Proteomics perturbations promoted by the protein kinase CK2 inhibitor quinalizarin Cinzia Franchin, Mauro Salvi, Giorgio Arrigoni, Lorenzo A. Pinna PII: DOI: Reference:
S1570-9639(15)00098-9 doi: 10.1016/j.bbapap.2015.04.002 BBAPAP 39564
To appear in:
BBA - Proteins and Proteomics
Received date: Revised date: Accepted date:
29 January 2015 25 March 2015 5 April 2015
Please cite this article as: Cinzia Franchin, Mauro Salvi, Giorgio Arrigoni, Lorenzo A. Pinna, Proteomics perturbations promoted by the protein kinase CK2 inhibitor quinalizarin, BBA - Proteins and Proteomics (2015), doi: 10.1016/j.bbapap.2015.04.002
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ACCEPTED MANUSCRIPT Proteomics perturbations promoted by the protein kinase CK2 inhibitor quinalizarin.
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Cinzia Franchin1,2, Mauro Salvi1, Giorgio Arrigoni1,2*, Lorenzo A. Pinna1* of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy 2Proteomics Center of Padova University, Via G. Orus 2/B, 35129 Padova, Italy * To whom correspondence should be addressed:
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Dr. Giorgio Arrigoni: Tel: +39 0498217449 Fax: +39 049 8217468 Mail:
[email protected] SC R
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1Department
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Prof. Lorenzo A. Pinna: Tel: +39 049 8276108 Fax: +39 049 8276363 Mail:
[email protected] ABSTRACT
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A SILAC analysis performed on HEK-293T cells either treated or not for 3 h with the CK2 inhibitor quinalizarin (QZ) led to the quantification of 53 phosphopeptides whose amount was reduced by 50% or more by QZ. These phosphopeptides include altogether 69
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phosphoresidues, a large proportion of which (almost 50%) are generated by CK2, while the others do not conform to the CK2 consensus. Intriguingly QZ treatment also promotes a 50% or more increase of 108 phosphopeptides including altogether 117 phosphoresidues, 30% of which conform to the CK2 consensus. Here we disclose two mechanisms by which the level of certain phosphosites can be increased rather than decreased by QZ: one relies on the uneven dephosphorylation rate of phosphoresidues close to each other, causing, upon CK2 blockage, the decrease/disappearance of bis-phosphorylated peptides paralleled by the rise of one of its two singly phosphorylated derivatives; the other reflects the increased cellular concentration of a subset of proteins whose expression is substantially up-regulated by QZ treatment. These proteins do not include CK2 itself, whose subunits level is unaffected by QZ. They do include instead a number of substrates whose phosphorylation is increased upon QZ treatment, as well as several kinase/phosphatase regulators and a large number of components of the ribosomal and proteasomal machinery, a circumstance that may partially account for side
ACCEPTED MANUSCRIPT effects of QZ not directly executed by CK2. Especially remarkable is the finding that all the components of the proteasomal catalytic core and of the PA28 complex committed to the degradation of the non-ubiquitinated proteins are increased, while those of the regulatory
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complex 19S conferring the ability to degrade the ubiquitinated proteins are unaffected.
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1. INTRODUCTION
Pharmacological down-regulation of protein kinases by cell permeable, selective inhibitors has become an invaluable tool for studies aimed at dissecting signal transduction pathways
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under physiological and pathological conditions [1, 2]. Several of these low molecular weight compounds have also provided leads for the development of drugs, especially in the field of
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cancer therapy. The interpretation of the effects observed upon cell treatment with kinase inhibitors, however, is not as straightforward as it could appear, being hampered by a number of factors related to the actual specificity of the inhibitor and the pleiotropicity of the
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kinase(s) it targets. A simplistic view, based on a number of utopian assumptions would imply
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that i) the inhibitor is endowed with an absolute selectivity toward the kinase under scrutiny alone; ii) under the experimental conditions adopted, indirect effects are negligible; iii) all the
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targets of the kinases are equally and promptly responsive to its blockage. A more realistic scenario however has to take into account on one side off-target effects of the inhibitor, whose selectivity toward the kinase of interest is never absolute especially in the case of ATP site-
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directed compounds (as the majority of kinase inhibitors are), on the other indirect/secondary effects mediated by kinases/phosphatases functionally interconnected with the primary target, and by the different turnover of the phosphosites affected, reflecting in variable responsiveness to kinase down-regulation. Enlightening insights into such an intricate situation can come from quantitative analyses of the proteome and phosphoproteome of cells treated with kinase inhibitors. To assess the potentials of such an approach we have recently performed a quantitative comparison between the phosphoproteomes of HEK-293T cells either treated or not with a fairly specific inhibitor of protein kinase CK2 (an acronym derived from the misnomer “casein kinase-2”) [3]. The choice fell on CK2 for a number of reasons: it is one of the most pleiotropic protein kinases, with hundreds of substrates already known [4, 5]; its unique consensus sequence (S/T-x-x-E/D/pS) provides a reliable criterion for the identification of its putative targets; its activity is not dependent on second messengers nor on other signaling molecules, a
ACCEPTED MANUSCRIPT circumstance that minimizes the contribution of endogenous stimuli which otherwise could interfere with the pharmacological effect of the inhibitor employed. Last but not least CK2 is implicated in several pathological situations [6-8] with special reference to neoplasia, where
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its abnormally elevated level generates the phenomenon of “non oncogene addiction” [9] by
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inducing a number of cellular responses – notably ability to escape apoptosis – which altogether are favoring the establishment and the progression of malignancy. Short time
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treatment of HEK-293T cells with the fairly specific inhibitor quinalizarin (QZ) resulted in drastic reduction of a minor proportion of phosphosites generated by CK2, largely belonging to proteins implicated in apoptosis and cell survival [3], suggesting that these are more
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readily responsive to CK2 down-regulation than the average. However many phosphosites not conforming to the CK2 consensus were also decreased by QZ treatment, while, paradoxically,
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a number of phosphosites larger than those decreased underwent a substantial increase upon QZ treatment, two circumstances consistent with off-target and intricate collateral effects of QZ.
