CK2 Substrates Research Articles

CK2 phosphorylation of CMTR1 promotes RNA cap formation and influenza virus infection

The RNA cap methyltransferase CMTR1 methylates the first transcribed nucleotide of RNA polymerase II transcripts, impacting gene expression mechanisms, including during innate immune responses. Using mass spectrometry, we identify a multiply phosphorylated region of CMTR1 (phospho-patch [P-Patch]), which is a substrate for the kinase CK2 (casein kinase II). CMTR1 phosphorylation alters intramolecular interactions, increases recruitment to RNA polymerase II, and promotes RNA cap methylation. P-Patch phosphorylation occurs during the G1 phase of the cell cycle, recruiting CMTR1 to RNA polymerase II during a period of rapid transcription and RNA cap formation. CMTR1 phosphorylation is required for the expression of specific RNAs, including ribosomal protein gene transcripts, and promotes cell proliferation. CMTR1 phosphorylation is also required for interferon-stimulated gene expression. The cap-snatching virus, influenza A, utilizes host CMTR1 phosphorylation to produce the caps required for virus production and infection. We present an RNA cap methylation control mechanism whereby CK2 controls CMTR1, enhancing co-transcriptional capping.

The substrate quality of CK2 target sites has a determinant role on their function and evolution

Most biological processes are regulated by signaling modules that bind to short linear motifs. For protein kinases, substrates may have full or only partial matches to the kinase recognition motif, a property known as "substrate quality." However, it is not clear whether differences in substrate quality represent neutral variation or if they have functional consequences. We examine this question for the kinase CK2, which has many fundamental functions. We show that optimal CK2 sites are phosphorylated at maximal stoichiometries and found in many conditions, whereas minimal substrates are more weakly phosphorylated and have regulatory functions. Optimal CK2 sites tend to be more conserved, and substrate quality is often tuned by selection. For intermediate sites, increases or decreases in substrate quality may be deleterious, as we demonstrate for a CK2 substrate at the kinetochore. The results together suggest a strong role for substrate quality in phosphosite function and evolution. A record of this paper's transparent peer review process is included in the supplemental information.

Casein kinase II promotes piRNA production through direct phosphorylation of USTC component TOFU-4

Piwi-interacting RNAs (piRNAs) are genomically encoded small RNAs that engage Piwi Argonaute proteins to direct mRNA surveillance and transposon silencing. Despite advances in understanding piRNA pathways and functions, how the production of piRNA is regulated remains elusive. Here, using a genetic screen, we identify casein kinase II (CK2) as a factor required for piRNA pathway function. We show that CK2 is required for the localization of PRG-1 and for the proper localization of several factors that comprise the ‘upstream sequence transcription complex’ (USTC), which is required for piRNA transcription. Loss of CK2 impairs piRNA levels suggesting that CK2 promotes USTC function. We identify the USTC component twenty-one-U fouled-up 4 (TOFU-4) as a direct substrate for CK2. Our findings suggest that phosphorylation of TOFU-4 by CK2 promotes the assembly of USTC and piRNA transcription. Notably, during the aging process, CK2 activity declines, resulting in the disassembly of USTC, decreased piRNA production, and defects in piRNA-mediated gene silencing, including transposons silencing. These findings highlight the significance of posttranslational modification in regulating piRNA biogenesis and its implications for the aging process. Overall, our study provides compelling evidence for the involvement of a posttranslational modification mechanism in the regulation of piRNA biogenesis.

Exploring Protein Kinase CK2 Substrate Recognition and the Dynamic Response of Substrate Phosphorylation to Kinase Modulation

Protein kinase CK2 (formerly known as casein kinase 2 or II), a ubiquitous and constitutively active enzyme, is widely recognized as one of the most pleiotropic serine/threonine kinases. It plays a critical role in numerous signaling pathways, with hundreds of bona fide substrates. However, despite considerable research efforts, our understanding of the entire CK2 substratome and its functional associations with the majority of these substrates is far from being completely deciphered. In this context, we aim to provide an overview of how CK2 recognizes its substrates. We will discuss the pros and cons of the existing methods to manipulate CK2 activity in cells, as well as exploring the dynamic response of substrate phosphorylation to CK2 modulation.

Analyzing the interactome of human CK2β in prostate carcinoma cells reveals HSP70-1 and Rho guanin nucleotide exchange factor 12 as novel interaction partners.

