Lorenzo A. Pinna

INO - Istituto Nazionale di Ottica, Florens, Tuscany, Italy

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Publications (423)1696.24 Total impact

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    ABSTRACT: By derivatizing the purely competitive CK2 inhibitor N1-(4,5,6,7-tetrabromo-1H-benzimidazol-2-yl)-propane-1,3-diamine (K137) at its 3-amino position with a peptidic fragment composed by 3 to 4 glutamic or aspartic acid residues, a new family of bi-substrate inhibitors has been generated whose ability to simultaneously interact with both the ATP and the phosphoacceptor substrate binding sites has been probed by running mixed competition kinetics and by mutational mapping of the kinase residues implicated in substrate recognition. The most effective bi-substrate inhibitor, K137-E4, interacts with three functional regions of the kinase: the hydrophobic pocket close to the ATP binding site, the basic residues of the p+1 loop that recognizes the acidic determinant at position n+1 and the basic residues of alpha helix C that recognize the acidic determinant at position n+3. Compared to the parent inhibitor (K137), K137-E4 is several fold more potent (IC50 25 vs 130 nM) and more selective, failing to inhibit any other kinase as drastically as CK2 out of 140 enzymes, whereas 35 kinases are inhibited more potently than CK2 by K137. K137-E4 is unable to penetrate inside the cell and to inhibit endogenous CK2, its proapoptotic efficacy being negligible as compared to cell permeant inhibitors; however it readily inhibits ecto-CK2 on the outer cell surface, reducing the phosphorylation of several external phosphoproteins. Inhibition of ecto-CK2 by K137-E4 is accompanied by a slower migration of cancer cells as judged by wound healing assays. Based on the cellular responses to K137-E4, we conclude that ecto-CK2 is implicated in cell motility , while its contribution to the prosurvival role of CK2 is negligible.
    Biochemical Journal 09/2015; DOI:10.1042/BJ20141127 · 4.40 Impact Factor
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    ABSTRACT: CK2 is a multifunctional, pleiotropic protein kinase involved in the regulation of cell proliferation and survival. Since fibroblasts from Type 1 Diabetes patients (T1DM) with Nephropathy exhibit increased proliferation, we studied cell viability, basal CK2 expression and activity, and response to specific CK2 inhibitors TBB (4,5,6,7-tetrabenzotriazole) and CX4945, in fibroblasts from T1DM patients either with (T1DM+) or without (T1DM-) Nephropathy, and from healthy controls (N). We tested expression and phosphorylation of CK2-specific molecular targets. In untreated fibroblasts from T1DM+, the cell viability was higher than in both N and T1DM-. CK2 inhibitors significantly reduced cell viability in all groups, but more promptly and with a larger effect in T1DM+. Differences in CK2-dependent phosphorylation sites were detected. In conclusion, our results unveil a higher dependence of T1DM+ cells on CK2 for their survival, despite a similar expression and a lower activity of this kinase compared with those of normal cells.
    Growth factors (Chur, Switzerland) 09/2015; 33(4):1-8. DOI:10.3109/08977194.2015.1073725 · 3.39 Impact Factor
  • Luca Cesaro · Lorenzo Alberto Pinna
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    ABSTRACT: In the infancy of studies on protein phosphorylation the occurrence of clusters of three or more consecutive phosphoseryl residues in secreted and in cellular phosphoproteins was reported. Later however, while the reversible phosphorylation of Ser, Thr and Tyr residues was recognized to be the most frequent and general mechanism of cell regulation and signal transduction, the phenomenon of multi-phosphorylation of adjacent residues was entirely neglected. Now-a-days, in the post-genomic era, the availability of large phosphoproteomics database makes possible a comprehensive re-visitation of this intriguing aspect of protein phosphorylation, aimed at shedding light on both its mechanistic occurrence and its functional meaning. Here we describe an analysis of the human phosphoproteome disclosing the existence of more than 800 rows of 3 to >10 consecutive phosphoaminocids, composed almost exclusively of phosphoserine, while clustered phosphothreonines and phosphotyrosines are almost absent. A scrutiny of these phosphorylated rows supports the conclusion that they are generated through the major contribution of a few hierarchical protein kinases, with special reference to CK2. Also well documented is the combined intervention of CK1 and GSK3, the former acting as priming and primed, the latter as primed kinase. The by far largest proportion of proteins containing (pS)n clusters display a nuclear localization where they play a prominent role in the regulation of transcription. Consistently the molecular function of the by far largest majority of these proteins is ability to bind other macromolecules and/or nucleotides and metal ions. A “String” analysis performed under stringent conditions reveals that >80% of them are connected to each other by physical and/or functional links, and that this network of interactions mostly take place at nuclear level.
    Molecular BioSystems 07/2015; DOI:10.1039/C5MB00337G · 3.21 Impact Factor
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    ABSTRACT: The existence of extracellular phosphoproteins has been acknowledged for over a century. However, research in this area has been undeveloped largely because the kinases that phosphorylate secreted proteins have escaped identification. Fam20C is a kinase that phosphorylates S-x-E/pS motifs on proteins in milk and in the extracellular matrix of bones and teeth. Here, we show that Fam20C generates the majority of the extracellular phosphoproteome. Using CRISPR/Cas9 genome editing, mass spectrometry, and biochemistry, we identify more than 100 secreted phosphoproteins as genuine Fam20C substrates. Further, we show that Fam20C exhibits broader substrate specificity than previously appreciated. Functional annotations of Fam20C substrates suggest roles for the kinase beyond biomineralization, including lipid homeostasis, wound healing, and cell migration and adhesion. Our results establish Fam20C as the major secretory pathway protein kinase and serve as a foundation for new areas of investigation into the role of secreted protein phosphorylation in human biology and disease. Copyright © 2015 Elsevier Inc. All rights reserved.