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To gain a deeper insight into these unanticipated outcomes we have now performed a more
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detailed analysis of the data, implemented by quantitative proteomics information. The results, presented and discussed here, highlight the implication of protein phosphatases as
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critical modulators of CK2 signaling while disclosing an unanticipated potential of QZ to affect
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the proteasome machinery.
2. MATERIALS AND METHODS 2.1 Materials
All solvents and chemicals were purchased from Sigma (if not specified otherwise) and were of MS grade or equivalent. Total Akt1 and cdc37 antibodies were purchased from Santa Cruz Biotechnology. Phospho-Akt1 (S129) and phospho-cdc37 (S13) antibodies were from Abcam. Secondary antibodies conjugated to horseradish peroxidase were purchased from PerkinElmer. 2.2 Cell culture and stable isotope labeling HEK-293T cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) as described in [3]. Cells were either labeled with L-13C6–arginine and L-13C6-lysine (Cambridge Isotope Laboratories) supplemented with 1 mM L-glutamine, 1% penicillin/streptomycin, and 10%
ACCEPTED MANUSCRIPT dialyzed Fetal Bovine Serum (FBS, Invitrogen) or grown with conventional L-arginine and Llysine (heavy and light medium respectively). Labeled and unlabeled cells did not show any appreciable difference in terms of cell morphology and doubling time. Cells were maintained
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in culture for more than 10 cell doublings before the treatment with the inhibitor was
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performed.
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2.3 Cell treatment and protein extraction
3 heavy labeled and 3 unlabeled cell cultures (for a total of 6 biological replicates) were treated for 3 h with 50 M quinalizarin (QZ) dissolved in dimethyl sulfoxide (DMSO), while 6
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samples (3 heavy labeled and 3 unlabeled cell cultures) were used as control and treated with the same volume of DMSO without inhibitor. After the treatment cells were washed twice with
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Phosphate Buffered Saline (PBS) and once with 5 mM Tris-HCl, 250 mM sucrose, pH 7.5. For each sample 500 μl of lysis buffer (10 mM Tris-HCl pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA) supplemented with complete proteases inhibitors EDTA-free (1 tablet/10 ml, Roche),
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and phosphatases inhibitors cocktail I (1:100, Roche) was used to harvest the cells.
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Lysed cells were then sonicated and centrifuged for 15 min at 14000xg. The supernatant was subjected to ultracentrifugation at 100000xg for 1 h at 4 °C. The pellet (membrane rich
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fraction) was further dissolved in 100 μl of lysis buffer containing 50 mM Tris-HCl pH 7.5, 1% SDS, complete proteases inhibitors EDTA-free (Roche), and phosphatases inhibitors cocktail (Roche). Finally, soluble and membrane rich fractions were pooled and quantified using the
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Micro-Lowry Peterson’s Modification Total Protein Kit (Sigma). 2.4 SDS-PAGE and in-gel protein digestion 30 μg of proteins from heavy labeled cells were mixed with 30 μg of proteins from light cells to obtain 6 independent replicates, i.e. 3 “forward” experiments (heavy labeled treated cells mixed with light control cells) and 3 “reverse” experiments (heavy labeled control cells mixed with light treated cells). Samples were loaded onto a 4-12% polyacrylamide precast gel (NuPAGE Bis-Tris Gel, Invitrogen), and proteins separated at 200 V for 1 h. Gels were stained with SimplyBlue coomassie (Invitrogen), destained overnight in water and each gel lane was divided into 5 slices which were then subjected to in-gel protein digestion with sequencing grade modified trypsin (Promega) as described in [10]. Extracted peptides were dried under vacuum and kept at -80 °C until LC-MS/MS analysis was performed. 2.5 LC-MS/MS and data analysis
ACCEPTED MANUSCRIPT LC-MS/MS analyses were performed with a LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) online with a nano-HPLC Ultimate 3000 (Dionex – Thermo Fisher Scientific). Peptides were loaded into a 10 cm pico-frit column (75 μm I.D., New Objective) packed in90 min linear
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house with C18 material (ReproSil, 300 , 3 μm; Dr. Maisch HPLC GmbH).
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gradient of acetonitrile (ACN)/0.1% formic acid (from 0% to 50% ACN in 60 min) was used to perform the chromatographic separation at a flow rate of 250 nl/min. The instrument was
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operating in a data-dependent mode with a Top4 method (1 full MS scan at 60000 resolution in the Orbitrap, followed by MS/MS scans on the 4 most abundant ions in the linear trap). To increase the number of peptide and protein identifications, each sample was analyzed a first
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time, data obtained were searched with Mascot search engine (version 2.2.4, Matrix Science) and a static excluding list was created with Proteome Discoverer software (Thermo Fisher
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Scientific) based on all peptides that were positively identified. Each sample was then analyzed again under identical instrumental and chromatographic conditions, but with the application of the excluding list. A total of 60 LC-MS/MS runs were performed.
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Raw files were analyzed with the software Proteome Discoverer 1.4 (Thermo Fisher
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Scientific) connected to a Sequest HT search engine (Thermo Fisher Scientific). Data were searched against the human section of the Uniprot database (version 2013.11.13). Enzyme
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specificity was set to trypsin with up to 1 missed cleavage, while mass tolerance was set to 10 ppm and to 0.6 Da for parent mass and fragment ions respectively. Carbamidomethylation of cysteines was set as fixed modification and
13C6-Lys
and
13C6-Arg
were set as variable
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modifications, together with methionine oxidation. The algorithm Percolator was used to assess the reliability of peptide identifications: data were filtered to keep into account only peptides identified with a q value < 0.01 (99% confidence) and rank 1. Quantification of SILAC pairs was performed directly by Proteome Discoverer software and only unique peptides were considered for quantification purposes. Data were analyzed with a MudPIT protocol by merging (for each of the experimental replicates) all data obtained from the technical replicates (without and with the application of the excluding list). Proteins were considered as correctly identified if at least 2 unique peptides were quantified with individual q value < 0.01. Only proteins that were quantified in all 6 biological replicates were retained for further analysis. Finally a two-tailed Z test was performed and only proteins with a p value < 0.05 and with a fold change > 1.5 were considered as differentially expressed between control and treated samples. 2.6 Bioinformatic analysis
ACCEPTED MANUSCRIPT Data collected in this study were compared with the sequences extracted from the PhosphositePlus database (http://www.phosphosite.org). Further bioinformatic analyses to define cellular localization and protein functions were performed with David Bioinformatics
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Resources 6.7 [11] and STRING [12]. 2.7 Western blot analysis
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HEK-293T Cells were plated onto 6-well dishes and treated with 50 μM quinalizarin (QZ) or with vehicle (DMSO) as control for the indicated times. After incubation cells were detached, centrifuged, extensively washed with PBS and lysed by the addition of ice-cold buffer
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containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100 (v/v), protease inhibitor cocktail Complete (Roche) and phosphatase inhibitor Cocktail 2 and
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3 (Sigma). After 20 min incubation on ice, the lysate was centrifuged 10 min at 10000xg at 4 °C. The supernatant was collected and protein concentration was determined by the Bradford method. Equal amounts of protein were loaded on 12% SDS-PAGE, blotted on Immobilon-P
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membranes (Millipore), processed by western blot with the indicated antibody, and detected
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by chemiluminescence on a Kodak Image Station 440MM PRO. Quantitation of the signal was
3. RESULTS
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obtained by analysis with the Kodak 1D Image software.
