Staurosporine-induced conformational changes of cAMP-dependent protein kinase catalytic subunit explain inhibitory potential

Interleukin-2 tyrosine kinase, Itk, is an important member of the Tec family of non-receptor tyrosine kinases that play a central role in signaling through antigen receptors such as the T-cell receptor, B-cell receptor, and Fc⑀. Selective inhibition of Itk may be an important way of modulating many diseases involving heightened or inappropriate activation of the immune system. In addition to an unliganded nonphophorylated Itk catalytic kinase domain, we determined the crystal structures of the phosphorylated and nonphosphorylated kinase domain bound to staurosporine, a potent broad-spectrum kinase inhibitor. These structures are useful for the design of novel, highly potent and selective Itk inhibitors and provide insight into the influence of inhibitor binding and phosphorylation on the conformation of Itk.The Tec kinases are a family of five non-receptor tyrosine kinases that play a central role in signaling through antigenreceptors such as the T-cell receptor (TCR), 1 B-cell receptor, and Fc⑀ (1) and are essential for T-cell activation. Three members of the family, Itk, Rlk, and Tec, are activated downstream of antigen receptor engagement in T-cells and transmit signals to downstream effectors, including PLC-␥. A fourth member, Btk, appears to act independently of T-cell signaling and is essential for B-cell development and activation. Btk-deficient murine mast cells have reduced degranulation and decreased production of proinflammatory cytokines following Fc⑀RI crosslinking (2). Btk deletion in mice has a profound effect on B-cell proliferation induced by anti-IgM and inhibits immune responses to thymus-independent type II antigens (3, 4). A biological role for the final member of this family, Bmx, has not been identified.Itk is a key member of this family, and a number of factors point to the importance of this kinase in immune disease.Deletion of Itk in mice results in reduced TCR-induced proliferation and secretion of the cytokines IL-2, IL-4, IL-5, IL-10, and interferon-␥ (5-7). Also, the immunological symptoms of allergic asthma are attenuated in ItkϪ/Ϫ mice, and lung inflammation, eosinophil infiltration, and mucous production are drastically reduced in response to challenge with the allergen OVA (8). Furthermore, the Itk gene is reported to be more highly expressed in peripheral blood T-cells from patients with moderate or severe atopic dermatitis than in controls or patients with mild atopic dermatitis (9).In certain cell types, the role of Itk may be intricately linked with other members of the family. For example, in mast cells, Btk and Itk are both expressed and activated by Fc⑀RI crosslinking (10). Splenocytes from RlkϪ/Ϫ mice secrete half the IL-2 produced by wild type animals in response to TCR engagement (5), whereas the combined deletion of Itk and Rlk in mice leads to a profound inhibition of TCR-induced responses, including proliferation and production of the cytokines IL-2, IL-4, IL-5, and interferon-␥ (5, 7). Furthermore, intracellular signaling following TCR engagement is affected in Itk...

Section: Interactions In the Atp-binding Site Of Itk-the Active Site Ofsupporting

confidence: 59%

Crystal Structures of Interleukin-2 Tyrosine Kinase and Their Implications for the Design of Selective Inhibitors

Brown

Long

Vial

et al. 2004

“…Accordingly, comparative analysis with three so far reported main conformational states of protein kinases, "open," "intermediate," and "closed" (reviewed in Ref. 31) indicates that, except for the glycine loop, relative disposition of the N-and C-lobes in PKC represents intermediate lobe structures (32), (20 (31). In contrast, and as represented in this crystal structure, the tip of the glycine-rich loop (residues 389 -392) moves deep inside the phosphate-binding subsite, approaching staurosporine and assuming positions that would clash with the nucleotide phosphate moiety.…”

Section: Resultsmentioning

confidence: 99%

Catalytic Domain Crystal Structure of Protein Kinase C-θ (PKCθ)

