David W. Litchfield

Substrate-Assisted Catalysis by PARP10 Limits Its Activity to Mono-ADP-Ribosylation

ADP-ribosylation controls many processes, including transcription, DNA repair, and bacterial toxicity. ADP-ribosyltransferases and poly-ADP-ribose polymerases (PARPs) catalyze mono- and poly-ADP-ribosylation, respectively, and depend on a highly conserved glutamate residue in the active center for catalysis. However, there is an apparent absence of this glutamate for the recently described PARP6-PARP16, raising questions about how these enzymes function. We find that PARP10, in contrast to PARP1, lacks the catalytic glutamate and has transferase rather than polymerase activity. Despite this fundamental difference, PARP10 also modifies acidic residues. Consequently, we propose an alternative catalytic mechanism for PARP10 compared to PARP1 in which the acidic target residue of the substrate functionally substitutes for the catalytic glutamate by using substrate-assisted catalysis to transfer ADP-ribose. This mechanism explains why the novel PARPs are unable to function as polymerases. This discovery will help to illuminate the different biological functions of mono- versus poly-ADP-ribosylation in cells.

Solution structure of the tetrameric minimum transforming domain of p53.

We report the solution structure of the minimum transforming domain (residues 303–366) of human p53 (p53tet) determined by multidimensional NMR spectroscopy. This domain contains a number of important functions associated with p53 activity including transformation, oligomerization, nuclear localization and a phosphorylation site for p34/cdc2 kinase. p53tet forms a symmetric dimer of dimers that is significantly different from a recent structure reported for a shorter construct of this domain. Phosphorylation of Ser 315 has only minor structural consequences, as this region of the protein is unstructured. Modelling based on the p53tet structure suggests possible modes of interaction between adjacent domains in full-length p53 as well as modes of interaction with DNA.

EGF-Induced ERK Activation Promotes CK2-Mediated Disassociation of α-Catenin from β-Catenin and Transactivation of β-Catenin

Increased transcriptional activity of beta-catenin resulting from Wnt/Wingless-dependent or -independent signaling has been detected in many types of human cancer, but the underlying mechanism of Wnt-independent regulation remains unclear. We demonstrate here that EGFR activation results in disruption of the complex of beta-catenin and alpha-catenin, thereby abrogating the inhibitory effect of alpha-catenin on beta-catenin transactivation via CK2alpha-dependent phosphorylation of alpha-catenin at S641. ERK2, which is activated by EGFR signaling, directly binds to CK2alpha via the ERK2 docking groove and phosphorylates CK2alpha primarily at T360/S362, subsequently enhancing CK2alpha activity toward alpha-catenin phosphorylation. In addition, levels of alpha-catenin S641 phosphorylation correlate with levels of ERK1/2 activity in human glioblastoma specimens and with grades of glioma malignancy. This EGFR-ERK-CK2-mediated phosphorylation of alpha-catenin promotes beta-catenin transactivation and tumor cell invasion. These findings highlight the importance of the crosstalk between EGFR and Wnt pathways in tumor development.

Tra1p Is a Component of the Yeast Ada·Spt Transcriptional Regulatory Complexes

The yeast Ada and TBP class of Spt proteins interact in multiple complexes that are required for transcriptional regulation. We have identified Tra1p as a component of these complexes through tandem mass spectrometry analysis of proteins that associate with Ngg1p/Ada3p. TRA1 is an essential gene and encodes a 3744-amino acid protein that is a member of a group of proteins including the catalytic subunit of DNA-dependent protein kinase, ATM and TRRAP, with carboxyl-terminal regions related to phosphatidylinositol 3-kinases. The interaction between Tra1p and Ada/Spt components was verified by the reciprocal coimmunoprecipitation of Ada2p and Tra1p from whole cell extracts in one or more complexes containing Spt7p. Tra1p cofractionated with Ngg1p and Spt7p through consecutive chromatography on Mono Q, DNA-cellulose, and Superose 6 columns. Binding of Tra1p to DNA-cellulose required Ada components. The association of Tra1p with two Ada·Spt complexes was suggested by its cofractionation with Ngg1p and Spt7p in two peaks on the Mono Q column. In the absence of Ada2p, the elution profile of Tra1p shifted to a distinct peak. Despite the similarity of Tra1p to a group of putative protein kinases, we have not detected protein kinase activity within immunoprecipitates of Tra1p or the Ada·Spt complexes.

