Phosphorylation In Human Cells Research Articles

Histidine phosphorylation in human cells; a needle or phantom in the haystack?

It has been suggested that in mammalian cells histidine residues in proteins may become as frequently phosphorylated as serine, threonine and tyrosine, and may play a key role in mammalian signaling. Here we applied a robust workflow that earlier allowed us to detect histidine phosphorylation in bacteria unambiguously, to probe for histidine phosphorylation in four human cell lines. Initially, seemingly hundreds of protein histidine phosphorylations were picked up in all studied human cell lines. However, careful examination of the data, and several control experiments, led us to the conclusion that >99% of these initially assigned pHis sites were not genuine, and should be site localized to neighboring Ser/Thr residues. Nevertheless, our methods are selective enough to detect just a handful of genuine pHis sites in mammalian cells, representing well-known enzymatic intermediates. Consequently, we do not find any evidence in our data supporting that protein histidine phosphorylation plays a role in mammalian signaling.

Quantitative Mass Spectrometry Reveals a Proteome‐wide Role for Cyclin A and Cks1 in Multisite, Non‐Proline Directed Phosphorylation by CDK1

Cdk1 is a master kinase of mitotic cell division, phosphorylating thousands of substrates in a highly temporally regulated manner. How Cdk1 phosphorylates the right substrate at the right time remains an open question.Cdk1 activity requires complex formation with a cyclin subunit: cyclin A or cyclin B. Additionally, active Cdk1 is found in complex with a small adaptor protein called Cks1. Cyclin subunits activate Cdk1 and tune Cdk1 substrate choice through interactions with short linear motifs on substrates. While Cks1 is essential in all organisms tested to date, a general function for Cks1 in Cdk1 substrate choice has not been demonstrated.In this study, we investigated the roles of the non‐catalytic subunits of Cdk1 in substrate phosphorylation in human cells. We designed an in vitro assay in which we induced protein phosphorylation in fixed and permeabilized human lymphoblasts using active recombinant Cdk1 either in complex with cyclin A, cyclin B, or cyclin B+Cks1. Global phosphorylation levels were then measured using quantitative mass spectrometry.Our data demonstrate that, contrary to the textbook model of Cdk1 as a proline‐directed kinase, both cyclin A and Cks1 facilitate widespread Cdk1 phosphorylation of sites lacking a +1 proline motif. Our results also suggest a role for Cks1 in multisite phosphorylation. We propose a model whereby cyclin A and Cks1 have indispensable functions in promoting non‐proline directed, multisite phosphorylation of Cdk1 substrates to mediate dynamic, switch‐like changes in protein function required for mitotic entry, proper chromosome segregation and cell division.

CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation.

Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD.

Widespread arginine phosphorylation in human cells—a novel protein PTM revealed by mass spectrometry

Arginine phosphorylation (pArg) is recently discovered as a ubiquitous protein N-phosphorylation in bacteria. However, its prevalence and roles in mammalian cells remain largely unknown due to the lack of established workflow and the inherent lability of phosphoramidate (P–N) bond. Emerging evidences suggest that N-phosphorylation may extensively exist in eu-karyotes and play crucial roles. We report a phosphoproteomic workflow, which allows for the first time revealing the widespread occurrence of pArg in human cells by mass spectrometry. By virtue of this approach, we identified 152 high-confidence pArg sites derived from 118 proteins. Remarkably, the discovered pArg phosphorylation motif and gene ontology hint a possible cellular function of arginine phosphorylation which may regulate the favorability of propeptide convertase substrate. The obtained pArg dataset paves a way for a better understanding of the biological functions of eukaryotic pArg in the future.