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3.1 Identification of phosphosites promptly responsive to CK2 inhibition. The results of a SILAC experiment aimed at the identification of phosphosites whose amount is altered upon short-time treatment of HEK-293T cells with the CK2 inhibitor quinalizarin (QZ) [3] are summarized in Fig. 1. Altogether 3465 unique phosphopeptides were quantified, the by far largest proportion of which (almost 95%) were not significantly affected by QZ. Only less than 2% of the phosphosites were substantially decreased, while slightly more than 3% were actually increased. Interestingly the proportion of phosphosites conforming to the consensus sequence of CK2 in the whole phosphosite population (21.5%) rises to almost 50% if the subset of decreased phosphosites is considered. A less pronounced enrichment in CK2 phosphosites is observed in the subset of increased phosphosites (see Fig. 1). As reported elsewhere [3] most of the sites conforming to the CK2 consensus, either decreased or increased by QZ, were validated as bona fide CK2 targets. By sharp contrast a scrutiny of the PhosphositePlus database (http://www.phosphosite.org) reveals that none of the decreased phosphosites not conforming to the CK2 consensus are listed among the bona
ACCEPTED MANUSCRIPT fide targets of those kinases which are only moderately affected by QZ [13], namely, ERK8, Pim1/3, DYRK1A/3, Hipk2, AuroraB, AMPK, and MARKK3. These data while reinforcing the conclusion that CK2 is the primary target of QZ in cells, support the view that off-target
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and/or collateral effects of QZ also take place.
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While performing this analysis the possibility that CK2 sites could be generated through hierarchical phosphorylation, primed by previous phosphorylation of a residue at n+3
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position [14] was also considered. Indeed 17 out of the 784 sites conforming to the CK2 consensus were of this kind (S-x-x-pS). Only two of these however fall in the list of those sites which are decreased by QZ treatment, while the other 15 are not affected by the treatment
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with the inhibitor. Recently it has been shown that the optimal consensus for primed CK2 phosphorylation implies the presence of additional phosphoserines nearby, in addition to the
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one at position n+3 ([DE]-S-pS-[ADE]-pS-[DEH]-[DEpS]-[DEpS]), and that whenever the priming kinase is a Pro directed one phosphorylation would occur optimally at sequence with a canonical determinant at the n+3 position followed by a proline directed phosphorylation
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site at the n+5 position (S-x-x-D/E-x-pS-P) [15]. By screening our CK2 phosphosites for these
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two motifs we could not find any conforming to either of them. It should be born in mind however that the limited number of doubly/multiply phosphorylated peptides identified
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(probably due to a bias towards the mono-phosphorylated peptides in our enrichment protocol) may have hampered our search of CK2 phosphosites generated through hierarchical phosphorylation.
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The list reported in Tab. S1, supplementary material, includes 69 phosphosites whose decrement upon 3 h cell treatment with QZ is 50% or more. To note that this list includes only less than 5% of the whole set of CK2 targets quantified in our previous study [3] (31 out of 784). These are listed in Tab. 1 together with some annotations about the proteins they belong to. Many of these are implicated in apoptosis and/or cell survival, thus accounting for the observation that enhanced apoptosis is one of the most general and initial responses to cell treatment with CK2 inhibitors. It may be worthy to note in this respect that under our experimental conditions the phosphorylation of two widely used reporters of CK2 activity, pS129 of Akt1 and pS13 of cdc37 are inhibited by about 35% and 0%, respectively. Akt1 pS129 in fact, as shown in the supplementary material (Fig. S1), would require a more prolonged treatment with QZ in order to display 50% reduction in phosphorylation, while cdc37 pS13 dephosphorylation tends becoming detectable only after 24 h. This means that all the phosphosites listed in Tab. 1 are more responsive to CK2 blockage than Akt1 pS129, not to say of cdc37 pS13. Apparently
ACCEPTED MANUSCRIPT this does not reflect different bioavailability of the inhibitor inside the cell, since proteins whose phosphosites are readily reduced by QZ (Tab. 1) are localized to different compartments, notably cytosol, nucleoplasm and nuclei, nucleoli, mitochondria, Golgi
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apparatus, or are membrane/membrane-associated proteins or part of extracellular vesicular
Table 1
Tr/Ctrl
Kinase /Phosphatase Effector/Interactor
RPSESDKEDELDKVK
PKC;PKA
A-kinase anchor protein 12
GLAEVQQDGEAEEGATSDGEKKR
0,53
PKC;PKA
AP-3 complex subunit beta-1
EGDELEDNGKNFYESDDDQKEK
0,67
Protein Description
Sequence
AKAP12 #
A-kinase anchor protein 12
AKAP12 # AP3B1
CHMP2B #
Isoform 2 of Charged multivesicular body protein 2b
DTD1
D-tyrosyl-tRNA(Tyr) deacylase 1
0,51
SVKHDSIPAADTFEDLSDVEGGGSEPTQR
0,65
KSLSDSESDDSKSK
0,59
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Chromobox protein homolog 3
SAGEEEDGPVLTDEQK
ATISDEEIER
0,50
SASSGAEGDVSSEREP
0,52
DHSPTPSVFNSDEERYR
0,56
EVSSRPSTPGLSVVSGISATSEDIPNKIEDLR
0,65
KQAREESEESEAEPVQR
0,67
CK2 CK2
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CBX3 #
0,53
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ASMTL
ADP-ribosylation factor-like protein 6-interacting protein 4 N-acetylserotonin Omethyltransferase-like protein
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Gene Name
ARL6IP4
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exosomes.
