Laszlo Gyenis

An Unbiased Evaluation of CK2 Inhibitors by Chemoproteomics Characterization of Inhibitor Effects on CK2 and Identification of Novel Inhibitor Targets

Recently protein kinases have emerged as some of the most promising drug targets; and therefore, pharmaceutical strategies have been developed to inhibit kinases in the treatment of a variety of diseases. CK2 is a serine/threonine-protein kinase that has been implicated in a number of cellular processes, including maintenance of cell viability, protection of cells from apoptosis, and tumorigenesis. Elevated CK2 activity has been established in a number of cancers where it was shown to promote tumorigenesis via the regulation of the activity of various oncogenes and tumor suppressor proteins. Consequently the development of CK2 inhibitors has been ongoing in preclinical studies, resulting in the generation of a number of CK2-directed compounds. In the present study, an unbiased evaluation of CK2 inhibitors 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz), and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) was carried out to elucidate the mechanism of action as well as inhibitor specificity of these compounds. Utilizing a chemoproteomics approach in conjunction with inhibitor-resistant mutant studies, CK2α and CK2α′ were identified as bona fide targets of TBB, TBBz, and DMAT in cells. However, inhibitor-specific cellular effects were observed indicating that the structurally related compounds had unique biological properties, suggesting differences in inhibitor specificity. Rescue experiments utilizing inhibitor-resistant CK2 mutants were unable to rescue the apoptosis associated with TBBz and DMAT treatment, suggesting the inhibitors had off-target effects. Exploitation of an unbiased chemoproteomics approach revealed a number of putative off-target inhibitor interactions, including the discovery of a novel TBBz and DMAT (but not TBB) target, the detoxification enzyme quinone reductase 2 (QR2). The results described in the present study provide insight into the molecular mechanism of action of the inhibitors as well as drug specificity that will assist in the development of more specific next generation CK2 inhibitors.

Loss of Pannexin 1 Attenuates Melanoma Progression by Reversion to a Melanocytic Phenotype

Background: Panx1 is a channel-forming glycoprotein that regulates epidermal differentiation and proliferation. Results: Depletion of Panx1 in melanomas causes cell re-differentiation into a melanocytic-like phenotype and reduced tumorigenesis. Conclusion: Panx1 is up-regulated during melanoma progression promoting tumor growth and metastasis. Significance: This is the first report of Panx1 as a proto-oncogene establishing it as a potential target for melanoma treatment. Pannexin 1 (Panx1) is a channel-forming glycoprotein expressed in different cell types of mammalian skin. We examined the role of Panx1 in melanoma tumorigenesis and metastasis since qPCR and Western blots revealed that mouse melanocytes exhibited low levels of Panx1 while increased Panx1 expression was correlated with tumor cell aggressiveness in the isogenic melanoma cell lines (B16-F0, -F10, and -BL6). Panx1 shRNA knockdown (Panx1-KD) generated stable BL6 cell lines, with reduced dye uptake, that showed a marked increase in melanocyte-like cell characteristics including higher melanin production, decreased cell migration and enhanced formation of cellular projections. Western blotting and proteomic analyses using 2D-gel/mass spectroscopy identified vimentin and β-catenin as two of the markers of malignant melanoma that were down-regulated in Panx1-KD cells. Xenograft Panx1-KD cells grown within the chorioallantoic membrane of avian embryos developed tumors that were significantly smaller than controls. Mouse-Alu qPCR of the excised avian embryonic organs revealed that tumor metastasis to the liver was significantly reduced upon Panx1 knockdown. These data suggest that while Panx1 is present in skin melanocytes it is up-regulated during melanoma tumor progression, and tumorigenesis can be inhibited by the knockdown of Panx1 raising the possibility that Panx1 may be a viable target for the treatment of melanoma.

The emerging CK2 interactome: insights into the regulation and functions of CK2

Protein kinase CK2 represents a small family of protein serine/threonine kinases implicated in a variety of biological processes including events relating to cell proliferation and survival. Notably, CK2 displays oncogenic activity in mice and exhibits altered expression in several types of cancer. Accordingly, a detailed understanding of the cellular functions of CK2 and elucidation of the mechanisms by which CK2 is regulated in cells is expected to contribute to understanding its role in tumorigenesis with the prospect of novel approaches to therapy. While CK2 has traditionally been viewed as a tetrameric complex composed of two catalytic and two regulatory subunits, mounting evidence suggests that its subunits may have functions independent of tetrameric CK2 complexes. In mammals, as is the case in the budding yeast Saccharomyces cerevisiae, there are two isozymic forms of CK2, adding additional heterogeneity to the CK2 family. Studies in yeast and in human cells demonstrate that the different forms of CK2 interact with a large number of cellular proteins. To reveal new insights regarding the regulation and functions of different forms of CK2, we have examined the emerging interactomes for each of the CK2 subunits. Analysis of these interactomes for both yeast and human CK2 reinforces the view that this family of enzymes participates in a broad spectrum of cellular events. Furthermore, while there is considerable overlap between the interactomes of the individual CK2 subunits, notable differences in each of the individual interactomes provides additional evidence for functional specialization for the individual forms of CK2.