Strong anion exchange‐mediated phosphoproteomics reveals extensive human non‐canonical phosphorylation

Phosphorylation is a key regulator of protein function under (patho)physiological conditions, and defining site‐specific phosphorylation is essential to understand basic and disease biology. In vertebrates, the investigative focus has primarily been on serine, threonine and tyrosine phosphorylation, but mounting evidence suggests that phosphorylation of other “non‐canonical” amino acids also regulates critical aspects of cell biology. However, standard methods of phosphoprotein characterisation are largely unsuitable for the analysis of non‐canonical phosphorylation due to their relative instability under acidic conditions and/or elevated temperature. Consequently, the complete landscape of phosphorylation remains unexplored. Here, we report an unbiased phosphopeptide enrichment strategy based on strong anion exchange (SAX) chromatography (UPAX), which permits identification of histidine (His), arginine (Arg), lysine (Lys), aspartate (Asp), glutamate (Glu) and cysteine (Cys) phosphorylation sites on human proteins by mass spectrometry‐based phosphoproteomics. Remarkably, under basal conditions, and having accounted for false site localisation probabilities, the number of unique non‐canonical phosphosites is approximately one‐third of the number of observed canonical phosphosites. Our resource reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for high‐throughput exploration of non‐canonical phosphorylation in all organisms.

Epitope-imprinted mesoporous silica nanoparticles for specific recognition of tyrosine phosphorylation.

Tyrosine phosphorylation regulates the upstream signaling pathway but accounts for less than 0.1% of total phosphorylation in human cells. Herein, molecularly imprinted mesoporous materials were first synthesized to recognize the phosphorylated tyrosine residue from other phosphorylated residues.

Dimerization of the Pragmin Pseudo-Kinase Regulates Protein Tyrosine Phosphorylation

The pseudo-kinase and signaling protein Pragmin has been linked to cancer by regulating protein tyrosine phosphorylation via unknown mechanisms. Here we present the crystal structure of the Pragmin 906-1,368 amino acid C terminus, which encompasses its kinase domain. We show that Pragmin contains a classical protein-kinase fold devoid of catalytic activity, despite a conserved catalytic lysine (K997). By proteomics, we discovered that this pseudo-kinase uses the tyrosine kinase CSK to induce protein tyrosine phosphorylation in human cells. Interestingly, the protein-kinase domain is flanked by N- and C-terminal extensions forming anoriginal dimerization domain that regulates Pragmin self-association and stimulates CSK activity. A1329E mutation in the C-terminal extension destabilizes Pragmin dimerization and reduces CSK activation. These results reveal a dimerization mechanism by which a pseudo-kinase can induce protein tyrosine phosphorylation. Further sequence-structure analysis identified an additional member (C19orf35) of the superfamily of dimeric Pragmin/SgK269/PEAK1 pseudo-kinases.

Mitochondrial translation requires folate-dependent tRNA methylation

Folates enable the activation and transfer of one-carbon units for biosynthesis of purines, thymidine and methionine1–3. Antifolates are important immunosuppressive4 and anticancer agents5. In proliferating lymphocytes6 and human cancers7,8, folate enzymes localizing to the mitochondria are particularly strongly upregulated. This in part reflects the need for mitochondria to generate one-carbon units and export them to the cytosol for anabolic metabolism2,9. The full range of uses of folate-bound one-carbon units in the mitochondrial compartment itself, however, has not been thoroughly explored. Here we show that loss of catalytic activity of the mitochondrial folate enzyme serine hydroxymethyltransferase 2 (SHMT2), but not other folate enzymes, leads to defective oxidative phosphorylation due to impaired mitochondrial translation. We find that SHMT2, presumably by generating mitochondrial 5,10-methylenetetrahydrofolate, provides methyl donors for producing the taurinomethyluridine base at the wobble position of select mitochondrial tRNAs. Mitochondrial ribosome profiling reveals that SHMT2 knockout cells, due to lack of this modified base, suffer from defective translation with preferential mitochondrial ribosome stalling at certain lysine (AAG) and leucine (UUG) codons. This results in impaired respiratory chain enzyme expression. Stalling at these specific codons also occurs in certain mitochondrial inborn errors of metabolism. Disrupting whole-cell folate metabolism, by folate deficiency or antifolate therapy, also impairs the respiratory chain. In summary, mammalian mitochondria use folate-bound one-carbon units to methylate tRNA, and this modification is required for respiratory chain translation and thus oxidative phosphorylation.

