The cancer drug imatinib inhibits the tyrosine kinases c-Abl, c-Kit, and the PDGF receptor. Imatinib is less effective against c-Src, which is difficult to understand because residues interacting with imatinib in crystal structures of Abl and c-Kit are conserved in c-Src. The crystal structure of the c-Src kinase domain in complex with imatinib closely resembles that of Abl*imatinib and c-Kit*imatinib, and differs significantly from the inactive "Src/CDK" conformation of the Src family kinases. Attempts to increase the affinity of c-Src for imatinib by swapping residues with the corresponding residues in Abl have not been successful, suggesting that the thermodynamic penalty for adoption of the imatinib-binding conformation by c-Src is distributed over a broad region of the structure. Two mutations that are expected to destabilize the inactive Src/CDK conformation increase drug sensitivity 15-fold, suggesting that the free-energy balance between different inactive states is a key to imatinib binding.

The kinase domain of human epidermal growth factor receptor (HER) 3/ErbB3, a member of the EGF receptor (EGFR) family, lacks several residues that are critical for catalysis. Because catalytic activity in EGFR family members is switched on by an allosteric interaction between kinase domains in an asymmetric kinase domain dimer, HER3 might be specialized to serve as an activator of other EGFR family members. We have determined the crystal structure of the HER3 kinase domain and show that it appears to be locked into an inactive conformation that resembles that of EGFR and HER4. Although the crystal structure shows that the HER3 kinase domain binds ATP, we confirm that it is catalytically inactive but can serve as an activator of the EGFR kinase domain. The HER3 kinase domain forms a dimer in the crystal, mediated by hydrophobic contacts between the N-terminal lobes of the kinase domains. This N-lobe dimer closely resembles a dimer formed by inactive HER4 kinase domains in crystal structures determined previously, and molecular dynamics simulations suggest that the HER3 and HER4 N-lobe dimers are stable. The kinase domains of HER3 and HER4 form similar chains in their respective crystal lattices, in which N-lobe dimers are linked together by reciprocal exchange of C-terminal tails. The conservation of this tiling pattern in HER3 and HER4, which is the closest evolutionary homolog of HER3, might represent a general mechanism by which this branch of the HER receptors restricts ligand-independent formation of active heterodimers with other members of the EGFR family.EGFR ͉ human epidermal growth factor receptor 3 ͉ human epidermal growth factor receptor4 ͉ receptor oligomerization

ZAP-70, a cytoplasmic tyrosine kinase required for T cell antigen receptor signaling, is controlled by a regulatory segment that includes a tandem SH2 unit responsible for binding to immunoreceptor tyrosine-based activation motifs (ITAMs). The crystal structure of autoinhibited ZAP-70 reveals that the inactive kinase domain adopts a conformation similar to that of cyclin-dependent kinases and Src kinases. The autoinhibitory mechanism of ZAP-70 is, however, distinct and involves interactions between the regulatory segment and the hinge region of the kinase domain that reduce its flexibility. Two tyrosine residues in the SH2-kinase linker that activate ZAP-70 when phosphorylated are involved in aromatic-aromatic interactions that connect the linker to the kinase domain. These interactions are inconsistent with ITAM binding, suggesting that destabilization of this autoinhibited ZAP-70 conformation is the first step in kinase activation.

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The delivery of signals from the activated T cell antigen receptor (TCR) inside the cell relies on the protein tyrosine kinase ZAP-70 (-associated protein of 70 kDa). A recent crystal structure of inactive full-length ZAP-70 suggests that a central interface formed by the docking of the two SH2 domains of ZAP-70 onto the kinase domain is crucial for suppressing catalytic activity. Here we validate the significance of this autoinhibitory interface for the regulation of ZAP-70 catalytic activity and the T cell response. For this purpose, we perform in vitro catalytic activity assays and binding experiments using ZAP-70 proteins purified from insect cells to examine activation of ZAP-70. Furthermore, we use cell lines stably expressing wild-type or mutant ZAP-70 to monitor proximal events in T cell signaling, including TCR-induced phosphorylation of ZAP-70 substrates, activation of the MAP kinase pathway, and intracellular Ca 2؉ levels. Taken together, our results directly correlate the stability of the autoinhibitory interface with the activation of these key events in the T cell response.activation ͉ catalytic activity ͉ conformation ͉ ITAM T he protein tyrosine kinase ZAP-70 (-associated protein of 70 kDa) (1) participates in the initial events of T cell antigen receptor (TCR) signaling and thus is of critical importance for the adaptive immune response (2-5). On activation of the TCR, 2 tyrosines in the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 or cytoplasmic tails of the TCR are phosphorylated by a Src family kinase member. Through the binding of 2 SH2 (Src homology 2) domains at the N terminus of ZAP-70 to these ITAM phosphotyrosines, ZAP-70 is then specifically recruited to a stimulated TCR. These 2 SH2 domains of ZAP-70 are tightly associated as a tandem unit with a precise structural organization that enables high-affinity ITAM peptide binding (6). In the very closely related Syk (spleen tyrosine) kinase, the destabilization of this structural organization by mutation results in both uncoupling of the 2 SH2 units and a dramatic reduction in ITAM binding affinity (7). Recruitment of ZAP-70 to the TCR results in activation of the catalytic domain located at the C terminus of ZAP-70 and signal propagation by phosphorylation of its downstream target molecules, including the ZAP-70 substrates LAT (linker for the activation of T cells) and SLP-76 (SH2 domain-containing leukocyte phosphoprotein of 76 kDa). Both LAT and SLP-76 are crucial adaptor proteins that, once phosphorylated, provide a platform for the assembly of signaling molecules (8, 9). Ultimately, these signaling events produce effector secretory responses, modulate transcription, and trigger T cell proliferation and differentiation.ZAP-70 plays critical roles in pre-TCR and TCR signaling, thymocyte development, and peripheral T cell function. The loss of ZAP-70 catalytic activity results in profound immunodeficiency, and reduced activity of ZAP-70 may result in autoimmunity (4, 10). Furthermore, the expression of ZAP-70 in B c...

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