mTORC1 and CK2 coordinate ternary and eIF4F complex assembly

Ternary complex (TC) and eIF4F complex assembly are the two major rate-limiting steps in translation initiation regulated by eIF2α phosphorylation and the mTOR/4E-BP pathway, respectively. How TC and eIF4F assembly are coordinated, however, remains largely unknown. We show that mTOR suppresses translation of mRNAs activated under short-term stress wherein TC recycling is attenuated by eIF2α phosphorylation. During acute nutrient or growth factor stimulation, mTORC1 induces eIF2β phosphorylation and recruitment of NCK1 to eIF2, decreases eIF2α phosphorylation and bolsters TC recycling. Accordingly, eIF2β mediates the effect of mTORC1 on protein synthesis and proliferation. In addition, we demonstrate a formerly undocumented role for CK2 in regulation of translation initiation, whereby CK2 stimulates phosphorylation of eIF2β and simultaneously bolsters eIF4F complex assembly via the mTORC1/4E-BP pathway. These findings imply a previously unrecognized mode of translation regulation, whereby mTORC1 and CK2 coordinate TC and eIF4F complex assembly to stimulate cell proliferation.

Pin1: Intimate involvement with the regulatory protein kinase networks in the global phosphorylation landscape☆☆☆

BACKGROUND Protein phosphorylation is a universal regulatory mechanism that involves an extensive network of protein kinases. The discovery of the phosphorylation-dependent peptidyl-prolyl isomerase Pin1 added an additional layer of complexity to these regulatory networks. SCOPE OF REVIEW We have evaluated interactions between Pin1 and the regulatory kinome and proline-dependent phosphoproteome taking into consideration findings from targeted studies as well as data that has emerged from systematic phosphoproteomic workflows and from curated protein interaction databases. MAJOR CONCLUSIONS The relationship between Pin1 and the regulatory protein kinase networks is not restricted simply to the recognition of proteins that are substrates for proline-directed kinases. In this respect, Pin1 itself is phosphorylated in cells by protein kinases that modulate its functional properties. Furthermore, the phosphorylation-dependent targets of Pin1 include a number of protein kinases as well as other enzymes such as phosphatases and regulatory subunits of kinases that modulate the actions of protein kinases. GENERAL SIGNIFICANCE As a result of its interactions with numerous protein kinases and their substrates, as well as itself being a target for phosphorylation, Pin1 has an intricate relationship with the regulatory protein kinase and phosphoproteomic networks that orchestrate complex cellular processes and respond to environmental cues. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

Unbiased Functional Proteomics Strategy for Protein Kinase Inhibitor Validation and Identification of bona fide Protein Kinase Substrates: Application to Identification of EEF1D as a Substrate for CK2

Protein kinases have emerged as attractive targets for treatment of several diseases prompting large-scale phosphoproteomics studies to elucidate their cellular actions and the design of novel inhibitory compounds. Current limitations include extensive reliance on consensus predictions to derive kinase–substrate relationships from phosphoproteomics data and incomplete experimental validation of inhibitors. To overcome these limitations in the case of protein kinase CK2, we employed functional proteomics and chemical genetics to enable identification of physiological CK2 substrates and validation of CK2 inhibitors including TBB and derivatives. By 2D electrophoresis and mass spectrometry, we identified the translational elongation factor EEF1D as a protein exhibiting CK2 inhibitor-dependent decreases in phosphorylation in 32P-labeled HeLa cells. Direct phosphorylation of EEF1D by CK2 was shown by performing CK2 assays with EEF1D-FLAG from HeLa cells. Dramatic increases in EEF1D phosphorylation following λ–phosphatase treatment and phospho-EEF1D antibody recognizing EEF1D pS162 indicated phosphorylation at the CK2 site in cells. Furthermore, phosphorylation of EEF1D in the presence of TBB or TBBz is restored using CK2 inhibitor-resistant mutants. Collectively, our results demonstrate that EEF1D is a bona fide physiological CK2 substrate for CK2 phosphorylation. Furthermore, this validation strategy could be adaptable to other protein kinases and readily combined with other phosphoproteomic methods.

Diverse post-translational modifications of the pannexin family of channel-forming proteins.

The pannexin family of channel-forming proteins is composed of 3 distinct but related members called Panx1, Panx2, and Panx3. Pannexins have been implicated in many physiological processes as well as pathological conditions, primarily through their function as ATP release channels. However, it is currently unclear if all pannexins are subject to similar or different post-translational modifications as most studies have focused primarily on Panx1. Using in vitro biochemical assays performed on ectopically expressed pannexins in HEK-293T cells, we confirmed that all 3 pannexins are N-glycosylated to different degrees, but they are not modified by sialylation or O-linked glycosylation in a manner that changes their apparent molecular weight. Using cell-free caspase assays, we also discovered that similar to Panx1, the C-terminus of Panx2 is a substrate for caspase cleavage. Panx3, on the other hand, is not subject to caspase digestion but an in vitro biotin switch assay revealed that it was S-nitrosylated by nitric oxide donors. Taken together, our findings uncover novel and diverse pannexin post-translational modifications suggesting that they may be differentially regulated for distinct or overlapping cellular and physiological functions.