Dimerization of the Pragmin Pseudo-Kinase Regulates Protein Tyrosine Phosphorylation

The pseudo-kinase and signaling protein Pragmin has been linked to cancer by regulating protein tyrosine phosphorylation via unknown mechanisms. Here we present the crystal structure of the Pragmin 906- 1368 amino acids C-terminus, which encompasses its kinase domain. We show that Pragmin contains a classical protein kinase fold devoid of catalytic activity. A particular inhibitory triad, conserved in a Pragmin/SgK269/PEAK1/C19orf35 superfamily, tightly holds the catalytic lysine (K997) to prevent ATP binding. By proteomics, we discovered that this pseudo-kinase uses the tyrosine kinase CSK to induce protein tyrosine phosphorylation in human cells. Interestingly, the protein kinase domain is bordered by N- and C-terminal extensions forming an original dimerization domain that regulates Pragmin self-association and stimulates CSK activity. A1329E mutation in the C-terminal extension destabilizes Pragmin dimerization and reduces CSK activation. Thus, our results reveal a new dimerization mechanism by which a pseudo-kinase can induce protein tyrosine phosphorylation.

Characterization of the Potent, Selective Nrf2 Activator, 3-(Pyridin-3-Ylsulfonyl)-5-(Trifluoromethyl)-2H-Chromen-2-One, in Cellular and In Vivo Models of Pulmonary Oxidative Stress

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key regulator of oxidative stress and cellular repair and can be activated through inhibition of its cytoplasmic repressor, Kelch-like ECH-associated protein 1 (Keap1). Several small molecule disrupters of the Nrf2-Keap1 complex have recently been tested and/or approved for human therapeutic use but lack either potency or selectivity. The main goal of our work was to develop a potent, selective activator of NRF2 as protection against oxidative stress. In human bronchial epithelial cells, our Nrf2 activator, 3-(pyridin-3-ylsulfonyl)-5-(trifluoromethyl)-2H-chromen-2-one (PSTC), induced Nrf2 nuclear translocation, Nrf2-regulated gene expression, and downstream signaling events, including induction of NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme activity and heme oxygenase-1 protein expression, in an Nrf2-dependent manner. As a marker of subsequent functional activity, PSTC restored oxidant (tert-butyl hydroperoxide)-induced glutathione depletion. The compound's engagement of the Nrf2 signaling pathway translated to an in vivo setting, with induction of Nrf2-regulated gene expression and NQO1 enzyme activity, as well as restoration of oxidant (ozone)-induced glutathione depletion, occurring in the lungs of PSTC-treated rodents. Under disease conditions, PSTC engaged its target, inducing the expression of Nrf2-regulated genes in human bronchial epithelial cells derived from patients with chronic obstructive pulmonary disease, as well as in the lungs of cigarette smoke-exposed mice. Subsequent to the latter, a dose-dependent inhibition of cigarette smoke-induced pulmonary inflammation was observed. Finally, in contrast with bardoxolone methyl and sulforaphane, PSTC did not inhibit interleukin-1β-induced nuclear factor-κB translocation or insulin-induced S6 phosphorylation in human cells, emphasizing the on-target activity of this compound. In summary, we characterize a potent, selective Nrf2 activator that offers protection against pulmonary oxidative stress in several cellular and in vivo models.

Antibody-mediated neutralization of autocrine Gas6 inhibits the growth of pancreatic ductal adenocarcinoma tumors in vivo.

Gas6 and its receptors Axl, Mer and Tyro-3 (TAM) are highly expressed in human malignancy suggesting that signaling through this axis may be tumor-promoting. In pancreatic ductal adenocarcinoma (PDAC), Gas6 and the TAM receptor Axl are frequently co-expressed and their co-expression correlates with poor survival. A strategy was devised to generate fully human neutralizing antibodies against Gas6 using XenoMouse® technology. Hybridoma supernatants were selected based on their ability to inhibit Gas6 binding to the receptor Axl and block Gas6-induced Axl phosphorylation in human cells. Two purified antibodies isolated from the screened hybridomas, GMAB1 and GMAB2, displayed optimal cellular potency which was comparable to that of the soluble extracellular domain of the receptor Axl (Axl-Fc). In vivo characterization of GMAB1 was conducted using a pharmacodynamic assay that measured inhibition of Gas6-induced Akt activation in the mouse spleen. Treatment of mice with a single dose (100-1000 µg) of GMAB1 led to greater than 90% inhibition of Gas6-induced phosphorylated Akt (pAkt) for up to 72 hr. Based on the target coverage observed in the PD assay, the efficacy of GMAB1 was tested against human pancreatic adenocarcinoma xenografts. At doses of 50 µg and 150 µg, twice weekly, GMAB1 was able to inhibit 55% and 76% of tumor growth, respectively (p < 0.001 for both treatments vs. control Ig). When combined with gemcitabine, GMAB1 significantly inhibited tumor growth compared to either agent alone (p < 0.001). Together, the data suggest that Gas6 neutralization may be important as a potential strategy for the treatment of PDAC.

