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Structural characterization of proline-rich tyrosine kinase 2 (PYK2) reveals a unique (DFG-out) conformation and enables inhibitor design.

ABSTRACT: Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptor tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase inhibitors.

SUBMITTER: Han S

PROVIDER: S-EPMC2676051 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Structural characterization of proline-rich tyrosine kinase 2 (PYK2) reveals a unique (DFG-out) conformation and enables inhibitor design.

Han Seungil S Mistry Anil A Chang Jeanne S JS Cunningham David D Griffor Matt M Bonnette Peter C PC Wang Hong H Chrunyk Boris A BA Aspnes Gary E GE Walker Daniel P DP Brosius Arthur D AD Buckbinder Leonard L

The Journal of biological chemistry 20090225 19

Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical ...[more]

PMID: 19244237

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Project description:AimsWe previously demonstrated Proline rich tyrosine kinase 2 (Pyk2) plays important roles in regulating tumor progression, migration and invasion in hepatocellular carcinoma (HCC). In this study, we aimed to examine the role of proline rich tyrosine kinase 2 (Pyk2) on cisplatin resistance in HCC and to explore its underlying molecular mechanism.Methodology/principal findingsStable transfectants either overexpressing or suppressing Pyk2 were established in different HCC cell lines. MTT, colony formation and Annexin-V assays were employed to examine their in vitro responses to cisplatin. Xenograft ectopic and orthotopic nude mice models were generated to investigate the in vivo responses of them to cisplatin treatment. cDNA microarray was performed to identify Pyk2-induced genes which were further validated by quantitative real-time RT-PCR using clinical HCC samples. In vitro functional study demonstrated that Pyk2-overexpressing HCC transfectants exhibited relatively lower cytotoxicity, higher colony-forming ability and lower apoptosis to cisplatin compared with the control transfectants. Moreover, Pyk2 overexpressing HCC transfectants had a higher survival rate under cisplatin treatment by up-regulation of AKT phosphorylation. In vivo xenograft nude mice model demonstrated that Pyk2-overexpressing transfectants developed higher tolerance to cisplatin treatment together with less tumor necrosis and apoptosis. cDNA microarray analysis revealed that there were more than 4,000 genes differentially expressed upon overexpression of Pyk2. Several upregulated genes were found to be involved in drug resistance and invasion in cancers. Among them, the expression profiles of MDR1, GAGE1, STAT1 and MAP7 were significantly associated with the expression of Pyk2 in clinical HCC samples.ConclusionsOur results may suggest a new evidence of Pyk2 on promoting cisplatin resistance of HCC cells through preventing cell apoptosis, activation of AKT pathway and upregulation of drug resistant genes.

Dataset Information

Structural characterization of proline-rich tyrosine kinase 2 (PYK2) reveals a unique (DFG-out) conformation and enables inhibitor design.

Publications

Structural characterization of proline-rich tyrosine kinase 2 (PYK2) reveals a unique (DFG-out) conformation and enables inhibitor design.

Similar Datasets

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