CK2β is the non-catalytic modulating part of the S/T-protein kinase CK2. However, the overall function of CK2β is poorly understood. Here, we report on the identification of 38 new interaction partners of the human CK2β from lysates of DU145 prostate cancer cells using photo-crosslinking and mass spectrometry, whereby HSP70-1 was identified with high abundance. The KD value of its interaction with CK2β was determined as 0.57 μM by microscale thermophoresis, this being the first time, to our knowledge, that a KD value of CK2β with another protein than CK2α or CK2α' was quantified. Phosphorylation studies excluded HSP70-1 as a substrate or activity modulator of CK2, suggesting a CK2 activity independent interaction of HSP70-1 with CK2β. Co-immunoprecipitation experiments in three different cancer cell lines confirmed the interaction of HSP70-1 with CK2β in vivo. A second identified CK2β interaction partner was Rho guanin nucleotide exchange factor 12, indicating an involvement of CK2β in the Rho-GTPase signal pathway, described here for the first time to our knowledge. This points to a role of CK2β in the interaction network affecting the organization of the cytoskeleton.

A proteomic screen of Ty1 integrase partners identifies the protein kinase CK2 as a regulator of Ty1 retrotransposition

BackgroundTransposable elements are ubiquitous and play a fundamental role in shaping genomes during evolution. Since excessive transposition can be mutagenic, mechanisms exist in the cells to keep these mobile elements under control. Although many cellular factors regulating the mobility of the retrovirus-like transposon Ty1 in Saccharomyces cerevisiae have been identified in genetic screens, only very few of them interact physically with Ty1 integrase (IN).ResultsHere, we perform a proteomic screen to establish Ty1 IN interactome. Among the 265 potential interacting partners, we focus our study on the conserved CK2 kinase. We confirm the interaction between IN and CK2, demonstrate that IN is a substrate of CK2 in vitro and identify the modified residues. We find that Ty1 IN is phosphorylated in vivo and that these modifications are dependent in part on CK2. No significant change in Ty1 retromobility could be observed when we introduce phospho-ablative mutations that prevent IN phosphorylation by CK2 in vitro. However, the absence of CK2 holoenzyme results in a strong stimulation of Ty1 retrotransposition, characterized by an increase in Ty1 mRNA and protein levels and a high accumulation of cDNA.ConclusionOur study shows that Ty1 IN is phosphorylated, as observed for retroviral INs and highlights an important role of CK2 in the regulation of Ty1 retrotransposition. In addition, the proteomic approach enabled the identification of many new Ty1 IN interacting partners, whose potential role in the control of Ty1 mobility will be interesting to study.

Phosphoproteomics revealed cellular signals immediately responding to disruption of cancer amino acid homeostasis induced by inhibition of l-type amino acid transporter 1

BackgroundCancer-upregulated l-type amino acid transporter 1 (LAT1; SLC7A5) supplies essential amino acids to cancer cells. LAT1 substrates are not only needed for cancer rapid growth, but involved in cellular signaling. LAT1 has been proposed as a potential target for cancer treatment—its inhibitor, JPH203, is currently in clinical trials and targets biliary tract cancer (BTC). Here, we revealed to what extent LAT1 inhibitor affects intracellular amino acid content and what kind of cellular signals are directly triggered by LAT1 inhibition.MethodsLiquid chromatography assay combined with o-phthalaldehyde- and 9-fluorenyl-methylchloroformate-based derivatization revealed changes in intracellular amino acid levels induced by LAT1 inhibition with JPH203 treatment in three BTC cell lines. Tandem mass tag-based quantitative phosphoproteomics characterized the effect of JPH203 treatment on BTC cells, and suggested key regulators in LAT1-inhibited cells. We further studied one of the key regulators, CK2 protein kinase, by using Western blot, enzymatic activity assay, and co-immunoprecipitation. We evaluated anticancer effects of combination of JPH203 with CK2 inhibitor using cell growth and would healing assay.ResultsJPH203 treatment decreased intracellular levels of LAT1 substrates including essential amino acids of three BTC cell lines, immediately and drastically. We also found levels of some of these amino acids were partially recovered after longer-time treatment. Therefore, we performed phosphoproteomics with short-time JPH203 treatment prior to the cellular compensatory response, and revealed hundreds of differentially phosphorylated sites. Commonly downregulated phosphorylation sites were found on proteins involved in the cell cycle and RNA splicing. Our phosphoproteomics also suggested key regulators immediately responding to LAT1 inhibition. Focusing on one of these regulators, protein kinase CK2, we revealed LAT1 inhibition decreased phosphorylation of CK2 substrate without changing CK2 enzymatic activity. Furthermore, LAT1 inhibition abolished interaction between CK2 and its regulatory protein NOLC1, which suggests regulatory mechanism of CK2 substrate protein specificity controlled by LAT1 inhibition. Moreover, we revealed that the combination of JPH203 with CK2 inhibitor resulted in the enhanced inhibition of proliferation and migration of BTC cells.ConclusionThis study provides new perspectives on LAT1-dependent cellular processes and a rationale for therapeutics targeting reprogrammed cancer metabolism.