Phosphoinositides: lipidic essential actors in the intracellular traffic

Phosphoinositides (PPIn) are lipids involved in the vesicular transport of proteins between the different intracellular compartments. They act by recruiting and/or activating effector proteins and are thus involved in crucial cellular functions including vesicle budding, fusion and dynamics of membranes and regulation of the cytoskeleton. Although they are present in low concentrations in membranes, their activity is essential for cell survival and needs to be tightly controlled. Therefore, phosphatases and kinases specific of the various cellular membranes can phosphorylate/dephosphorylate their inositol ring on the positions D3, D4 and/or D5. The differential phosphorylation determines the intracellular localisation and the activity of the PPIn. Indeed, non-phosphorylated phosphatidylinositol (PtdIns) is the basic component of the PPIn and can be found in all eukaryotic cells at the cytoplasmic face of the ER, the Golgi, mitochondria and microsomes. It can get phosphorylated on position D4 to obtain PtdIns4P, a PPIn enriched in the Golgi compartment and involved in the maintenance of this organelle as well as anterograde and retrograde transport to and from the Golgi. PtdIns phosphorylation on position D3 results in PtdIns3P that is required for endosomal transport and multivesicular body (MVB) formation and sorting. These monophosphorylated PtdIns can be further phosphorylated to produce bisphophorylated PtdIns. Thus, PtdIns(4,5)P2, mainly produced by PtdIns4P phosphorylation, is enriched in the plasma membrane and involved in the regulation of actin cytoskeleton and endocytosis. PtdIns(3,5)P2, mainly produced by PtdIns3P phosphorylation, is enriched in late endosomes, MVBs and the lysosome/vacuole and plays a role in endosome to vacuole transport. PtdIns(3,4)P2 is absent in yeast, cells and mainly produced by PtdIns4P phosphorylation in human cells; PtdIns(3,4)P2 is localised in the plasma membrane and plays an important role as a second messenger by recruiting specific protein kinases (Akt and PDK1). Finally the triple phosphorylated PPIn, PtdIns(3,4,5)P3 also absent in yeast, is produced by the phosphorylation of PtdIns(3,4)P2 and localized at the plasma membrane of human cells where it binds proteins via their PH domain. Interaction partners include members of the Arf (ADP-ribosylation factors) family, PDK1 (Phosphoinositide Dependent Kinase 1) and Akt. Therefore this last PPIn is essential for the control of cell proliferation and its deregulation leads to the development of numerous cancers. In conclusion, the regulation of PPIn phosphorylation/dephosphorylation is complex and needs to be very precisely regulated. Indeed phosphatases and kinases allow the maintenance of the equilibrium between the different forms. PPIn play a crucial role in numerous cellular functions and a loss in their synthesis or regulation results in severe genetic diseases.

Specific phosphorylation of histone demethylase KDM3A determines target gene expression in response to heat shock.

Author SummaryHistone methylation regulates gene expression and can have drastic consequences for health if the process is defective. Histone lysine demethylases (KDMs) counteract the activity of methyl-transferases and remove methyl group(s) from histones. KDM3A is a H3K9me2/1 demethylase that performs diverse functions via the regulation of its target genes, which are involved in spermatogenesis, metabolism, and cell differentiation. However, the mechanisms underlying KDM3A regulation of specific genes at specific times are largely unknown. Here we found that a physiological stress—elevated temperature—induces KDM3A phosphorylation in human cells via the MSK1 kinase. This phosphorylated form of KDM3A directly interacts with the transcription factor Stat1, which enables Stat1 to recruit KDM3A to Stat1-binding sequences at the promoters of specific target genes. KDM3A then acts to demethylate H3K9me2/1 at these targets, thereby causing specific gene expression in response to the thermal stress. We conclude that heat shock can affect the expression of many genes in human cells via a novel activation mechanism that is centered around the phosphorylation of KDM3A.

