A new quinoline-based chemical probe inhibits the autophagy-related cysteine protease ATG4B.

The cysteine protease ATG4B is a key component of the autophagy machinery, acting to proteolytically prime and recycle its substrate MAP1LC3B. The roles of ATG4B in cancer and other diseases appear to be context dependent but are still not well understood. To help further explore ATG4B functions and potential therapeutic applications, we employed a chemical biology approach to identify ATG4B inhibitors. Here, we describe the discovery of 4-28, a styrylquinoline identified by a combined computational modeling, in silico screening, high content cell-based screening and biochemical assay approach. A structure-activity relationship study led to the development of a more stable and potent compound LV-320. We demonstrated that LV-320 inhibits ATG4B enzymatic activity, blocks autophagic flux in cells, and is stable, non-toxic and active in vivo. These findings suggest that LV-320 will serve as a relevant chemical tool to study the various roles of ATG4B in cancer and other contexts.

HER2/Neu-mediated activation of the ETS transcription factor ER81 and its target gene MMP-1.

In this study, we show that the ETS transcription factor ER81 directly binds to and activates the promoter of the matrix metalloproteinase gene, MMP-1. Further, the oncoprotein HER2/Neu synergizes with ER81 to stimulate MMP-1 transcription. The activation of ER81 by HER2/Neu is mediated by MAP kinases, which phosphorylate ER81 in its N-terminal activation domain. Four respective phosphorylation sites have been identified. Blocking phosphorylation at these sites decreases ER81 transcriptional activity, which can be further diminished by abolishment of phosphorylation at two non-MAP kinase sites. Altogether, our results reveal mechanisms of how phosphorylation of ER81 regulates the expression of target genes such as MMP-1, which may be important for many physiological processes from embryogenesis to adulthood as well as for tumor metastasis.

Functional specialization of CK2 isoforms and characterization of isoform-specific binding partners.

In mammals, protein kinase CK2 has two isozymic forms of its catalytic subunit, designated CK2alpha and CK2alpha'. CK2alpha and CK2alpha' exhibit extensive similarity within their catalytic domains but have completely unrelated C-terminal sequences. To systematically examine the cellular functions of each CK2 isoform in mammalian cells, we have generated human osteosarcoma U2-OS cell lines with the expression of active or inactive versions of each CK2 isoform under the control of an inducible promoter. Examination of these cell lines provides evidence for functional specialization of CK2 isoforms at the cellular level in mammals with indications that CK2alpha' is involved in the control of proliferation and/or cell survival. To understand the molecular basis for functional differences between CK2alpha and CK2alpha', we have undertaken studies to identify proteins that interact specifically with each isoform of CK2 and could contribute to the regulation of their independent functions. A novel pleckstrin-homology domain containing protein, designated CK2-interacting protein 1 (i.e. CKIP-1) was isolated using the yeast two hybrid system as a protein that interacts with CK2alpha but not CK2alpha'. When expressed in cells as a fusion with green fluorescent protein, CKIP-1 localizes to the cell membrane and to the nucleus. In this study, we present evidence from deletion analysis of CKIP-1 suggesting that a C-terminal region containing a putative leucine zipper has a role in regulating its nuclear localization. Collectively, our data supports a model whereby CKIP-1 is a non-enzymatic regulator of CK2alpha that regulates the cellular functions of CK2alpha by targeting or anchoring CK2alpha to specific cellular localization or by functioning as an adapter to integrate CK2alpha-mediated signaling events with components of other signal transduction pathways.

Identification and characterization of CKIP-1, a novel pleckstrin homology domain-containing protein that interacts with protein kinase CK2.

The catalytic subunits of protein kinase CK2, CK2alpha and CK2alpha', are closely related to each other but exhibit functional specialization. To test the hypothesis that specific functions of CK2alpha and CK2alpha' are mediated by specific interaction partners, we used the yeast two-hybrid system to identify CK2alpha- or CK2alpha'-binding proteins. We report the identification and characterization of a novel CK2-interacting protein, designated CKIP-1, that interacts with CK2alpha, but not CK2alpha', in the yeast two-hybrid system. CKIP-1 also interacts with CK2alpha in vitro and is co-immunoprecipitated from cell extracts with epitope-tagged CK2alpha and an enhanced green fluorescent protein fusion protein encoding CKIP-1 (i.e. EGFP-CKIP-1) when they are co-expressed. CK2 activity is detected in anti-CKIP-1 immunoprecipitates performed with extracts from non-transfected cells indicating that CKIP-1 and CK2 interact under physiological conditions. The CKIP-1 cDNA is broadly expressed and encodes a protein with a predicted molecular weight of 46,000. EGFP-CKIP-1 is localized within the nucleus and at the plasma membrane. The plasma membrane localization is dependent on the presence of an amino-terminal pleckstrin homology domain. We postulate that CKIP-1 is a non-enzymatic regulator of one isoform of CK2 (i.e. CK2alpha) with a potential role in targeting CK2alpha to a particular cellular location.