Writing and Erasing O-GlcNAc on Casein Kinase 2 Alpha Alters the Phosphoproteome.

O-GlcNAc is an essential carbohydrate modification that intersects with phosphorylation signaling pathways via crosstalk on protein substrates or by direct modification of the kinases that write the phosphate modification. Casein kinase 2 alpha (CK2α), the catalytic subunit of the ubiquitously expressed and constitutively active kinase CK2, is modified by O-GlcNAc, but the effect of this modification on the phosphoproteome in cells is unknown. Here, we apply complementary targeted O-GlcNAc editors, nanobody-OGT and -splitOGA, to selectively write and erase O-GlcNAc from a tagged CK2α to measure the effects on the phosphoproteome in cells. These tools effectively and selectively edit the Ser347 glycosite on CK2α. Using quantitative phosphoproteomics, we report 51 phosphoproteins whose enrichment changes as a function of editing O-GlcNAc on CK2α, including HDAC1, HDAC2, ENSA, SMARCAD1, and PABPN1. Specific phosphosites on HDAC1 Ser393 and HDAC2 Ser394, both reported CK2 substrates, are significantly enhanced by O-GlcNAcylation of CK2α. These data will propel future studies on the crosstalk between O-GlcNAc and phosphorylation.

De novo variants of CSNK2B cause a new intellectual disability-craniodigital syndrome by disrupting the canonical Wnt signaling pathway.

SummaryCSNK2B encodes for casein kinase II subunit beta (CK2β), the regulatory subunit of casein kinase II (CK2), which is known to mediate diverse cellular pathways. Variants in this gene have been recently identified as a cause of Poirier-Bienvenu neurodevelopmental syndrome (POBINDS), but functional evidence is sparse. Here, we report five unrelated individuals: two of them manifesting POBINDS, while three are identified to segregate a new intellectual disability-craniodigital syndrome (IDCS), distinct from POBINDS. The three IDCS individuals carried two different de novo missense variants affecting the same codon of CSNK2B. Both variants, NP_001311.3; p.Asp32His and NP_001311.3; p.Asp32Asn, lead to an upregulation of CSNK2B expression at transcript and protein level, along with global dysregulation of canonical Wnt signaling. We found impaired interaction of the two key players DVL3 and β-catenin with mutated CK2β. The variants compromise the kinase activity of CK2 as evident by a marked reduction of phosphorylated β-catenin and consequent absence of active β-catenin inside nuclei of the patient-derived lymphoblastoid cell lines (LCLs). In line with these findings, whole-transcriptome profiling of patient-derived LCLs harboring the NP_001311.3; p.Asp32His variant confirmed a marked difference in expression of genes involved in the Wnt signaling pathway. In addition, whole-phosphoproteome analysis of the LCLs of the same subject showed absence of phosphorylation for 313 putative CK2 substrates, enriched in the regulation of nuclear β-catenin and transcription of the target genes. Our findings suggest that discrete variants in CSNK2B cause dominant-negative perturbation of the canonical Wnt signaling pathway, leading to a new craniodigital syndrome distinguishable from POBINDS.

Inhibition of Protein Kinase CK2 Affects Thymidylate Synthesis Cycle Enzyme Level and Distribution in Human Cancer Cells.

Thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT) constitute the thymidylate synthesis cycle providing thymidylate for DNA synthesis and repair. Our previous studies indicated that TS and DHFR are the substrates of protein kinase CK2. This work has been aimed at the elucidation of the effect of CK2 activity on cell cycle progression, thymidylate synthesis enzyme expression and localization, and the role of CK2-mediated TS phosphorylation in in vitro di- and trimolecular complex formation. The results were obtained by means of western blot, confocal microscopy, flow cytometry, quantitative polymerase chain reaction (QPCR), quartz crystal microbalance with dissipation monitoring (QCM-D), and microthermophoresis (MST). Our research indicates that CK2 inhibition does not change the levels of the transcripts; however, it affects the protein levels of DHFR and TS in both tested cell lines, i.e., A549 and CCRF-CEM, and the level of SHMT1 in CCRF-CEM cells. Moreover, we show that CK2-mediated phosphorylation of TS enables the protein (pTS) interaction with SHMT1 and leads to the stability of the tri-complex containing SHMT1, DHFR, and pTS. Our results suggest an important regulatory role of CK2-mediated phosphorylation for inter- and intracellular protein level of enzymes involved in the thymidylate biosynthesis cycle.