Phosphorylation status of human RNA-binding protein 8A in cells and its inhibitory regulation by Magoh.

The RNA-binding protein 8A (RBM8A)-mago-nashi homolog, proliferation-associated (Magoh) complex is a component of the exon junction complex (EJC) required for mRNA metabolism involving nonsense-mediated mRNA decay (NMD). RBM8A is a phosphorylated protein that plays some roles in NMD. However, the detailed status and mechanism of the phosphorylation of RBM8A is not completely understood. Therefore, in this study, we analyzed in detail RBM8A phosphorylation in human cells. Accordingly, analysis of the phosphorylation status of RBM8A protein in whole-cell lysates by using Phos-tag gels revealed that the majority of endogenous RBM8A was phosphorylated throughout the cell-cycle progression. Nuclear and cytoplasmic RBM8A and RBM8A in the EJC were also found to be mostly phosphorylated. We also screened the phosphorylated serine by mutational analysis using Phos-tag gels to reveal modifications of serine residues 166 and 168. A single substitution at position 168 that concomitantly abolished the phosphorylation of serine 166 suggested the priority of kinase reaction between these sites. Furthermore, analysis of the role of the binding protein Magoh in RBM8A phosphorylation revealed its inhibitory effect invitro and invivo. Thus, we conclude that almost all synthesized RBM8A proteins are rapidly phosphorylated in cells and that phosphorylation occurs before the complex formation with Magoh.

Abstract 5158: Generation of a fully human Gas6 neutralizing antibody with anti-tumor activity in vivo.

Abstract Growth arrest specific 6 (Gas6) is a vitamin-K dependent protein that is involved in the regulation of a wide array of cellular activities, including adhesion, migration, immune evasion, and apoptosis. Gas6 as well as its receptors Axl, Mer, and Tyro3 are highly expressed in multiple malignancies suggesting that signaling through this axis may be tumor promoting in human disease. A strategy was devised to generate fully human neutralizing antibodies against Gas6 using XenoMouse technology. Hybridoma supernatants were first screened for their ability to bind Gas6, then for their ability to inhibit Gas6 binding to the Axl receptor and block Gas6-induced Axl phosphorylation in human cells. Two purified antibodies isolated from the screened hybridomas, 1D9.1 and 1G9.1, maintained potency in all 3 assays and also inhibited Gas6-induced cellular migration and proliferation. In the scratch wound migration assay, the antibodies inhibited both basal and Gas6-induced wound closure to a greater extent than the soluble extracellular domain of the receptor Axl (Axl- Fc). In the proliferation assay, the antibody 1D9.1 completely inhibited Gas6-induced proliferation with a half maximal effective concentration (EC50) of 0.15nM. In vivo characterization of one of the antibodies, 1D9.1, was conducted using a pharmacodynamic (PD) assay that measured the ability of the antibody to inhibit Gas6-induced Akt activation in the mouse spleen. Treatment of mice with a single dose (100-1000 μg) of 1D9.1 led to greater than 90% inhibition of Gas6-induced phosphorylated Akt (pAkt) for up to 72 hours. Based on the target coverage observed in the PD assay, we tested the efficacy of 1D9.1 against Panc-1 human pancreatic adenocarcinoma xenografts implanted in athymic nu/nu female mice. At doses of 50 and 150 μg, twice weekly, the Gas6 neutralizing antibody 1D9.1 was able to inhibit 55% and 76% of tumor growth, respectively. (p&amp;lt;0.001 for both treatments vs. control Ig). When combined with gemcitabine, 1D9.1 was able to inhibit tumor growth to a greater extent than either agent alone (p&amp;lt;0.001 vs. either monotherapy). Together, the data suggest that Gas6 neutralization should be further explored as a potential strategy for the treatment of pancreatic cancer. Citation Format: Gordon E. Moody, Brian Belmontes, Chadwick King, Shuying Liu, Stephanie Masterman, Chris Murawsky, Trace Tsuruda, Wei Wang, Robert Radinsky, Pedro J. Beltran. Generation of a fully human Gas6 neutralizing antibody with anti-tumor activity in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5158. doi:10.1158/1538-7445.AM2013-5158