Functional proteomics strategy for validation of protein kinase inhibitors reveals new targets for a TBB-derived inhibitor of protein kinase CK2.

CK2 is a constitutively active protein kinase with key regulatory roles in many cellular signaling events which has been implicated in several human diseases. To investigate its roles in biological events and potential as a therapeutic target, several potent CK2 inhibitors have been developed including TBB and its derivatives that have been employed in many studies. Despite the utility of these compounds, a precise understanding of their mode of action within cells remains incomplete. In fact, cells are typically treated with inhibitor concentrations (>5 μM) that are orders of magnitude higher than their in vitro inhibitory constants (<0.05 μM). Accordingly, we hypothesized that CK2 inhibitors could have off-target effects that are not recognized when inhibitors are profiled using panels of recombinant protein kinases. To address this issue, we combined structural modeling with inhibitor-affinity purification and proteomics to test the specificity of derivatives of TBB using whole cell lysates of HeLa cells. While these studies confirmed that CK2 does bind to the immobilized inhibitor, several other abundant ATP/GTP-binding proteins were also identified and confirmed. These results suggest that highly abundant nucleotide binding proteins may limit the bioavailability of the free inhibitor and interactions with CK2 in the cellular environment. This article is part of a Special Issue entitled: From protein structures to clinical applications.

Peroxide-mediated oxidation and inhibition of the peptidyl-prolyl isomerase Pin1

Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase that plays a critical role in mediating protein conformational changes involved in signaling processes related to cell cycle control. Pin1 has also been implicated as being neuroprotective in aging-related neurodegenerative disorders including Alzheimers disease where Pin1 activity is diminished. Notably, recent proteomic analysis of brain samples from patients with mild cognitive impairment revealed that Pin1 is oxidized and also displays reduced activity. Since the Pin1 active site contains a functionally critical cysteine residue (Cys113) with a low predicted pK(a), we hypothesized that Cys113 is sensitive to oxidation. Consistent with this hypothesis, we observed that treatment of Pin1 with hydrogen peroxide results in a 32Da mass increase, likely resulting from the oxidation of Cys113 to sulfinic acid (Cys-SO(2)H). This modification results in loss of peptidyl-prolyl isomerase activity. Notably, Pin1 with Cys113 substituted by aspartic acid retains activity and is no longer sensitive to oxidation. Structural studies by X-ray crystallography revealed increased electron density surrounding Cys113 following hydrogen peroxide treatment. At lower concentrations of hydrogen peroxide, oxidative inhibition of Pin1 can be partially reversed by treatment with dithiothreitol, suggesting that oxidation could be a reversible modification with a regulatory role. We conclude that the loss of Pin1 activity upon oxidation results from oxidative modification of the Cys113 sulfhydryl to sulfenic (Cys-SOH) or sulfinic acid (Cys-SO(2)H). Given the involvement of Pin1 in pathological processes related to neurodegenerative diseases and to cancer, these findings could have implications for the prevention or treatment of disease.

Chemical proteomics and functional proteomics strategies for protein kinase inhibitor validation and protein kinase substrate identification: applications to protein kinase CK2.

Since protein kinases have been implicated in numerous human diseases, kinase inhibitors have emerged as promising therapeutic agents. Despite this promise, there has been a relative lag in the development of unbiased strategies to validate both inhibitor specificity and the ability to inhibit target activity within living cells. To overcome these limitations, our efforts have been focused on the development of systematic strategies that employ chemical and functional proteomics. We utilized these strategies to evaluate small molecule inhibitors of protein kinase CK2, a constitutively active kinase that has recently emerged as target for anti-cancer therapy in clinical trials. Our chemical proteomics strategies used ATP or CK2 inhibitors immobilized on sepharose beads together with mass spectrometry to capture and identify binding partners from cell extracts. These studies have verified that interactions between CK2 and its inhibitors occur in complex mixtures. However, in the case of CK2 inhibitors related to 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), our work has also revealed off-targets for the inhibitors. To complement these studies, we devised functional proteomics approaches to identify proteins that exhibit decreases in phosphorylation when cells are treated with CK2 inhibitors. To identify and validate those proteins that are direct substrates for CK2, we have also employed mutants of CK2 with decreased inhibitor sensitivity. Overall, our studies have yielded systematic platforms for studying CK2 inhibitors which we believe will foster efforts to define the biological functions of CK2 and to rigorously investigate its potential as a candidate for molecular-targeted therapy. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).