Molecular Plasticity of Crystalline CK2α' Leads to KN2, a Bivalent Inhibitor of Protein Kinase CK2 with Extraordinary Selectivity.

CK2α and CK2α' are paralogous catalytic subunits of CK2, which belongs to the eukaryotic protein kinases. CK2 promotes tumorigenesis and the spread of pathogenic viruses like SARS-CoV-2 and is thus an attractive drug target. Efforts to develop selective CK2 inhibitors binding offside the ATP site had disclosed the αD pocket in CK2α; its occupation requires large conformational adaptations of the helix αD. As shown here, the αD pocket is accessible also in CK2α', where the necessary structural plasticity can be triggered with suitable ligands even in the crystalline state. A CK2α' structure with an ATP site and an αD pocket ligand guided the design of the bivalent CK2 inhibitor KN2. It binds to CK2 with low nanomolar affinity, is cell-permeable, and suppresses the intracellular phosphorylation of typical CK2 substrates. Kinase profiling revealed a high selectivity of KN2 for CK2 and emphasizes the selectivity-promoting potential of the αD pocket.

Targeting of Protein Kinase CK2 in Acute Myeloid Leukemia Cells Using the Clinical-Grade Synthetic-Peptide CIGB-300.

Protein kinase CK2 has emerged as an attractive therapeutic target in acute myeloid leukemia (AML), an advent that becomes particularly relevant since the treatment of this hematological neoplasia remains challenging. Here we explored for the first time the effect of the clinical-grade peptide-based CK2 inhibitor CIGB-300 on AML cells proliferation and viability. CIGB-300 internalization and subcellular distribution were also studied, and the role of B23/nucleophosmin 1 (NPM1), a major target for the peptide in solid tumors, was addressed by knock-down in model cell lines. Finally, pull-down experiments and phosphoproteomic analysis were performed to study CIGB-interacting proteins and identify the array of CK2 substrates differentially modulated after treatment with the peptide. Importantly, CIGB-300 elicited a potent anti-proliferative and proapoptotic effect in AML cells, with more than 80% of peptide transduced cells within three minutes. Unlike solid tumor cells, NPM1 did not appear to be a major target for CIGB-300 in AML cells. However, in vivo pull-down experiments and phosphoproteomic analysis evidenced that CIGB-300 targeted the CK2α catalytic subunit, different ribosomal proteins, and inhibited the phosphorylation of a common CK2 substrates array among both AML backgrounds. Remarkably, our results not only provide cellular and molecular insights unveiling the complexity of the CIGB-300 anti-leukemic effect in AML cells but also reinforce the rationale behind the pharmacologic blockade of protein kinase CK2 for AML-targeted therapy.

Preclinical efficacy of CIGB-300, an anti-CK2 peptide, on breast cancer metastasic colonization

CK2 is a serine/threonine kinase that is overexpressed in breast cancer and its inhibition is associated to reduced tumor growth and disease progression. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to protein kinase CK2 catalytic subunit alpha and to CK2 substrates thus preventing the enzyme activity. Our aim was to evaluate the potential therapeutic benefits of CIGB-300 on breast cancer disease using experimental models with translational relevance. We demonstrated that CIGB-300 reduces breast cancer cell growth in MDA-MB-231, MCF-7 and F3II cells, exerting a pro-apoptotic action and cell cycle arrest. We also found that CIGB-300 decreased cell adhesion, migration and clonogenic capacity of malignant cells. Effect on experimental breast cancer lung metastasis was evaluated after surgical removal of primary F3II tumors or after tail vein injection of tumor cells, also we evaluated CIGB-300 effect on spontaneous lung metastasis in an orthotopic model. Systemic CIGB-300 treatment inhibited breast cancer colonization of the lung, reducing the size and number of metastatic lesions. The present preclinical study establishes for the first time the efficacy of CIGB-300 on breast cancer. These encouraging results suggest that CIGB-300 could be used for the management of breast cancer as an adjuvant therapy after surgery, limiting tumor metastatic spread and thus protecting the patient from distant recurrence.