HIRIP3
HIRA-interacting protein 3
SLKESEQESEEEILAQKK
0,62
HUWE1 #
Isoform 2 of E3 ubiquitin-protein ligase HUWE1
GSGTASDDEFENLR
0,56
LARP7
La-related protein 7
SRPTSEGSDIESTEPQK
0,49
LARP7
La-related protein 7
DIEISTEEEKDTGDLKDSSLLK
0,67
LEO1 #
Isoform 2 of RNA polymeraseassociated protein LEO1
KLTSDEEGEPSGKR
0,62
NDRG1 #
Protein NDRG1
TASGSSVTSLDGTR
0,66
PDCD4 #
Isoform 2 of Programmed cell death protein 4
FVSEGDGGR
0,44
PPP1R2
Protein phosphatase inhibitor 2
IQEQESSGEEDSDLSPEER
0,50
KETESEAEDNLDDLEK
0,65
RGEGDAPFSEPGTTSTQRPSSPETATK
0,19
SCFESSPDPELK
0,39
SRRM1 SRRM2 SRRM2
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GAPVD1 #
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HIRIP3
Isoform 3 of Pre-mRNA 3'-endprocessing factor FIP1 GTPase-activating protein and VPS9 domain-containing protein 1 HIRA-interacting protein 3
FIP1L1
Serine/arginine repetitive matrix protein 1 Serine/arginine repetitive matrix protein 2 Serine/arginine repetitive matrix protein 2
SSRP1 #
FACT complex subunit SSRP1
EGMNPSYDEYADSDEDQHDAYLER
0,67
SUB1 #
Activated RNA polymerase II transcriptional coactivator p15
ELVSSSSSGSDSDSEVDKK
0,65
SURF2
Surfeit locus protein 2
DLGSTEDGDGTDDFLTDKEDEKAKPPR
0,67
TMF1
TATA element modulatory factor
SVSEINSDDELSGK
0,51
CDKN2A
JUN kinase; MAP4K1 PP1
FER
ACCEPTED MANUSCRIPT Some of the proteins listed in Tab. 1, whose phosphosites are generated by CK2 and substantially decreased by QZ, provide clues to account for indirect effects of the inhibitor, initially mediated by CK2 but ultimately affecting phosphosites generated by other kinases. To
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note in particular A-kinase anchor protein 12 and the inhibitor-2 of protein phosphatase 1.
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Although it is not known if and how the activity of these proteins may be influenced by phosphorylation at these residues, it should be noted that both have the potential to affect the
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phosphorylation level of many others, thus providing a rationale for the alteration of phosphosites not directly targeted by CK2. The same may apply to other proteins listed in Tab.1, notably PDCD4 (a regulator of Jun kinase and MAP4K1 [16, 17]), and TMF1 which
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interacts with the Tyr kinase FER [18]. In principle all these proteins have the potential to affect the phosphorylation of other proteins once their own phosphorylation by CK2 is
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decreased.
More in general the variable responsiveness to QZ of phosphosites generated by endogenous CK2 highlights the crucial role protein phosphatases play as modulators of the signaling
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propagating from this constitutively active kinase. Not only in fact 3 h treatment with QZ does
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not significantly affect the majority of CK2 targets, but even those phosphosites which undergo a substantial decrease respond differently, as illustrated in Tab. 1. These data, while
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being consistent with the concept that on the average the phosphate incorporated by CK2 turns over quite slowly [19] (see also Fig. S1) additionally show that there are exceptions to this rule, explainable assuming special susceptibility of individual phosphoresidues to
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dephosphorylation.
3.2 Phosphosites up-regulated: the implication of protein phosphatases. A somewhat paradoxical outcome of our analysis was that quite a number of phosphopeptides are increased, rather than being decreased, by QZ treatment (see Tab. S2, supplementary material). This did not entirely come as a surprise, a similar effect of kinase inhibition having been described also elsewhere [20, 21]. In an attempt to disclose the rationale underlying such a phenomenon, two possibilities have been considered: the contribution of uneven dephosphorylation of multi-phosphorylated sites, on one side, and an increased concentration of phosphorylatable protein substrates, on the other. The former possibility was initially suggested by the observation that while bisphosphorylated peptides are quite abundant among those which are decreased by QZ, reaching 30% of total, their proportion drops to 8% among the repertoire of increased phosphopeptides. Such a disproportion becomes striking if the scrutiny is limited to peptides
ACCEPTED MANUSCRIPT whose phosphoresidues conform to the CK2 consensus: 11 bis-phosphorylated peptides out of a total of 26 are decreased, while only one is present among 34 which are increased. We reasoned that this could reflect a different dephosphorylation rate of individual
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phosphoresidues present in the same bis-phosphorylated peptide, producing, upon CK2
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blockage, the preferential accumulation of one of the two singly phosphorylated peptides. The yield of this latter therefore would be increased upon CK2 inhibition in parallel with the
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decrease or disappearance of the bis-phosphorylated peptide. A paradigmatic example of the real occurrence of this mechanism is provided by the peptide encompassing residues Ser627 and Ser629 of AKAP12, whose bis-phosphorylated form is halved by QZ (see Tab. 1) while its
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derivative mono-phosphorylated at Ser627 actually increases 1.6-fold (Tab. S2). This opposite behavior is consistent with the mechanism schematically depicted in Fig. 2A, implying that
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pSer629 is more prone to dephosphorylation than pSer627, thus causing, upon blockage of CK2 (equally affecting both phosphoresidues) the decrease of the bis-phosphorylated peptide accompanied by the rise of the peptide singly phosphorylated at Ser627. By the same
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mechanism the bis-phosphorylated peptide derived from SRRM2 (including two adjacent
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phosphoserines at positions 322 and 323) is drastically reduced upon QZ treatment whereas its derivative singly phosphorylated at Ser323 increases. In this case CK2 is responsible for
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the phosphorylation of just one of the two phosphoresidues, Ser322. Consequently CK2 downregulation reflects in the rise of the peptide singly phosphorylated at Ser323, the one which is not targeted by CK2 itself (see Fig. 2B).