Expression and regulation of protein kinase CK2 during the cell cycle.

There are indications from genetic, biochemical and cell biological studies that protein kinase CK2 (formerly casein kinase II) has a variety of functions at different stages in the cell cycle. To further characterize CK2 and its potential roles during cell cycle progression, one of the objectives of this study was to systematically examine the expression of all three subunits of CK2 at different stages in the cell cycle. To achieve this objective, we examined levels of CK2alpha, CK2alpha' and CK2beta on immunoblots as well as CK2 activity in samples prepared from: (i) elutriated populations of MANCA (Burkitt lymphoma) cells, (ii) serum-stimulated GL30-92/R (primary human fibroblasts) cells and (iii) drug-arrested chicken bursal lymphoma BK3A cells. On immunoblots, we observed a significant and co-ordinate increase in the expression of CK2alpha and CK2alpha' following serum stimulation of quiescent human fibroblasts. By comparison, no major fluctuations in CK2 activity were detected during any other stages during the cell cycle. Furthermore, we did not observe any dramatic differences between the relative levels of CK2alpha to CK2alpha' during different stages in the cell cycle. However, we observed a significant increase in the amount of CK2beta relative to CK2alpha in cells arrested with nocodazole. We also examined the activity of CK2 in extracts or in immunoprecipitates prepared from drug-arrested cells. Of particular interest is the observation that the activity of CK2 is not changed in nocodazole-arrested cells. Since CK2 is maximally phosphorylated in these cells, this result suggests that the phosphorylation of CK2 by p34cdc2 does not affect the catalytic activity of CK2. However, the activity of CK2 was increased by incubation with p34cdc2 in vitro. Since this activation was independent of ATP we speculate that p34cdc2 may have an associated factor that stimulates CK2 activity. Collectively, the observations that relative levels of CK2beta increase in mitotic cells, that CK2alpha and CK2beta are phosphorylated in mitotic cells and that p34cdc2 affects CK2 activity in vitro suggest that CK2 does have regulatory functions associated with cell division.

Fibroblast growth factor-2 decreases metabolic coupling and stimulates phosphorylation as well as masking of connexin43 epitopes in cardiac myocytes.

Cardiac gap junction (GJ) channels, composed of connexins, allow electrical and metabolic couplings between cardiomyocytes, properties important for coordinated action of the heart as well as tissue homeostasis and control of growth and differentiation. Fibroblast growth factor-2 (FGF-2) is an endogenous growth-promoting protein, believed to participate in the short- and long-term responses of the heart to injury. We have examined short-term effects of FGF-2 on cardiac myocyte GJ-mediated metabolic coupling, using cultures of neonatal rat cardiomyocytes. FGF-2 decreased coupling between cardiomyocytes assessed by scrape dye loading as well as microinjection and dye transfer within 30 minutes of administration. Genistein blocked the effects of FGF-2. To determine the mechanism, we next assessed the effect of FGF-2 on expression, distribution, and phosphorylation of connexin43 (Cx43), which is a major cardiomyocyte connexin. FGF-2 did not affect Cx43 mRNA or protein accumulation and synthesis, and it did not change Cx43 localization at sites of intercellular contact as assessed by immunostaining with a polyclonal anti-Cx43 antibody raised against a synthetic peptide containing residues 346 to 363 of Cx43. FGF-2, however, decreased staining intensity at sites of intermyocyte contact when a monoclonal anti-Cx43 antibody was used, suggesting a localized masking of epitope(s) recognized by the monoclonal but not the polyclonal antibody. These epitopes appear to reside within residues 261 to 270 of Cx43, as indicated by full quenching of monoclonal antibody staining with synthetic peptides. In addition, FGF-2 induced a more than twofold increase in Cx43 phosphorylation. Phosphoamino acid analysis indicated increased phosphorylation of Cx43 on serine residues. Although tyrosine phosphorylation of Cx43 was not detected in either treated or control cells, a fraction of Cx43 was immunoprecipitated with anti-phosphotyrosine-specific antibodies in FGF-2-treated myocytes, suggesting interaction (and hence coprecipitation) with phosphotyrosine-containing protein(s). In conclusion, we have identified Cx43 and intercellular communication as targets of FGF-2-triggered and tyrosine phosphorylation-dependent signal transduction in cardiac myocytes. It is suggested that phosphorylation of Cx43 on serine induced by FGF-2 contributes to decreased metabolic coupling between cardiomyocytes.