Abstract 6232: Preclinical efficacy of CIGB-300, an anti-CK2 peptide, on breast cancer metastasic colonization

Abstract CK2 is a serine/threonine kinase that is overexpressed in breast cancer and its inhibition is associated to reduced tumor growth and disease progression. With more than three hundred substrates, this kinase is involved in a large number of cellular functions, such as replication, cell proliferation, differentiation, regulation of apoptosis and angiogenesis. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to the phospho-acceptor domain of CK2 substrates impairing the correct phosphorylation by the enzyme. Our aim was to evaluate the potential therapeutic benefits of CIGB-300 on breast cancer disease using experimental models with translational relevance. We demonstrated that CIGB-300 reduces breast cancer cell growth in MDA-MB-231, MCF-7 and F3II cells, exerting a pro-apoptotic action. We found that CIGB-300 increased Bax and decreased Bcl-2 expression, so the intrinsic pathway was involved in the proapoptotic effect of CIGB-300 in breast cancer cells. Moreover, CIGB-300 inhibits the CK2-mediated phosphorylation of B23, such phosphorylation impairment was correlated with the ability of CIGB-300 to induce nucleolar disassembly. Also, we investigate whether CIGB-300 affects the sensitivity of tumor cells to chemotherapeutic agents. We evaluated the effects of the combination of CIGB-300 and cisplatin on cell viability and apoptosis. We demonstrated that CIGB-300 sensitize breast cancer cells to cisplatin, reducing cell viability and increasing apoptosis. In vivo, the effect on experimental breast cancer lung metastasis was evaluated after surgical removal of primary F3II tumors or after tail vein injection of tumor cells. Systemic CIGB-300 treatment inhibited breast cancer colonization of the lung, reducing the size and number of metastatic lesions. The present preclinical study establishes for the first time the efficacy of CIGB-300 on breast cancer. These encouraging results suggest that CIGB-300 could be used for the management of breast cancer as an adjuvant therapy after surgery, limiting tumor metastatic spread and thus protecting the patient from distant recurrence. Citation Format: Maria F. Gottardo, Johanna E. Sidabra, Carla S. Capobianco, Juan Garona, Silvio Perea Rodriguez, Daniel F. Aonso, Hernan G. Farina. Preclinical efficacy of CIGB-300, an anti-CK2 peptide, on breast cancer metastasic colonization [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6232.

Protein Kinase CK2 in Cancer Energetics.

Protein kinase CK2 (formerly known as casein kinase 2) is abnormally elevated in many cancers. This may increase tumor aggressiveness through CK2-dependent phosphorylation of key proteins in several signaling pathways. In this work, we have compiled evidence from the literature to suggest that CK2 also modulates a metabolic switch characteristic of cancer cells that enhances resistance to death, due to either drugs or to a microenvironment deficient in oxygen or nutrients. Concurrently, CK2 may help to preserve mitochondrial activity in a PTEN-dependent manner. PTEN, widely recognized as a tumor suppressor, is another CK2 substrate in the PI3K/Akt signaling pathway that promotes cancer viability and aerobic glycolysis. Given that CK2 can regulate Akt as well as two of its main effectors, namely mTORC1 and β-catenin, we comprehensively describe how CK2 may modulate cancer energetics by regulating expression of key targets and downstream processes, such as HIF-1 and autophagy, respectively. Thus, the specific inhibition of CK2 may lead to a catastrophic death of cancer cells, which could become a feasible therapeutic strategy to beat this devastating disease. In fact, ATP-competitive inhibitors, synthetic peptides and antisense oligonucleotides have been designed as CK2 inhibitors, some of them used in preclinical models of cancer, of which TBB and silmitasertib are widely known. We will finish by discussing a hypothetical scenario in which cancer cells are “addicted” to CK2; i.e., in which many proteins that regulate signaling pathways and metabolism-linked processes are highly dependent on this kinase.

CIGB-300 anticancer peptide regulates the protein kinase CK2-dependent phosphoproteome.