Enzymatic Site‐Specific Functionalization of Protein Methyltransferase Substrates with Alkynes for Click Labeling

Posttranslational modifications of proteins are key to essentially all regulatory processes in cells. Many different modifications, including methylation, have been described for core histones, the protein components of nucleosomes. The modifications occur preferentially on the N-terminal tails and are thought to control the interaction with proteins associated with the regulation of chromatin structure and gene transcription. Recent studies have demonstrated that methylation of the side chains of lysine and arginine residues of core histones are associated with specific functional states of promoters. For example, methylation of histone H3 at lysine 9 (H3K9) is a negative mark for gene transcription, and trimethylation of histone H3 at lysine 4 (H3K4) is a marker for transcribed promoters. Methylation at H3K4 is interconnected with other histone modifications, including dimethylation of histone H3 at arginine 2 (H3R2), a transcriptionally negative mark which inhibits methylation of H3K4. 7] Protein methyltransferases (MTases) transfer the activated methyl group from the cofactor S-adenosyl-l-methionine (AdoMet or SAM) mainly to lysine and arginine side chains in their protein substrates. These enzymes are often sequence-specific; for example, mixed-lineage leukaemia (MLL) histone MTase complexes trimethylate H3K4. Methylation of lysine residues is a dynamic, reversible modification involving MTases and demethylases. The main protein MTase substrates described are core histones and a few proteins associated with gene transcription. Comprehensive analyses of MTase substrates are lacking, at least in part because the methyl group is a poor reporter. Antibodies seem to recognize methylated amino acids only in a context-dependent manner, that is, in combination with the underlying peptide sequence. Therefore we thought to develop alternative methods to identify MTase substrates. Recently, we reported on synthetic double-activated AdoMet analogues with allylic and propargylic methyl group replacements for site-specific DNA modification by DNA MTases. Such analogues also function as cofactors for small molecule MTases. Compared to aziridinium-based AdoMet analogues, these cofactors have the advantage that strong product inhibitors are not formed during the MTasecatalyzed reaction. When an amino function was appended to the propargylic side chain, it was possible to couple Nhydroxysuccinimde (NHS)-activated reporters to the modified DNA in a second step. Since introduction of amino groups is generally not productive for the analysis of proteins, we designed the new AdoMet-based cofactor AdoEnYn (1; Scheme 1), in which

Phosphorylation of the cytoskeletal protein talin by protein kinase C

Talin, a component of the focal contact of cultured cells, is an in vitro substrate for protein kinase C. Immunoprecipitation confirms that talin is the phosphorylated protein. Phosphorylation is dependent on both phosphatidylserine and calcium and reaches a level of incorporation of 0.8 mol phosphate/mol protein. Phosphoamino acid analysis demonstrates the presence of phosphoserine and phosphothreonine, but no phosphotyrosine. Two dimensional mapping of tryptic peptides, and V8 peptides reveals the existence of multiple phosphorylation sites. The identification of talin as a substrate for protein kinase C implicates talin as a potential regulator of focal contact organization and perhaps cell morphology.