ALS-Associated Ataxin 2 PolyQ Expansions Enhance Stress-Induced Caspase 3 Activation and Increase TDP-43 Pathological Modifications

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease caused by the loss of motor neurons. The degenerating motor neurons of ALS patients are characterized by the accumulation of cytoplasmic inclusions containing phosphorylated and truncated forms of the RNA-binding protein TDP-43. Ataxin 2 intermediate-length polyglutamine (polyQ) expansions were recently identified as a risk factor for ALS; however, the mechanism by which they contribute to disease is unknown. Here, we show that intermediate-length ataxin 2 polyQ expansions enhance stress-induced TDP-43 C-terminal cleavage and phosphorylation in human cells. We also connect intermediate-length ataxin 2 polyQ expansions to the stress-dependent activation of multiple caspases, including caspase 3. Caspase activation is upstream of TDP-43 cleavage and phosphorylation since caspase inhibitors block these pathological modifications. Analysis of the accumulation of activated caspase 3 in motor neurons revealed a striking association with ALS cases harboring ataxin 2 polyQ expansions. These findings indicate that activated caspase 3 defines a new pathological feature of ALS with intermediate-length ataxin 2 polyQ expansions. These results provide mechanistic insight into how ataxin 2 intermediate-length polyQ expansions could contribute to ALS--by enhancing stress-induced TDP-43 pathological modifications via caspase activation. Because longer ataxin 2 polyQ expansions are associated with a different disease, spinocerebellar ataxia 2, these findings help explain how different polyQ expansions in the same protein can have distinct cellular consequences, ultimately resulting in different clinical features. Finally, since caspase inhibitors are effective at reducing TDP-43 pathological modifications, this pathway could be pursued as a therapeutic target in ALS.

Phosphorylation of human Argonaute proteins affects small RNA binding

Argonaute (Ago) proteins are highly conserved between species and constitute a direct-binding platform for small RNAs including short-interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi interacting RNAs (piRNAs). Small RNAs function as guides whereas Ago proteins are the actual mediators of gene silencing. Although the major steps in RNA-guided gene silencing have been elucidated, not much is known about Ago-protein regulation. Here we report a comprehensive analysis of Ago2 phosphorylation in human cells. We find that the highly conserved tyrosine Y529, located in the small RNA 5′-end-binding pocket of Ago proteins can be phosphorylated. By substituting Y529 with a negatively charged glutamate (E) mimicking a phosphorylated tyrosine, we show that small RNA binding is strongly reduced. Our data suggest that a negatively charged phospho-tyrosine generates a repulsive force that prevents efficient binding of the negatively charged 5′ phosphate of the small RNA.

Acetaldehyde stimulates FANCD2 monoubiquitination, H2AX phosphorylation, and BRCA1 phosphorylation in human cells in vitro: Implications for alcohol-related carcinogenesis