Casein-kinase CK2 is a Ser/Thr protein kinase that fosters cell survival and proliferation of malignant cells. The CK2 holoenzyme, formed by the association of two catalytic alpha/alpha' (CK2α/CK2α') and two regulatory beta subunits (CK2β), phosphorylates diverse intracellular proteins partaking in key cellular processes. A handful of such CK2 substrates have been identified as targets for the substrate-binding anticancer peptide CIGB-300. However, since CK2β also contains a CK2 phosphorylation consensus motif, this peptide may also directly impinge on CK2 enzymatic activity, thus globally modifying the CK2-dependent phosphoproteome. To address such a possibility, firstly, we evaluated the potential interaction of CIGB-300 with CK2 subunits, both in cell-free assays and cellular lysates, as well as its effect on CK2 enzymatic activity. Then, we performed a phosphoproteomic survey focusing on early inhibitory events triggered by CIGB-300 and identified those CK2 substrates significantly inhibited along with disturbed cellular processes. Altogether, we provided here the first evidence for a direct impairment of CK2 enzymatic activity by CIGB-300. Of note, both CK2-mediated inhibitory mechanisms of this anticancer peptide (i.e., substrate- and enzyme-binding mechanism) may run in parallel in tumor cells and help to explain the different anti-neoplastic effects exerted by CIGB-300 in preclinical cancer models.

The Combination of the CIGB-300 Anticancer Peptide and Cisplatin Modulates Proteins Related to Cell Survival, DNA Repair and Metastasis in a Lung Cancer Cell Line Model

Background:CIGB-300 is a pro-apoptotic peptide that abrogates CK2-mediated phosphorylation, and can elicit synergistic interaction in vitro and in vivo when combined with certain anticancer drugs.Objective:The combination of CIGB-300 with cisplatin is studied through data mining and expressionbased proteomics to reveal the molecular basis of this interaction. Cisplatin resistance-associated proteins, which have also been reported as CK2 substrates, were first identified by bioinformatic analyses.Methods:Data from these analyses suggested that the cisplatin resistance phenotype could be directly improved by inhibiting CK2 phosphorylation on specific substrates. Furthermore, 157 proteins were differentially modulated on the NCI-H125 lung cancer cell line in response to CIGB-300, cisplatin or both drugs as determined by LC-MS/MS.Results:The expression of 28 cisplatin resistance-associated proteins was changed when cisplatin was combined with CIGB-300. Overall, the proteins identified are also related to cell survival, cell proliferation and metastasis. Furthermore, the CIGB-300 regulated proteome revealed proteins that were initially involved in the mechanism of action of CIGB-300 and cisplatin as single agents.Conclusion:This is the first report describing the protein array modulated by combining CIGB-300 and cisplatin that will support the rationale for future clinical settings based on a multi-target cancer therapy.

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach.

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

Lymphocyte Phosphatase-Associated Phosphoprotein Is a Substrate of Protein Kinase CK2.

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a molecular partner of CD45 phosphatase that plays a key role in the regulation of antigen-specific activation of lymphocytes. The functions of LPAP still remain unknown. We believe that studying LPAP phosphorylation pathways could shed light on its functions. In this work, we studied the phosphorylation of LPAP ectopically expressed in non-lymphoid cells in order to determine the effect of LPAP interaction partners on its phosphorylation. We found that phosphorylation at Ser153 and Ser163 in non-hematopoietic HEK293 cells was conserved, while phosphorylation at Ser99 and Ser172 was almost absent. The pattern of LPAP phosphorylation in K562 erythroid and U937 myeloid cells expressing endogenous CD45 protein was similar to that observed in T and B lymphocytes. We demonstrated for the first time that LPAP is a substrate for protein kinase CK2 that phosphorylates it at Ser153, presumably ensuring LPAP resistance to degradation.

DNA mismatch repair activity of MutLα is regulated by CK2-dependent phosphorylation of MLH1 (S477).

MutLα, a heterodimer consisting of MLH1 and PMS2, is a key player of DNA mismatch repair (MMR), yet little is known about its regulation. In this study, we used mass spectrometry to identify phosphorylated residues within MLH1 and PMS2. The most frequently detected phosphorylated amino acid was serine 477 of MLH1. Pharmacological treatment indicates‎ that Casein kinase II (CK2) could be responsible for the phosphorylation of MLH1 at serine 477 in vivo. In vitro kinase assay verified MLH1 as a substrate of CK2. Most importantly, using in vitro MMR assay we could demonstrate that p-MLH1S477 lost MMR activity. Moreover, we found that levels of p-MLH1S477 varied during the cell cycle. In summary, we identified that phosphorylation of MLH1 by CK2 at amino acid position 477 can switch off MMR activity in vitro. Since CK2 is overexpressed in many tumors and is able to inactivate MMR, the new mechanism here described could have an important impact on tumors overactive in CK2.