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Seemingly the same mechanism, relying on uneven dephosphorylation of bis-phosphorylated peptides, accounts for the increase of the other peptides listed in Tab. 2, all of which share the presence of a phosphorylated residue whose occupancy increases upon CK2 inhibition by QZ, and of one or more additional phosphoacceptor sites conforming to the CK2 consensus which have been found in our study or have been reported elsewhere to undergo phosphorylation. To note that Tab. 2 includes a substantial proportion of the phosphopeptides increased upon QZ treatment (39 out if 108, see supplementary material, Tab. S2). For all of these the hypothesis that their increment is taking place through a mechanism like that depicted in Fig. 2, is consistent with the experimental information available. Table 2 Uniprot Accession
Gene Name
Protein Description
Sequence
Tr/Ctrl
B4DR64
CTPS1
CTP synthase 1
S*GSSSPDSEITELKFPSINHD
1,5
B4E2T8
CANX
Calnexin
QKSDAEEDGGTVS*QEEEDRKPK
2,1
B7Z1L3
PGRMC1
Membrane-associated progesterone receptor component 1
EGEEPT*VYSDEEEPKDESAR
1,6
ACCEPTED MANUSCRIPT SMC4
Structural maintenance of chromosomes protein 4
TESPAT*AAET*ASEELDNR
5,0
D6RAF8
HNRNPD
Heterogeneous nuclear ribonucleoprotein D0
IDAS*KNEEDEGHSNSSPR
1,5
D6REM6
MATR3
Matrin-3
RDS*FDDRGPSLNPVLDYDHGSR
2,8
E9PQN2
BCLAF1
Bcl-2-associated transcription factor 1
SQEEPKDTFEHDPSES*IDEFNK
2,2
F5H3J2
NASP
Nuclear autoantigenic sperm protein
LVPSQEETKLS*VEESEAAGDGVDTK
2,5
F5H3J2
NASP
Nuclear autoantigenic sperm protein
LVPS*QEETKLS*VEESEAAGDGVDTK
2,0
F5H7B0
SKIV2L
Helicase SKI2W
AS*SLEDLVLK
2,0
H0YJ03
PSMA3
Proteasome subunit alpha type-3
ES*LKEEDESDDDNM
1,5
K7ES59
PTBP1
Polypyrimidine tract-binding protein 1
O15173
PGRMC2
O43399-6 O43719
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IP
T
C9JWF0
1,6
Membrane-associated progesterone receptor component 2
LLKPGEEPS*EYTDEEDTKDHNKQD
1,5
TPD52L2
Isoform 6 of Tumor protein D54
NSAT*FKSFEDR
1,6
HTATSF1
HIV Tat-specific factor 1
RSDSVSASER
2,5
O43765
SGTA
Small glutamine-rich tetratricopeptide repeatcontaining protein alpha
SRTPSAS*NDDQQE
1,5
P04637-7
TP53
Isoform 7 of Cellular tumor antigen p53
ALPNNT*SSSPQPK
1,7
P07814
EPRS
Bifunctional glutamate/proline--tRNA ligase
EYIPGQPPLSQSS*DSSPTR
1,8
P38432
COIL
Coilin
NSSEKLPTELSK(EE)
1,5
P55327-2
TPD52
Isoform 2 of Tumor protein D52
NSPT*FKSFEEKVENLK
1,7
P55327-2
TPD52
Isoform 2 of Tumor protein D52
NSPTFKS*FEEKVENLK
1,9
Q02952
AKAP12
A-kinase anchor protein 12
RPSES*DKEDELDKVK
1,6
Q08170
SRSF4
Serine/arginine-rich splicing factor 4
SESSQREGRGESENAGTNQETR
4,0
Q13185
CBX3
Chromobox protein homolog 3
RKSLS*DS*ESDDSK
1,8
Q13442
PDAP1
28 kDa heat- and acid-stable phosphoprotein
S*LDSDES*EDEEDDYQQK
1,5
Q16513-5
PKN2
Isoform 5 of Serine/threonine-protein kinase N2
ASS*LGEIDESSELR
1,8
Q2TBE0-3
CWF19L2
Isoform 3 of CWF19-like protein 2
STFAGSPERESIHILSVDEK
1,5
Q7Z6P5
MCM3
DNA replication licensing factor MCM3
DGDSYDPYDFSDT*EEEMPQVHTPK
2,0
Q8NEF9
SRFBP1
Serum response factor-binding protein 1
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D
MA
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ELKT*DSSPNQAR
1,5
T*DADSESDNSDNNTIFVQGLGEGVSTDQVGEFFK
1,9
Protein IWS1 homolog
FHS*SDSEEEEHKK
1,5
Isoform 2 of 5'-3' exoribonuclease 2
KAEDSDS*EPEPEDNVR
2,0
AC
AVTIANSPSKPS*EKDSVVSLESQK
Isoform Short of TATA-binding proteinassociated factor 2N
Q92804-2
TAF15
Q96ST2
IWS1
Q9H0D6-2
XRN2
Q9H8G2
CAAP1
Caspase activity and apoptosis inhibitor 1
S*VNEILGLAESSPNEPK
1,8
Q9NR30-2
DDX21
Isoform 2 of Nucleolar RNA helicase 2
KKEEPS*QNDISPK
2,1
Q9NZ63
C9orf78
Uncharacterized protein C9orf78
RRGDS*ESEEDEQDSEEVR
1,5
Q9UQ35
SRRM2
Serine/arginine repetitive matrix protein 2
RGEGDAPFSEPGTTSTQRPS*SPETATK
1,5
Q9UQ35
SRRM2
Serine/arginine repetitive matrix protein 2
S*RTSPITR
1,5
Q9UQ35
SRRM2
Serine/arginine repetitive matrix protein 2
SPSVS*SPEPAEK
4,2
Q9Y2W1
THRAP3
Thyroid hormone receptor-associated protein 3
FS*GEEGEIEDDESGTENREEKDNIQPTTE
1,5
3.3 QZ promotes the up-regulation of proteasomal and ribosomal proteins level. Another factor potentially accounting for up-regulated phosphorylation of certain sites upon QZ treatment could be the increased cellular concentration of some protein substrates. CK2 has been reported to both hamper and foster protein degradation, depending on the
ACCEPTED MANUSCRIPT circumstances. Many caspase sites, e.g., have been shown to become refractory to cleavage upon phosphorylation by CK2 of residues nearby (reviewed by [22]) and the phosphorylation of -catenin by CK2 correlates to the ability to escape fragmentation [23]. On the other hand
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many examples are known of proteins (e.g. PTEN, IKK, and PML), which are committed to
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degradation by CK2 [24-26]. We have therefore examined if QZ treatment was significantly altering the level of individual cellular proteins, reasoning that these could become more or
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less susceptible to phosphorylation. As reported in the supplementary material (Tab. S3-9) our SILAC analysis led to the identification of more than a thousand proteins that could be quantified in all 6 biological replicates. We found that under the experimental conditions of
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our SILAC analysis the by far largest amount of proteins underwent no significant quantitative alteration. While however there was no significant decrease of any protein, one hundred
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proteins displayed a significantly increased expression level, with a fold change ranging between 1.5 to almost 9.0, as compared to control untreated cells. These altered proteins (p value < 0.05) are listed in Tab. S3 of supplementary material, while all details regarding
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protein and peptide identification and quantification for all 6 biological replicates are
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reported in Tab. S4-9, supplementary material. Among the significantly altered proteins, 9 show an increased concentration that correlates with the increased occupancy of their
Table 3 Gene Name
Protein Description
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Uniprot Accession
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phosphosites (see Tab. 3), suggesting a cause-effect relationship between the two events.