According to a recent IARC Working Group report, alcohol consumption is causally related to an increased risk of cancer of the upper aerodigestive tract, liver, colorectum, and female breast [R. Baan, K. Straif, Y. Grosse, B. Secretan, F. El Ghissassi, V. Bouvard, A. Altieri, V. Cogliano, Carcinogenicity of alcoholic beverages, Lancet Oncol. 8 (2007) 292–293]. Several lines of evidence indicate that acetaldehyde (AA), the first product of alcohol metabolism, plays a very important role in alcohol-related carcinogenesis, particularly in the esophagus. We previously proposed a model for alcohol-related carcinogenesis in which AA, generated from alcohol metabolism, reacts in cells to generate DNA lesions that form interstrand crosslinks (ICLs) [J.A. Theruvathu, P. Jaruga, R.G. Nath, M. Dizdaroglu, P.J. Brooks, Polyamines stimulate the formation of mutagenic 1,N2-propanodeoxyguanosine adducts from acetaldehyde, Nucleic Acids Res. 33 (2005) 3513–3520]. Since the Fanconi anemia–breast cancer associated (FANC–BRCA) DNA damage response network plays a crucial role in protecting cells against ICLs, in the present work we tested this hypothesis by exposing cells to AA and monitoring activation of this network. We found that AA exposure results in a concentration-dependent increase in FANCD2 monoubiquitination, which is dependent upon the FANC core complex. AA also stimulated BRCA1 phosphorylation at Ser1524 and increased the level of γH2AX, with both modifications occurring in a dose-dependent manner. However, AA did not detectably increase the levels of hyperphosphorylated RPA34, a marker of single-stranded DNA exposure at replication forks. These results provide the initial description of the AA–DNA damage response, which is qualitatively similar to the cellular response to mitomycin C, a known DNA crosslinking agent. We discuss the mechanistic implications of these results, as well as their possible relationship to alcohol-related carcinogenesis in different human tissues.

Inhibition of Heat Shock Induction of Heat Shock Protein 70 and Enhancement of Heat Shock Protein 27 Phosphorylation by Quercetin Derivatives

Inhibitors of heat-induced heat shock protein 70 (HSP70) expression have the potential to enhance the therapeutic effectiveness of heat-induced radiosensitization of tumors. Among known small molecule inhibitors, quercetin has the advantage of being easily modified for structure-activity studies. Herein, we report the ability of five monomethyl and five carbomethoxymethyl derivatives of quercetin to inhibit heat-induced HSP70 expression and enhance HSP27 phosphorylation in human cells. While quercetin and several derivatives inhibit HSP70 induction and enhance HSP27 phosphorylation at Ser78, other analogues selectively inhibit HSP70 induction without enhancing HSP27 phosphorylation that would otherwise aid in cell survival. We also show that good inhibitors of HSP70 induction are also good inhibitors of both CK2 and CamKII, kinases that are known to activate HSP70 expression by phosphorylation of heat shock transcription factor 1. Derivatives that show poor inhibition of either or both kinases are not good inhibitors of HSP70 induction, suggesting that quercetin's effectiveness is due to its ability to inhibit both kinases.

Dissociation of Cohesin from Chromosome Arms and Loss of Arm Cohesion during Early Mitosis Depends on Phosphorylation of SA2

Cohesin is a protein complex that is required to hold sister chromatids together. Cleavage of the Scc1 subunit of cohesin by the protease separase releases the complex from chromosomes and thereby enables the separation of sister chromatids in anaphase. In vertebrate cells, the bulk of cohesin dissociates from chromosome arms already during prophase and prometaphase without cleavage of Scc1. Polo-like kinase 1 (Plk1) and Aurora-B are required for this dissociation process, and Plk1 can phosphorylate the cohesin subunits Scc1 and SA2 in vitro, consistent with the possibility that cohesin phosphorylation by Plk1 triggers the dissociation of cohesin from chromosome arms. However, this hypothesis has not been tested yet, and in budding yeast it has been found that phosphorylation of Scc1 by the Polo-like kinase Cdc5 enhances the cleavability of cohesin, but does not lead to separase-independent dissociation of cohesin from chromosomes. To address the functional significance of cohesin phosphorylation in human cells, we have searched for phosphorylation sites on all four subunits of cohesin by mass spectrometry. We have identified numerous mitosis-specific sites on Scc1 and SA2, mutated them, and expressed nonphosphorylatable forms of both proteins stably at physiological levels in human cells. The analysis of these cells lines, in conjunction with biochemical experiments in vitro, indicate that Scc1 phosphorylation is dispensable for cohesin dissociation from chromosomes in early mitosis but enhances the cleavability of Scc1 by separase. In contrast, our data reveal that phosphorylation of SA2 is essential for cohesin dissociation during prophase and prometaphase, but is not required for cohesin cleavage by separase. The similarity of the phenotype obtained after expression of nonphosphorylatable SA2 in human cells to that seen after the depletion of Plk1 suggests that SA2 is the critical target of Plk1 in the cohesin dissociation pathway.