminal tail. The latter become binding sites for SH2, Gab1, and PTB binding domains of a variety of transducer and adaptor proteins. The structure of the unphosphorylated kinase domain and C-terminal tail has been determined in the apo form and also in complex with a staurosporine analog (10). These structures both show an inactive conformation of the kinase domain where helix C is displaced, the Lys1110-Glu1127 salt bridge is not formed, and the A-loop obstructs the substrate binding site. Part of the A-loop also passes through the phosphate binding site of ATP and disallows proper positioning of helix C. It can, however, interact with the inhibitor, which is interesting for designing selective inhibitors. There is also an extra a-helix (helix A) at the N-terminus of the kinase domain that interacts with helix C. It has been suggested that this helix might have a role in the activation of the kinase domain when an extracellular ligand binds, because of the fact that a concerted movement of helices A and C will be required (10). Activating mutations in the A-loop and the JM region, like in the cases of Abl and cKit, are found in patients with various forms of cancer. The C-terminal tail structure shows that residues 1349-1352, 1353-1356 and 1356-1359 have similar conformations to peptides bound to SH2, PTB, and Grb2SH2 domains, respectively. The binding of these proteins to the first two motifs would be prevented by clashes with the C-terminal lobe of the kinase, but the third motif is accessible. In the structure the tyrosine residues in these motifs are unphosphorylated. Phosphorylation would presumably make them all accessible for binding and thus downstream signaling.
The Eph receptors consist of an extracellular portion containing a ligand-binding domain, a cysteine-rich domain, and two fibronectin III repeats, and an intracellular portion containing a JM region, a tyrosine kinase domain, a sterile-a-motif (involved in oligomerization), and a PDZ binding motif. Like most other receptor tyrosine kinases, the catalytic domain of the Eph receptors is regulated by the JM region. In the structure of EphB2 (14), the unphosphorylated JM
region contains a helix that interacts extensively with the N-terminal lobe of the kinase domain. The interactions cause distortion of helix C in the N-terminal lobe, which means that the conserved glutamate side chain from this structural element is slightly displaced from the active site and the kinase is inactive. The JM region probably also blocks the A-loop from adopting an active conformation. The overall conformation of the kinase domain is closed and resembles that of an active conformation, despite the fact that helix C is bent and the A-loop has an inactive fold. Phosphorylation of the tyrosine residues in the JM region would keep it away from the kinase domain owing to steric and electrostatic forces as observed for cKit. Helix C and the A-loop could then adopt an active conformation and the JM region would be available to interact with signaling proteins that have pTyr binding sites. However, the structure of the isolated kinase domain of the homologous EphA2 shows that in the absence of the JM region, helix C can still be bent and the kinase remains in an inactive conformation (63). This suggests that there is an inherent structural flexibility in helix C of the Eph receptor kinases that facilitates autoinhibition. The A-loop in both of these structures is disordered in the crystals, indicating that it is exposed to solvent and flexible. It is possible that the JM region can stabilize the autoinhibited conformation of the Eph receptor kinase, whereas phosphorylation of the A-loop in the absence of the JM region destabilizes the bent conformation of helix C and shifts the equilibrium toward the active conformation.
In contrast to other members of the receptor tyrosine kinase (RTK) family, there is evidence that epidermal growth factor receptors (EGFRs) exist as preformed dimers and form higher order oligomers, and heterooligomers with other members of the subfamily (ErbB2, ErbB3, ErbB4) for signaling (70,71). Autophosphorylation of tyrosine residues in the cytoplasmic part of the receptor provides sites for the binding of SH2 proteins and signaling. EGFR (and ErB2 and ErbB4) does not require phosphorylation of residues in the kinase domain for activity. In addition, the EGFR subfamily members have a dimerization domain between the kinase domain and the C-terminal phosphorylation sites.
The structure shows an active conformation for the kinase domain despite the lack of phosphorylation (72). The A-loop superimposes very well with those of activated kinase structures, such as trisphosphorylated IRK and monophosphory-lated Lck. Although Tyr845 is in the same position as tyrosines in other kinases that need to be phosphorylated for activity, mutation of this residue in EGFR has no effect on the activity (73). This may be because of the position of a neighbouring glutamate side chain (Glu848), which could structurally mimic the phosphate group of a phosphorylated A-loop tyrosine, stabilizing the active conformation of the A-loop. In the crystals there is a large packing interface between two kinase domains with part of the C-terminal tail sandwiched between them. It is possi ble that this type of interaction has a role in the regulation of the activity of the kinase. A C-terminal motif (Leu-Val-Ile) has previously been shown to have a role in the regulation of the phosphorylation of substrate tyrosines, but it interacts tightly with the C-terminal lobe and is not available for binding in the conformation observed in the crystal structure. These residues may become more accessible on disruption of the dimer, an event that has been observed in vitro on the addition of ATP. The binding of ATP would require a change in the relative orientation of the N- and C-terminal lobes, which would disturb the dimer interaction seen in the crystals.