Xu¹,

Chaudhary

Olland³

et al. 2004

135

137

A member of the novel protein kinase C (PKC) subfamily, PKC, is an essential component of the T cell synapse and is required for optimal T cell activation and interleukin-2 production. Selective involvement of PKC in TCR signaling makes this enzyme an attractive therapeutic target in T cell-mediated disease processes. In this report we describe the crystal structure of the catalytic domain of PKC at 2.0-Å resolution. Human recombinant PKC kinase domain was expressed in bacteria as catalytically active phosphorylated enzyme and co-crystallized with its subnanomolar, ATP site inhibitor staurosporine. The structure follows the classic bilobal kinase fold and shows the enzyme in its active conformation and phosphorylated state. Inhibitory interactions between conserved features of staurosporine and the ATP-binding cleft are accompanied by closing of the glycine-rich loop, which also maintains an inhibitory arrangement by blocking the phosphate recognition subsite. The two major phosphorylation sites, Thr-538 in the activation loop and Ser-695 in the hydrophobic motif, are both occupied in the structure, playing key roles in stabilizing active conformation of the enzyme and indicative of PKC autocatalytic phosphorylation and activation during bacterial expression. The PKC-staurosporine complex represents the first kinase domain crystal structure of any PKC isotypes to be determined and as such should provide valuable insight into PKC specificity and into rational drug design strategies for PKC selective leads. Inhibitors of PKC1 are currently being used in clinical trials for various types of cancer, and a PKC␤ inhibitor is being used in clinical trials for diabetes-related retinopathy (1).PKC and PKB/AKT kinase domains are related by sequence homology; however, there are key structural differences in the regulatory domains and second messenger cofactor requirements. PKB/AKT contains an N-terminal pleckstrin homology domain regulated by phosphoinositide second messengers, a central catalytic kinase domain, and a C-terminal regulatory region facilitating key protein-protein interactions with signal-

“…rhZAP70 327-606 yielded diffractionquality crystals in the presence of staurosporine and AMP-PCP (see "Experimental Procedures"). Staurosporine, a microbial alkaloid from Streptomyces, is a nonselective, ATP-competitive inhibitor of many kinases (23)(24)(25)(26)(27). In the TR-FRET assay, staurosporine inhibited rhZAP70 327-606 with an IC 50 of 55.8 nM (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Jin

Pluskey²,

Petrella

et al. 2004

The ZAP-70 tyrosine kinase plays a critical role in T cell activation and the immune response and therefore is a logical target for immunomodulatory therapies. Although the crystal structure of the tandem Src homology-2 domains of human ZAP-70 in complex with a peptide derived from the subunit of the T cell receptor has been reported (Hatada, M. H., Lu, X., Laird, E. R., Green, J., Morgenstern, J. P., Lou, M., Marr, C. S., Phillips, T. B., Ram, M. K., Theriault, K., Zoller, M. J., and Karas, J. L. (1995) Nature 377, 32-38), the structure of the kinase domain has been elusive to date. We crystallized and determined the three-dimensional structure of the catalytic subunit of ZAP-70 as a complex with staurosporine to 2.3 Å resolution, utilizing an active kinase domain containing residues 327-606 identified by systematic N-and C-terminal truncations. The crystal structure shows that this ZAP-70 kinase domain is in an active-like conformation despite the lack of tyrosine phosphorylation in the activation loop. The unique features of the ATP-binding site, identified by structural and sequence comparison with other kinases, will be useful in the design of ZAP-70-selective inhibitors.The zeta-associated protein, 70 kDa (ZAP-70), 1 a Syk family tyrosine kinase associated with the subunit of the T cell receptor, is primarily expressed in T and NK cells and plays an essential role in signaling through the T cell receptor (TCR) (2, 3). TCR-mediated activation of T cells is crucial to the immune response. Transplant rejection and diseases, such as allergic responses and autoimmune disorders, stem from a failure to adequately modulate T cell activation. In humans, ZAP-70 gene mutations have been identified that confer lower ZAP-70 protein expression levels or catalytically inactive ZAP-70 proteins (4 -6). ZAP-70 deficiency results in the absence of mature CD8 ϩ T cells and the prevention of TCR-mediated activation of CD4 ϩ T cells, and it can lead to severe combined immunodeficiency. Peptides that block the association of ZAP-70 with the subunit (7,8) and peptides that antagonize ZAP-70 tyrosine kinase activity (9) block T cell activation in vitro. These studies indicate that ZAP-70 antagonists could be useful immunomodulatory therapeutic agents.ZAP-70 contains two N-terminal SH2 (Src homology domain 2) domains and a C-terminal kinase domain. During T cell activation, the binding of ZAP-70 SH2 domains to the phosphorylated subunit on the activated TCR complex causes a colocalization with the Lck tyrosine kinase that phosphorylates ZAP-70 on Tyr 493 in the activation loop (10, 11). Also, ZAP-70 autophosphorylates multiple tyrosines in the region between the SH2 domains and the kinase domain (10, 12), including the binding sites for additional SH2-containing signaling proteins such as SLP-76, Lat, Lck, PLC␥1, Vav, Shc, Ras-GAP, and Abl (reviewed in Refs. 8, 13, and 15). ZAP-70-mediated activation of these downstream effectors leads to the release of intracellular calcium stores, and the transcription of interleukin-2 and other ...