The Three-dimensional Structure of the ZAP-70 Kinase Domain in Complex with Staurosporine

Jin, Lei; Pluskey, Scott; Petrella, Eugene C.; Cantin, Susan; Gorga, Joan C.; Rynkiewicz, Michael J.; Pandey, Pramod; Strickler, J. Rudi; Babine, Robert E.; Weaver, David T.; Seidl, Katherine J.

doi:10.1074/jbc.m407096200

Cited by 58 publications

(59 citation statements)

References 51 publications

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“…AMP-PNP and MgCl 2 reduced background precipitation but were not necessary for crystal growth, nor were they visible in the final electron density. Similar behavior was reported for Zap-70 incubated with AMP-PCP (22). For preparation of Syk-Gleevec crystals, Gleevec was added to the protein to a final concentration of 1 mM 1 h before crystallization using the strategy outlined above.…”

Section: Methodsmentioning

confidence: 92%

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A Novel Mode of Gleevec Binding Is Revealed by the Structure of Spleen Tyrosine Kinase

Atwell

Adams

Badger

et al. 2004

Journal of Biological Chemistry

182

159

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Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine 3 phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine.Syk family members include two human proteins, Syk (spleen tyrosine kinase) and its closest relative Zap-70 (70-kDa chain-associated protein 1). Syk operates downstream of the B-cell receptor in B-cells, the IgE receptor Fc⑀RI in mast cells, Fc␥R in macrophages (2), and other receptors (3). Zap-70 performs a similar function in T-cell receptor signaling (4). Syk is expressed in a wide range of cell types, although its function is best understood in hematopoietic cells. Knock-out mouse studies have shown that Syk is essential for lymphocyte development (2). Syk has attracted particular interest as a therapeutic target for treatment of asthma, because Syk-deficient mast cells do not degranulate in response to Fc⑀RI aggregation (5, 6). Analyses of three-dimensional structures of the kinase domains of the insulin receptor kinase (7,8), fibroblast growth factor receptor 1 (9), and Lck (10) gave rise to a model of conformational transition upon activation in kinases. In this model, the preactivated conformation is characterized by activation loop occlusion of the ATP-and/or substrate-binding sites ("loop in"), effectively preventing substrate access. Phosphorylation stabilizes an open conformation of the activation loop ("loop out") that does not occlude the substrate-binding sites and is compatible with catalysis (11, 12). In the loop-out conformation, the activation loop also forms a platform for peptide substrate docking (12). In addition to permitting access to the su...

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Section: Methodsmentioning

confidence: 92%

“…Unphosphorylated Syk (17) or Syk mutated at the activation loop tyrosines retains substantial catalytic activity (32)(33)(34)(35). The recently published structure of the isolated Zap-70 kinase catalytic domain suggests that it too adopts an active conformation (22) in the absence of phosphorylation.…”

Section: Discussionmentioning

confidence: 99%

A Novel Mode of Gleevec Binding Is Revealed by the Structure of Spleen Tyrosine Kinase

Atwell

Adams

Badger

et al. 2004

Journal of Biological Chemistry

182

159

View full text Add to dashboard Cite

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“…For example, when the structure of Chk2 is compared with those of Zap (PDB code: 1U59) and EGFR (PDB code: 1XKK), it is self evident that full access to this pocket is impeded by two bulkier methionine residues in the latter two enzymes whereas the analogous residues are Leu277 and Leu301 in Chk2. 40,41 Leu301 in Chk2 corresponds to the ''gatekeeper'' residue in many kinases, 42 which has been found to form contacts with bound inhibitors and is poorly conserved. Accordingly, differences in this position appear to account for the selectivity of many kinase inhibitors by modulating access to the hydrophobic pocket.…”

Section: Implications For Drug Designmentioning

confidence: 99%

Crystal structure of checkpoint kinase 2 in complex with NSC 109555, a potent and selective inhibitor

et al. 2008

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Checkpoint kinase 2 (Chk2), a ser/thr kinase involved in the ATM-Chk2 checkpoint pathway, is activated by genomic instability and DNA damage and results in either arrest of the cell cycle to allow DNA repair to occur or apoptosis if the DNA damage is severe. Drugs that specifically target Chk2 could be beneficial when administered in combination with current DNAdamaging agents used in cancer therapy. Recently, a novel inhibitor of Chk2, NSC 109555, was identified that exhibited high potency (IC 50 = 240 nM) and selectivity. This compound represents a new chemotype and lead for the development of novel Chk2 inhibitors that could be used as therapeutic agents for the treatment of cancer. To facilitate the discovery of new analogs of NSC 109555 with even greater potency and selectivity, we have solved the crystal structure of this inhibitor in complex with the catalytic domain of Chk2. The structure confirms that the compound is an ATP-competitive inhibitor, as the electron density clearly reveals that it occupies the ATPbinding pocket. However, the mode of inhibition differs from that of the previously studied structure of Chk2 in complex with debromohymenialdisine, a compound that inhibits both Chk1 and Chk2. A unique hydrophobic pocket in Chk2, located very close to the bound inhibitor, presents an opportunity for the rational design of compounds with higher binding affinity and greater selectivity.

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“…We were surprised to find that in the inactive full-length structure of ZAP-70, the kinase domain did not deviate significantly from the structure of the active, isolated kinase domain (29) in the regions involved in tandem SH2 docking. Instead, the structure of inactive ZAP-70 revealed an extensive network of hydrogen-bonding interactions that appear to rigidify the hinge region connecting the 2 lobes of the kinase domain (Fig.…”

mentioning

confidence: 99%

Stability of an autoinhibitory interface in the structure of the tyrosine kinase ZAP-70 impacts T cell receptor response

Deindl

Kadlecek

Cao

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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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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The Three-dimensional Structure of the ZAP-70 Kinase Domain in Complex with Staurosporine

Cited by 58 publications

References 51 publications

A Novel Mode of Gleevec Binding Is Revealed by the Structure of Spleen Tyrosine Kinase

A Novel Mode of Gleevec Binding Is Revealed by the Structure of Spleen Tyrosine Kinase

Crystal structure of checkpoint kinase 2 in complex with NSC 109555, a potent and selective inhibitor

Stability of an autoinhibitory interface in the structure of the tyrosine kinase ZAP-70 impacts T cell receptor response

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