Inducible Expression of Protein Kinase CK2 in Mammalian Cells EVIDENCE FOR FUNCTIONAL SPECIALIZATION OF CK2 ISOFORMS

Protein kinase CK2 (formerly casein kinase II) exhibits elevated expression in a variety of cancers, induces lymphocyte transformation in transgenic mice, and collaborates with Ha-Ras in fibroblast transformation. To systematically examine the cellular functions of CK2, human osteosarcoma U2-OS cells constitutively expressing a tetracycline-regulated transactivator were stably transfected with a bidirectional plasmid encoding either catalytic isoform of CK2 (i.e. CK2α or CK2α′) together with the regulatory CK2β subunit in order to increase the cellular levels of either CK2 isoform. To interfere with either CK2 isoform, cells were also transfected with kinase-inactive CK2α or CK2α′ (i.e. GK2α (K68M) or CK2α′(K69M)) together with CK2β. In these cells, removal of tetracycline from the growth medium stimulated coordinate expression of catalytic and regulatory CK2 subunits. Increased expression of active forms of CK2α or CK2α′ resulted in modest decreases in cell proliferation, suggesting that optimal levels of CK2 are required for optimal proliferation. By comparison, the effects of induced expression of kinase-inactive CK2α differed significantly from the effects of induced expression of kinase-inactive CK2α′. Of particular interest is the dramatic attenuation of proliferation that is observed following induction of CK2α′(K69M), but not following induction of CK2α(K68M). These results provide evidence for functional specialization of CK2 isoforms in mammalian cells. Moreover, cell lines exhibiting regulatable expression of CK2 will facilitate efforts to systematically elucidate its cellular functions.

A CK2-dependent mechanism for activation of the JAK-STAT signaling pathway

JAK-STAT signaling is involved in the regulation of cell survival, proliferation, and differentiation. JAK tyrosine kinases can be transiently activated by cytokines or growth factors in normal cells, whereas they become constitutively activated as a result of mutations that affect their function in tumors. Specifically, the JAK2V617F mutation is present in the majority of patients with myeloproliferative disorders (MPDs) and is implicated in the pathogenesis of these diseases. In the present study, we report that the kinase CK2 is a novel interaction partner of JAKs and is essential for JAK-STAT activation. We demonstrate that cytokine-induced activation of JAKs and STATs and the expression of suppressor of cytokine signaling 3 (SOCS-3), a downstream target, are inhibited by CK2 small interfering RNAs or pharmacologic inhibitors. Endogenous CK2 is associated with JAK2 and JAK1 and phosphorylates JAK2 in vitro. To extend these findings, we demonstrate that CK2 interacts with JAK2V617F and that CK2 inhibitors suppress JAK2V617F autophosphorylation and downstream signaling in HEL92.1.7 cells (HEL) and primary cells from polycythemia vera (PV) patients. Furthermore, CK2 inhibitors potently induce apoptosis of HEL cells and PV cells. Our data provide evidence for novel cross-talk between CK2 and JAK-STAT signaling, with implications for therapeutic intervention in JAK2V617F-positive MPDs.

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.

Phosphorylation regulates the stability of the regulatory CK2β subunit

Protein kinase CK2 is a protein serine/threonine kinase that exhibits elevated expression in a number of cancers and displays oncogenic activity in mice. The regulatory CK2β subunit has a central role in assembly of functional tetrameric CK2 complexes where it participates in modulation of catalytic activity and substrate specificity. Since overexpression of CK2β results in elevated levels of CK2 activity, we investigated the molecular mechanisms that control its degradation since perturbations in these pathways could contribute to elevated CK2 in cancer. In this study, we demonstrate that CK2β is degraded by a proteasome-dependent pathway and that it is ubiquitinated. We have also investigated the role of phosphorylation and a putative destruction box in regulating its stability in cells. Importantly, replacement of three serine residues within the autophosphorylation site of CK2β with glutamic acid residues resulted in a significant decrease in its degradation indicating that autophosphorylation is involved in regulating its stability. Notably, although the autophosphorylation site of CK2β is remarkably conserved between species, this is the first functional role ascribed to this site. Furthermore, based on these results, we speculate that alterations in the phosphorylation or dephosphorylation of the regulatory CK2β subunit could underlie the elevated expression of CK2 that is observed in cancer cells.