Granzyme B induces apoptosis and cyclin A-associated cyclin-dependent kinase activity in all stages of the cell cycle.

Granzyme B rapidly induces apoptosis in the presence of the pore-forming protein perforin. We have examined the cell cycle restriction of this apoptosis by separating Jurkat cells into fractions representing different stages of the cell cycle by centrifugal elutriation. Cells were susceptible to apoptosis from G1 through to G2/M, with no significant resistance detected at any stage. Similarly, cells arrested at G1/S or G2/M with either hydroxyurea or nocodazole were slightly more sensitive than asynchronously growing cells. Granzyme B induces Cdc2 kinase activity and requires its induction for apoptosis. Cyclin-dependent kinase (CDK) activity is regulated by phosphorylation and association with cyclins that also control subcellular localization of the CDK/cyclin complexes. Cdc2 associates with both cyclin A, which is synthesized at G1 and S, and cyclin B, which is produced later during S and G2 before G2/M transition. We find that the CDK activity induced by granzyme B is associated primarily with cyclin A in both asynchronous and G1/S-arrested cells, while cyclin B-associated kinase activity is minimal. Because cyclin A is also able to associate with Cdk2, a kinase that is important for G1/S transition, we examined the activation of this CDK during granzyme B-induced apoptosis and find that Cdk2 is induced as rapidly as Cdc2. In conclusion, we have identified a lack of cell cycle restriction of granzyme B-induced apoptosis and the rapid activation of both cyclin A/Cdc2 and cyclin A/Cdk2 kinase activity.

Analysis of interactions between the subunits of protein kinase CK2.

Protein kinase CK2, which was formerly known as casein kinase II, is a highly conserved protein serine/threonine kinase implicated in the control of cell proliferation through its phosphorylation of regulatory nuclear proteins. The enzyme consists of catalytic (alpha and (or) alpha') subunits and beta subunits that modulate the activity of the catalytic subunits. These subunits are arranged in homotetrameric (i.e., alpha 2 beta 2 or alpha' 2 beta 2) or heterotetrameric (i.e., alpha alpha' beta 2) complexes. We previously demonstrated using the yeast two-hybrid system that alpha (or alpha') subunits can interact with beta subunits but not other alpha (or alpha') subunits. By comparison, beta subunits can interact with alpha (or alpha') and with beta subunits, suggesting that the protein kinase CK2 holoenzyme forms because of the ability of beta subunits to dimerize, bringing two heterodimers (alpha beta or alpha' beta) into a tetrameric complex. In the present study, we used the yeast two-hybrid system to examine the domains of interactions between the alpha and beta subunits of protein kinase CK2. These studies indicate that the ability of beta to interact with alpha resides within the carboxy-terminal domain of beta. By comparison, our studies suggest that individual domains of alpha are not sufficient for interactions with beta.

The protein kinase from mitotic human cells that phosphorylates Ser-209 on the casein kinase II beta-subunit is p34cdc2.

Casein kinase II is a highly conserved enzyme that is essential for viability. In cells, the casein kinase II beta-subunit is phosphorylated at an autophosphorylation site and at a site (Ser-209) that is maximally phosphorylated in mitotic cells. To identify protein kinase activities that phosphorylate Ser-209, we fractionated extracts from mitosis-arrested human Burkitt lymphoma MANCA cells. A single Ser-209 kinase activity was detected following each fractionation step. The Ser-209 kinase was purified to a specific activity of approx. 250 nmol/min per mg and efficiently phosphorylated histone H1, a synthetic peptide containing Ser-209 (Ser-209 peptide), myelin basic protein and casein. Immunoblot analysis demonstrated that all fractions containing Ser-209 kinase activity contained p34cdc2. Furthermore, depletion of the Ser-209 kinase activity with p13suc1-Sepharose and anti-p34cdc2 antiserum demonstrated conclusively that the isolated Ser-209 kinase is p34cdc2. These studies provide strong biochemical evidence that p34cdc2 is the enzyme that phosphorylates Ser-209 on the beta-subunit of CKII in mitotic cells. In addition, these results indicate that the Ser-209 peptide can be utilized as a specific reagent for the assay of p34cdc2 activity in mitotic extracts, since no other Ser-209 peptide kinase activities were detected.