Sequence
Tr/Ctrl Phosphopeptide
Tr/Ctrl Protein
D6REM6
MATR3
Matrin-3
RDSFDDRGPSLNPVLDYDHGSR
2,8
1,5
D6REM6
MATR3
Matrin-3
SYSPDGKESPSDKK
1,6
1,5
D6REM6
MATR3
Matrin-3
SYSPDGKESPSDKK
1,6
1,5
E9PL71
EEF1D
Elongation factor 1-delta
KPATPAEDDEDDDIDLFGSDNEEEDK EAAQLREER
1,6
E9PSF4
RPS3
40S ribosomal protein S3
DEILPTTPISEQK
2,0
G3V2D6
HNRNPC
EAEEGEDDRDSANGEDDS
1,5
H0YJ03
PSMA3
ESLKEEDESDDDNM
1,5
P09651-2
HNRNPA1
SESPKEPEQLR
1,6
P30050
RPL12
IGPLGLSPK
1,5
Q00839-2
HNRNPU
AKSPQPPVEEEDEHFDDTVVCLDTYN CDLHFK
1,6
Q9NR30-2
DDX21
KKEEPSQNDISPK
2,1
Heterogeneous nuclear ribonucleoproteins C1/C2 Proteasome subunit alpha type-3 Isoform A1-A of Heterogeneous nuclear ribonucleoprotein A1 60S ribosomal protein L12 Isoform Short of Heterogeneous nuclear ribonucleoprotein U Isoform 2 of Nucleolar RNA helicase 2
1,7 1,6 2,8 1,6 1,6
6,0 1,6
2,0
ACCEPTED MANUSCRIPT Also worthy to note is the presence among proteins whose expression is increased by QZ of one protein kinase (DNA-PK/PRKDC) and at least 7 proteins (EEF1E1, ELAVL1, KIF5B, PSMA3, RBMX, RPS3, RPS7) that interact with and/or modulate the activity of protein kinases
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and phosphatases. These are highlighted by short comments in Tab. S3. Altogether these
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proteins are good candidates for causing some of the collateral effects observed upon cell treatment with QZ.
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A functional analysis of proteins whose expression is up-regulated by QZ reveals that most of them are constituents of the ribosome and the proteasome (see Fig. 3). The possibility that this phenomenon reflects an off-target effect of QZ cannot be ruled out. In this connection it
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should be noted that a similar rise of ribosomal, though not of proteasomal proteins, has been reported to occur upon cell treatment with a cyclic peptide which is expected to partially
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abrogates CK2 signaling by a substrate directed mechanism. What is really remarkable anyway is the finding that the proteasomal proteins increased by QZ exclusively belong to the catalytic core (complex 20S) and to the complex PA28. Indeed, as
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depicted schematically in Fig. 4A, all the proteins of these two elements, with just one
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exception (PSMA5, which was found increased with a fold change of 1.4, just below the selected cutoff of 1.5), are increased by QZ, while none of those of the regulatory complex 19S,
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whose association with the catalytic core confers to the proteasome the ability to degrade ubiquitinated proteins [28], are significantly altered. This would mean that in the presence of QZ, as also depicted in Fig. 4B and C, only the elements committed with the degradation of the
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non-ubiquitinated polypeptides are increased [29], while the pathway leading to degradation of the ubiquitinated peptides remains unaffected, being limited by the regulatory complex 19S, whose elements are insensitive to QZ treatment. While the mechanism by which short time cell treatment with QZ leads to such a dramatic rearrangement of the proteasome subunits composition remains a matter of conjecture, a working hypothesis worthy of consideration is that CK2 plays a crucial role in shunting the proteasomal activity from nonubiquitinated to ubiquitinated protein degradation pathway. 3.4 The cellular level and phosphorylation of CK2 subunits are not altered by QZ. An important point related to the quantification of proteins in cells either treated or not with QZ was to make sure that the inhibitor does not modify the level of CK2 itself. Such an alteration in fact would bias the interpretation of the phosphoproteomics analysis by varying the amount of the catalyst. All the 3 subunits composing CK2 holoenzyme, the two catalytic ones, α and α’, and the regulatory one, β, were among the proteins detected and quantified in
ACCEPTED MANUSCRIPT our study, and none of them underwent any significant change upon QZ treatment. This corroborates the concept that the proteomics and phosphoproteomics alterations observed in our study are not due to an increase or decrease of the kinase which represents the primary
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target of QZ. lso to note is that two phosphosites belonging to the β-subunit of CK2, pSer205
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and pSer209, have been detected and quantified in our previous study [3], and neither of them is altered by QZ. Both these phosphorylations have been previously reported to occur in
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the cell, Ser205 being a notorious target for cyclin dependent kinases [30], while no information is available about the kinase(s) responsible for the phosphorylation of Ser209. Its location 4 residues downstream from Ser205, makes it a potential target for CK1 once Ser205
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has been phosphorylated [14]. Although the bis-phosphorylated peptide including both pSer205 and pSer209 was not found in our study it is possible that it escaped detection for a
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number of reasons, e.g. fast dephosphorylation of pSer205 once it has primed the phosphorylation of Ser209. Rather it looks intriguing that, having successfully identified and quantified these two facultative phosphosites of CK2β, we have missed the typical
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autophosphorylation site, affecting its two N-terminal serines (Ser2 and Ser3). Such an
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autophosphorylation is a very fast and stoichiometric event occurring every time CK2 is assayed in vitro under optimal conditions. It appears to have no functional consequences and