Dimerization of the fibroblast growth factor receptor (FGFR) requires the binding of heparin sulphate proteoglycans in addition to the growth factor. The subsequent activation allows autophosphorylation of up to seven tyrosines in the cytosolic domain, including two in the kinase domain that are critical for activation, and one in the C-terminal tail that is a site for binding of phospho-lipase. The crystal structure of the unphosphorylated kinase domain of FGFR1 shows an autoinhibited state, where interactions between the conserved DFG motif at the N-terminus of the A-loop and residues from helix C hold the kinase in a rather open conformation, although less open than the inactive IRK structure (9). The path of the A-loop in FGFR1 lies halfway between that of an active conformation like in trisphosphorylated IRK and an inactive conformation like in unphosphorylated IRK or Abl. The DFG motif is in an active conformation, but the binding of ATP is blocked because the P-loop has folded down into the ATP binding site. The C-terminal part of the A-loop blocks the substrate binding site, but the unphosphorylated tyrosines of the A-loop do not bind in the substrate site in the way that was observed for IRK. Structures of FGFR1 have also been determined in complex with inhibitors (74). It is interesting to note that the most selective of these induced conformational changes in the P-loop. As for Abl kinase, the ability of some kinase domains and not others to adapt to the binding of certain inhibitors contributes to the selectivity of these compounds.
3.2.8. Vascular Endothelial Growth Factor Receptor and Tie2
The Tie receptor PTKs (Tie1 and Tie2, or Tek) as well as the vascular endothelial growth factor receptor (VEGFR) PTKs have a role in normal vascular development. They also have a critical role in a number of diseases, such as ischemic coronary artery disease, cancer, diabetic retinopathy, and rheumatoid arthritis. The VEGFRs are believed to act in the early stages of vascular development, whereas the Tie receptors are involved in vascular remodeling and maturation.
Tie2 is tightly regulated by agonistic and antagonistic extracellular ligands. Activation requires phosphorylation in response to the binding of agonists. The structure of the unphosphorylated Tie2 kinase domain has been determined using a construct that also contains the KID and the C-terminal tail, but does not contain the JM region (29). The structure was solved in four different crystal forms and all of them show the same conformation, which rules out the possibility that the structures of features like the P-loop, the C-terminal tail, and the A-loop would be owing to crystal packing artefacts, because the different crystal forms have different crystal packing arrangements. This structure shows some novel means of self-regulation including the P-loop, which binds in the ATP binding cleft and blocks the binding of this cofactor, and the C-terminal tail, which partially blocks the substrate binding site. The A-loop adopts an active-like conformation, despite the fact that it is not phosphorylated, but the DFG motif has an inactive conformation similar to that seen for IRK, which also contributes to block the binding of ATP. The KID is ordered in Tie2 and forms 2 short a-helical segments that pack against the C-terminal lobe (Fig. 8). The C-terminal tail packs under the KID, runs along aI, aF, and aE and ends near the substrate binding site. Two tyrosines in this tail (Tyr1101, Tyr1112) are buried in this structure, but are known to interact with a number of proteins containing SH2 and PTB domains when phosphorylated. Deletion of the C-terminus has been shown to significantly enhance activity of Tie2 (32). Helix C from the N-terminal lobe is shifted compared to an active PTK conformation, not dramatically, but enough to prevent the formation of the Glu872 to Lys855 salt bridge required for the correct positioning of the a and P phosphates of ATP for catalysis.
Phosphorylation of Tyr1212 in the VEGFR2 (KDR) C-terminal tail is also crucial for the activation of this receptor and subsequent VEGFR2-mediated angiogenesis (75). Other tyrosine residues that become phosphorylated are Tyr1054 and Tyr1059 in the activation loop, and Tyr799 and Tyr1173 that are binding sites for PI3 kinase. The structure of KDR phosphorylated on Tyr1059 (67) has an open, inactive conformation very similar to that of FGFR1 (9). The A-loop is largely disordered, but the positions of the P-loop and helix C superimpose well with those of FGFR1. The start and end portions of the KID are visible in the electron density and form a two-stranded P-sheet between them. Phe935, which is conserved in many KIDs, is important for the structural integrity of this motif. Therefore, this structural feature may be well conserved among other receptor PTKs. The structure of KDR phosphorylated on Tyr1054 and Tyr1059 has also been determined in complex with a small-molecule inhibitor (76) and revealed that the DFG motif is displaced to allow binding of the compound in a very similar fashion to what is observed for the binding of Gleevec to Abl kinase. The relative orientation of the N- and C-terminal lobes is very similar to that observed for Abl, but slightly more closed than in the apo-structure. The position of helix C is also similar in Abl. However, the P-loop in
KDR adopts an active-like conformation, suggesting that the unusual conformation of this loop in Abl is very specific to Abl in complex with Gleevec.
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