Phosphorylation of casein kinase II by p34cdc2. Identification of phosphorylation sites using phosphorylation site mutants in vitro.

The alpha and beta subunits of casein kinase II are dramatically phosphorylated in cells that are arrested in mitosis (Litchfield, D. W., Lüscher, B., Lozeman, F. J., Eisenman, R. N., and Krebs, E.G. (1992) J. Biol. Chem. 267, 13943-13951). Comparative phosphopeptide mapping experiments indicated that the mitotic phosphorylation sites on the alpha subunit of casein kinase II can be phosphorylated in vitro by p34cdc2. In the present study, we have demonstrated that a glutathione S-transferase fusion protein encoding the C-terminal 126 amino acids of the alpha subunit is phosphorylated by p34cdc2 at the same sites as intact casein kinase II, indicating that the mitotic phosphorylation sites are localized within the C-terminal domain of alpha. Four residues within this domain, Thr-344, Thr-360, Ser-362, and Ser-370, conform to the minimal consensus sequence for p34cdc2 phosphorylation. Synthetic peptides corresponding to regions of alpha that contain each of these residues are phosphorylated by p34cdc2 at these sites. Furthermore, alterations in the phosphorylation of the glutathione S-transferase proteins encoding the C-terminal domain of alpha are observed when any of the four residues are mutated to alanine. When all four residues are mutated to alanine, the fusion protein is no longer phosphorylated by p34cdc2 at any of the sites that are phosphorylated in mitotic cells. These results indicate that Thr-344, Thr-360, Ser-362, and Ser-370 are the sites on the alpha subunit of casein kinase II that are phosphorylated in mitotic cells.

DNase I hypersensitivity sites and nuclear protein binding on the fatty acid synthase gene: identification of an element with properties similar to known glucose-responsive elements.

We have shown previously that fatty acid synthase (FAS) gene expression is positively regulated by glucose in rat adipose tissue and liver. In the present study, we have identified in the first intron of the gene a sequence closely related to known glucose-responsive elements such as in the L-pyruvate kinase and S14 genes, including a putative upstream stimulatory factor/major late transcription factor (USF/MLTF) binding site (E-box) (+ 292 nt to + 297 nt). Location of this sequence corresponds to a site of hypersensitivity to DNase I which is present in the liver but not in the spleen. Moreover, using this information from a preliminary report of the present work, others have shown that a + 283 nt to + 303 nt sequence of the FAS gene can confer glucose responsiveness to a heterologous promoter. The protein binding to this region has been investigated in vitro by a combination of DNase I footprinting and gel-retardation experiments with synthetic oligonucleotides and known nuclear proteins. DNase I footprinting experiments using a + 161 nt to + 405 nt fragment of the FAS gene demonstrate that a region from + 290 nt to + 316 nt is protected by nuclear extracts from liver and spleen. This region binds two ubiquitous nuclear factors, USF/MLTF and the CAAT-binding transcription factor/nuclear factor 1 (CTF/NF1). Binding of these factors is similar in nuclear extracts from liver which does or does not express the FAS gene as observed for glucose-responsive elements in the L-pyruvate kinase and S14 genes. This suggests a posttranslational modification of a factor of the complex after glucose stimulation.

Image analysis as a tool for quantitative enzyme determination at the cellular level: application for monocytic differentiation of the UM-384 cell line.

Image analysis has been used to determined enzyme activity at the cellular level in individual smeared cells. The counterstains used to visualize smeared cells were chosen to avoid overlap with the chromogene. The amount of the reaction product was quantified by computerised scanning cytophotometry when the conditions of incubation, time and temperature of the reaction, and substrate concentration varied. Under optimal conditions for time, temperature and substrate concentration, a linear relationship was found between enzyme activity determined on smeared cells and in cell lysate. Using these defined conditions, differentiation of UM-384 cells was studied by measuring enzyme activity. After a monocytic differentiation process, induced by sodium butyrate, non-specific esterase cell activity was compared either with differentiation markers (HLA-DR, plasminogen activator inhibitor type 2 and lysozyme) or with markers of proliferation (DNA content) or functional properties (nitroblue tetrazolium reduction and phagocytosis). The results show that, using image analysis, non-specific esterase seems to be a useful means for the assessment of monocytic differentiation whereas myeloperoxidase is not. More generally, quantification of enzyme activity at the cellular level using image analysis can be applied to the study of the differentiation process and may help in the classification of leukemic cells.