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it takes place through a “pseudo-intramolecular” (cis) mechanism which implies the association of two or more CK2 tetrameric holoenzyme molecules, being suppressed by conditions and/or mutations that prevent interactions between the tetrameric holoenzymes
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[31]. CK2 autophosphorylation at its -subunit therefore, is considered a reliable criterion to probe the occurrence both in vitro and in vivo of CK2 supramolecular complexes which have been suggested to cooperate to the regulation of its activity [32-35]. Our failure to detect any phosphopeptide accounting for the autophosphorylation site of CK2 may suggest therefore that in non-manipulated HEK-293T cells under the conditions of our experiment, the by far predominant form of CK2 holoenzyme is the one which is not associated with other CK2 tetramers to generate oligomeric structures. 4. DISCUSSION Quinalizarin (1,2,5,8-tetrahydroxyanthraquinone), shortened here as QZ, was firstly identified as a potential CK2 inhibitor by a computer aided virtual screening of the MMS (Molecular Modeling Section) database, based on the crystal structure of CK2 catalytic subunit. Its in vitro selectivity was profiled (at 1 μM inhibitor concentration) against a panel of 75 protein kinases, showing that only CK2 activity was suppressed with just few other kinases, notably
ACCEPTED MANUSCRIPT Erk8, AuroraB, AMPK, MARKK3, DYRKs 1A and 3, Pim-1 and -3 and HIPK2 partially inhibited (40-20%) [13]. Our present analysis, based on a quantitative phosphoproteomics study performed with HEK-293T cells either treated or not for a short time (3 h) with 50 μM QZ [3]
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shows that almost half of the 69 phosphosites which are decreased by QZ treatment are
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generated by CK2 as judged from their fulfillment of the CK2 consensus (S/T-x-x-E/D) and/or from their belonging to repertoires of bona fide CK2 sites. By contrast none of the other
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phosphosites decreased by QZ is among those reported in PhosphositePlus as targets of the aforementioned kinases moderately inhibited by QZ. These data confirm that CK2 is the primary target of QZ also in living cells but they also disclose the occurrence of off-target
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effects probably mediated by as yet unidentified kinase(s) not included in the panel used for profiling the in vitro selectivity of QZ [13], and/or of collateral events possibly triggered by
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CK2 inhibition, but executed by different kinases. The presence among CK2 phosphosites of several residues belonging to signaling molecules, with special reference to inhibitor-2 of protein phosphatase 1 and A-kinase anchoring protein-12 (AKAP12), provide plausible
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examples of how such indirect effects may take place.
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Also to note is the strikingly variable efficacy of QZ on phosphosites generated by CK2. Those which are affected represent a tiny minority, with more than 600 quantified CK2
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phosphosites undergoing no significant alteration. Even the 31 CK2 phosphosites that are significantly decreased (a kind of “tip of the iceberg”) display variable responsiveness to QZ, with fold changes ranging between 1.5 (the minimal one for being considered) and 5.0. This
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highlights on one side the overall remarkable stability of CK2 phosphosites, in accordance with other studies [19], on the other the crucial relevance of each individual site’s turnover (and therefore of the complementary phosphatase(s)) in determining the timeliness of CK2 signaling and responsiveness to CK2 blockage. The scenario becomes even more complicated if we now take into account the phosphosites that are increased by QZ treatment, and which are even more numerous than those which are decreased. Although this intriguing phenomenon had been already observed (e.g. [20, 21]) no attempt was done up to now to provide a rational explanation for it, except generically arguing about “indirect” effects. Our present analysis discloses at least two mechanisms which can account for the increase in phosphorylation upon cell treatment with a kinase inhibitor: one based on the uneven dephosphorylation of two phosphoresidues close to each other, the other promoted by the increased concentration of some phosphorylatable protein substrates. As far as the former mechanism is concerned the novel paradigm that should be taken into
ACCEPTED MANUSCRIPT account is that a singly phosphorylated peptide can be generated not only by phosphorylation of its non phosphorylated residue, but also by preferential dephosphorylation of a phosphoresidue nearby, assuming both residues were previously phosphorylated. This
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mechanism is exemplified in our case by two bis-phosphorylated peptides, belonging to
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AKAP12 and SRRM2 both present in the list of phosphopeptides decreased by QZ, whose singly phosphorylated derivatives are instead listed among those which are increased (see
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Tab. S1 and S2 and Fig. 2A and B). It is quite conceivable moreover that the same mechanism contributes to increase the level of the singly phosphorylated peptides listed in Tab. 2 either conforming or not to the CK2 consensus, but sharing anyway the presence of a bona fide CK2
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site nearby. Although in these cases the corresponding bis-phosphorylated peptides escaped detection in our SILAC analysis, it is quite expectable that upon QZ treatment the CK2
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phosphosite (or one of the two if both conform to the CK2 consensus) is preferentially dephosphorylated, thus contributing to increase the yield of the singly phosphorylated derivative.
