Structural Basis for Chk1 Inhibition by UCN-01

Zhao, Baoguang; Bower, Michael J.; McDevitt, Patrick; Zhao, Huizhen; Davis, Stephen T.; Johanson, Kyung; Green, Susan M.; Concha, N.O.; Zhou, Bin‐Bing S.

doi:10.1074/jbc.m201233200

Cited by 201 publications

(135 citation statements)

References 21 publications

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“…Since part of the activation loop is interacting with a symmetry-related molecule in the crystal, it is impossible to determine from this structure whether the loop was in an apparently active conformation in solution prior to crystal packing or whether crystal packing induced the observed conformation. However, a similar conformation of the nonphosphorylated activation loop is observed in the crystal structure of the Chk1-staurosporine complex (33). There is a strong correlation between the phosphorylation of full-length ZAP-70 and its activation (3, 7, 11, 21, 22, 34 -36).…”

Section: Resultsmentioning

confidence: 65%

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

Jin

Pluskey²,

Petrella

et al. 2004

Journal of Biological Chemistry

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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 ...

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Section: Resultsmentioning

confidence: 65%

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

Jin

Pluskey²,

Petrella

et al. 2004

Journal of Biological Chemistry

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“…Others have reported that UCN-01 inhibited cdks resulting in cell cycle arrest or apoptosis (De Azevedo et al 1996;Brusselbach et al 1998;Gersher 2000;Kohn et al 2002;Senderowicz 2002;Zhao et al 2002). In colon cancer, UCN-01 was shown to inhibit p53 up-regulation, chk2 and chk1, although the action of UCN-01 was determined to be independent of p53 (Yu et al 2002).…”

Section: Discussionmentioning

confidence: 99%

UCN‐01 alters phosphorylation of Akt and GSK3β and induces apoptosis in six independent human neuroblastoma cell lines

Shankar¹,

Krupski²,

Parashar³

et al. 2004

Journal of Neurochemistry

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In this study we evaluated UCN-01, a small molecule that inhibits protein kinases by interacting with the ATP-binding site, as a potential anti-cancer agent for neuroblastoma. UCN-01 was effective at inducing apoptosis in six neuroblastoma cell lines with diverse cellular and genetic phenotypes. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), terminal deoxynucleotidyl transferase-mediated dUTP nickend-labeling (TUNEL) assays, detection of active caspase-3 and cleaved poly ADP-ribose polymerase (PARP) confirmed that UCN-01 induced apoptosis. Cell cycle analysis determined that the UCN-01 treated cells accumulated in S phase by 16 h. Unlike vinblastine and docetaxel that increased survivin expression, UCN-01 treatment did not increase X-linked inhibitor of apoptosis protein (XIAP) and survivin levels. Analysis of specific phosphoepitopes on chk1/2, Akt, and GSK3b following UCN-01 treatment determined that there was no significant change in phospho-chk1/2. However, there was decreased immunoreactivity at Ser473 and Thr308 of Akt and Ser9 of GSK3b by 4 h indicating that the Akt survival pathway and downstream signalling was compromised. Thus, UCN-01 was effective at inducing apoptosis in neuroblastoma cell lines. Keywords: Akt, apoptosis, GSK3b, neuroblastomas, staurosporine, survivin, 7-hydroxystaurosporine (UCN-01). Neuroblastoma is the most common malignancy of infancy and constitutes 50% of all childhood cancers (Gale et al. 1982;Young et al. 1986;Taylor and Locker 1990;Brodeur and Castleberry 1997). Some neuroblastomas may be cured by minimal therapy; however, there is no effective treatment for many advanced and metastatic neuroblastomas (Brodeur and Castleberry 1997). In addition, present treatments for neuroblastoma have significant morbidity (Brodeur and Castleberry 1997). Among the specific biological abnormalities that correlate with poor clinical outcome and aggressiveness of the tumor are nuclear DNA content, near diploidy or tetraploidy (Taylor et al. 1988;Taylor and Locker 1990;Gestblom et al. 1995), deletion of the short arm of chromosome 1 (1p) (Westermann and Schwab 2002), N-myc amplification (Taylor and Locker 1990), loss of Bcl-2 (Reed et al. 1991), insensitivity to radiation (Halperin et al. 1999), and increased expression of the inhibitor-of-apoptosis (IAP) family member, survivin (Adida et al. 1998;Azuhata et al. 2001). While these factors predict therapeutic outcome, there are specific biological determinants, such as components of the apoptotic cascade, that may be prognostic indicators for drug resistance (Emran et al. 2002;Gallo et al. 2003;Iolascon et al. 2003). Therefore, new and novel therapies with a biological rationale are urgently needed for this fatal disease.Members of the IAP family bind to and inhibit caspases and suppress cell death in cancer. Presently, there are eight mammalian IAP family members that include survivin, neuronal apoptosis inhibitor protein (NAIP), BIR-repeatcontaining ubiquitin-conjugating enzyme (BRUCE), livin, ILP-2, cIAP-1, cIAP-...

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“…For example, UCN-01 was first identified as protein kinase C (PKC) inhibitor but later found to inhibit both Chk1 and Chk2. 35,36 Caffeine, on the other hand, can inhibit ATM/ATR, which are upstream regulators of Chk1 and Chk2 kinases. 37,38 Here, we have used an RNA interference (RNAi)-mediated gene knockdown approach to investigate the comparative roles of Wee1, Chk1, and Myt1 kinases in the G 2 checkpoint pathway in Hela versus normal human mammary epithelial cells (HMEC), which lack or express functional p53 alleles, respectively.…”

confidence: 99%

Knockdown of Chk1, Wee1 and Myt1 by RNA Interference Abrogates G2 Checkpoint and Induces Apoptosis

Wang¹,

Decker²,

Sebolt–Leopold³

2004

Cancer Biology & Therapy

124

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Structural Basis for Chk1 Inhibition by UCN-01

Cited by 201 publications

References 21 publications

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

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

UCN‐01 alters phosphorylation of Akt and GSK3β and induces apoptosis in six independent human neuroblastoma cell lines

Knockdown of Chk1, Wee1 and Myt1 by RNA Interference Abrogates G2 Checkpoint and Induces Apoptosis

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