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Another mechanism by which QZ can promote an increased phosphorylation of some residues
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is disclosed by the scrutiny of a subset of proteins whose amount is increased by QZ treatment. These represent a minority of the proteins quantified in our study, just 100 (see
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Tab. S3) out of about a thousand, which were quantified in all 6 biological replicates. The amount of all the other proteins is in fact unaffected by 3 h QZ treatment. To note that 9 of the proteins whose amount is increased by QZ include 11 sites whose phosphorylation is
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increased as well (see Tab. 3). It is quite expectable in this case that the increased level of phosphorylation rather than being due to altered efficiency of kinases and/or phosphatases is, at least partially, promoted by the increased cellular concentration of the protein substrate. The list of proteins whose expression is increased by QZ deserves attention in two other respects. Firstly it includes molecules that are expected to affect the quantitative composition of the phosphoproteome, either for being kinases themselves (e.g. DNAPK) or for being kinase/phosphatase regulators (e.g. EEF1E1, ELAVL1, KIF5B, PSMA3, RBMX, RPS3, RPS7). It is expectable therefore that their increase contributes to some extent to the quantitative alterations of the phosphoproteome not directly attributable to CK2 inhibition. Secondly, as highlighted in Fig. 3, the augmented proteins are not randomly distributed among cellular compartments but they are concentrated into the ribosome and the proteasome. Even more striking is the observation that all the proteasomal proteins which are increased belong to the catalytic core and to the PA28 subunit, whose association is instrumental to the degradation
ACCEPTED MANUSCRIPT of non ubiquitinated proteins [29], while none of the proteins composing the 19S subunit, specifically targeting to degradation the ubiquitinated proteins [28], is increased by QZ. Such a situation, schematically depicted in Fig. 4, makes expectable that in the presence of QZ the
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functionality of the proteasome is shifted from the degradation of ubiquitinated proteins to
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that of non-ubiquitinated ones. Interestingly this ubiquitin independent degradation route has been recently shown to play a relevant role in cell viability, becoming up-regulated under
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conditions of oxidative stress where protein damage occurs (reviewed in [29]). Whether its enhancement by QZ is mediated by CK2 inhibition or represents the outcome of off-target effects of QZ is not clear. If the implication of CK2 in the reshuffling of proteasomal
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functionality is confirmed, this will add a new unanticipated gear to the multifarious machinery by which CK2 exerts its control over protein degradation, authorizing the
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speculation that under conditions where the level of CK2 is abnormally high (as in general observed in cancer cells) the balance between degradation of ubiquitinated vs nonubiquitinated proteins is up-regulated. However the possibility should be also considered that
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QZ operates by a CK2-independent mechanism leading to the accumulation of
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damaged/unfolded proteins which, in turn, up-regulate the 20S proteasome degradation
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machinery. Acknowledgments
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The authors whish to thank the “Cassa di risparmio di Padova e Rovigo” (Cariparo) Holding for funding the acquisition of the LTQ-Orbitrap XL mass spectrometer. G.A was supported by a grant from the University of Padova (CPDA082784). The financial support from AIRC (grant IG10312) to LAP is gratefully acknowledged.
Legends of Figures and Tables Table 1. Phosphopeptides decreased upon inhibition with QZ. The table lists the phosphopeptides attributable to CK2 that were found significantly decreased upon treatment of HEK-293T cells with the inhibitor QZ (drawn from Tab. S1, supplementary material). In red phosphosites identified in [3]; underlined residues belong to the category of putative CK2 sites. The symbol # indicates proteins implicated in cell death/survival (references in Tab. S1, supplementary material). Table 2. Phosphopeptides increased upon treatment with QZ and bearing additional phosphoacceptor sites conforming to the CK2 consensus. In red identified phosphosites drawn from Tab. S2 in order to include only phosphopeptides that bear additional CK2 sites within the same peptide sequence. In bold phosphoacceptor sites conforming to the CK2 consensus. The symbol * indicates putative CK2 sites reported in PhopshositePlus as phosphorylated.
ACCEPTED MANUSCRIPT Table 3. Phosphopeptides with increased phosphorylation belonging to proteins whose expression level is also increased upon QZ treatment. In red identified phosphosites (see Tab. S2). Underlined phosphosites conforming to the CK2 consensus sequence.
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Figure 1. Schematic picture of the main outcomes of the phosphoproteomics study.
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The pie-charts summarize the results of a the SILAC experiment described in [3]. 3465 unique phosphopeptides were quantified, more than 20% of which conform to the CK2 consensus sequence and were randomly validated as bona fide CK2 substrates. Almost 95% of the quantified sites were not significantly affected by a 3 h treatment with QZ. Less than 2% of the phosphosites were decreased, almost 50% of which conforms to the CK2 consensus sequence. About 3% of the phosphosites were increased and almost 30% of these conform to the CK2 consensus.
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Figure 2. Schematic representation of the mechanism accounting for the generation of mono-phosphorylated derivatives by uneven dephosphorylation of bis-phosphorylated peptides.
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The different dephosphorylation rate of individual phosphoresidues present in the bisphosphorylated peptide from AKAP12 (A) or from SRRM2 (B) promotes upon CK2 blockage the preferential accumulation of one of the two singly phosphorylated peptides.
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Figure 3. STRING analysis of proteins whose expression level is significantly increased by the treatment with QZ.
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Functional/physical interactions between the proteins are displayed. Output data were filtered to have the highest possible interaction score (>0.9). The proteins are mainly clustered in the two functional categories of ribosome and proteasome.
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Figure 4. Schematic representation of the proteasomal proteins identified in the study and their implication in ubiquitin-dependent and –independent degradation pathways. (A) Schematic representation of the proteasomal complex. Proteins with an increased expression level upon QZ treatment are colored in blue and belong to the core complex 20S. Proteins belonging to the regulatory complex 19S are colored in green if they were identified in this study but their expression level was not significantly affected by the treatment with QZ. Proteins colored in grey were not identified in our SILAC analysis. (B) The proteasomal complex formed by the subunits 20S and 19S is responsible for the degradation of ubiquitinated proteins. (C) The combination of the core complex 20S with the complex PA28 (whose proteins show an increased expression level upon QZ treatment and are colored in blue) is responsible for the degradation of non-ubiquitinated proteins.
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Figure 4
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The CK2 inhibitor quinalizarin (QZ) causes the overexpression of 100 proteins QZ also promotes the increase of 108 phosphosites Some of these are accounted for by augmented proteins, others by uneven loss of P Most over-expressed proteins belong to ribosomes and to the proteasome Increased proteasomal proteins are committed to ubiquitin-independent degradation
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-