    Cell 06/2015; 161(7):1619-1632. DOI:10.1016/j.cell.2015.05.028 · 32.24 Impact Factor
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    ABSTRACT: Enhancement of cellular senescence in tumours triggers a stable cell growth arrest and activation of an antitumour immune response that can be exploited for cancer therapy. Currently, there are only a limited number of targeted therapies that act by increasing senescence in cancers, but the majority of them are not selective and also target healthy cells. Here we developed a chemogenomic screening to identify compounds that enhance senescence in PTEN-deficient cells without affecting normal cells. By using this approach, we identified casein kinase 2 (CK2) as a pro-senescent target. Mechanistically, we show that Pten loss increases CK2 levels by activating STAT3. CK2 upregulation in Pten null tumours affects the stability of Pml, an essential regulator of senescence. However, CK2 inhibition stabilizes Pml levels enhancing senescence in Pten null tumours. Taken together, our screening strategy has identified a novel STAT3-CK2-PML network that can be targeted for pro-senescence therapy for cancer.
    Nature Communications 06/2015; 6:7227. DOI:10.1038/ncomms8227 · 11.47 Impact Factor
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    ABSTRACT: Fam20C is an atypical kinase implicated in bio-mineralization and phosphate homeostasis disorders, and has recently been shown to account for the activity of an orphan enzyme ("genuine casein kinase", G-CK) previously characterized for its ability to phosphorylate casein and a plethora of secreted proteins at serine residues specified by the S-x-E/pS motif. Fam20C/G-CK activity is only appreciable in the presence of high Mn(2+) concentration (>1mM), and is negligible if Mn(2+) is replaced by physiological Mg(2+) concentrations. Here we show that sphingosine (but not its biological precursor ceramide) not only stimulates several-fold Fam20C activity in the presence of Mn(2+), but also confers a comparable activity to Fam20C assayed with Mg(2+). Activation by sphingosine is evident using a variety of substrates, and is accounted for by both higher Vmax and decreased Km(ATP), as judged from kinetics run with the β(28-40) substrate peptide and a physiological substrate, BMP-15. Sphingosine also protects Fam20C from thermal inactivation. Consistent with the in vitro results, by treating Fam20C expressing HEK293T cells with myriocin, a potent inhibitor of the sphingosine biosynthetic pathway, the activity of Fam20C released into the conditioned medium is substantially decreased corroborating the concept that sphingosine (or related metabolite(s)) is a co-factor required by Fam20C to optimally display its biological functions. None of the small molecule kinase inhibitors tested so far were able to inhibit Fam20C. Interestingly however fingolimod, an immunosuppressive drug structurally related to sphingosine, used for the treatment of multiple sclerosis, is a powerful activator of Fam20C, both wild type and its pathogenic, loss of function, T268M mutant. This article is part of a Special Issue entitled:Inhibitors of Protein Kinases. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta 04/2015; DOI:10.1016/j.bbapap.2015.04.023 · 4.66 Impact Factor
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    ABSTRACT: Protein kinase CK2 is a tetrameric enzyme composed of two catalytic (α/α') and two regulatory (β) subunits. It has a global prosurvival function, especially in cancer, and represents an attractive therapeutic target. Most CK2 inhibitors available so far are ATP-competitive compounds; however, the possibility to block only the phosphorylation of few substrates has been recently explored, and a compound composed of a Tat cell-penetrating peptide and an active cyclic peptide, selected for its ability to bind to the CK2 substrate E7 protein of human papilloma virus, has been developed [Perea et al., Cancer Res. 2004; 64:7127-7129]. By using a similar chimeric peptide (CK2 modulatory chimeric peptide, CK2-MCP), we performed a study to dissect its molecular mechanism of action and the signaling pathways that it affects in cells. We found that it directly interacts with CK2 itself, counteracting the regulatory and stabilizing functions of the β subunit. Cell treatment with CK2-MCP induces a rapid decrease of the amount of CK2 subunits, as well as of other signaling proteins. Concomitant cell death is observed, more pronounced in tumor cells and not accompanied by apoptotic events. CK2 relocalizes to lysosomes, whose proteases are activated, while the proteasome machinery is inhibited. Several sequence variants of the chimeric peptide have been also synthesized, and their effects compared to those of the parental peptide. Intriguingly, the Tat moiety is essential not only for cell penetration but also for the in vitro efficacy of the peptide. We conclude that this class of chimeric peptides, in addition to altering some properties of CK2 holoenzyme, affects several other cellular targets, causing profound perturbations of cell biology.:Inhibitors of Protein Kinases. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta 04/2015; DOI:10.1016/j.bbapap.2015.04.026 · 4.66 Impact Factor
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    ABSTRACT: In eukaryotic protein synthesis the translation initiation factor 3 (eIF3) is a key player in the recruitment and assembly of the translation initiation machinery. Mammalian eIF3 consists of 13 subunits, including the loosely associated eIF3j subunit that plays a stabilizing role in the eIF3 complex formation and interaction with the 40S ribosomal subunit. By means of both co-immunoprecipitation and mass spectrometry analyses we demonstrate that the protein kinase CK2 interacts with and phosphorylates eIF3j at Ser127. Inhibition of CK2 activity by CX-4945 or down-regulation of the expression of CK2 catalytic subunit by siRNA cause the dissociation of j-subunit from the eIF3 complex as judged from glycerol gradient sedimentation. This finding proves that CK2-phosphorylation of eIF3j is a prerequisite for its association with the eIF3 complex. Expression of Ser127Ala-eIF3j mutant impairs both the interaction of mutated j-subunit with the other eIF3 subunits and the overall protein synthesis. Taken together our data demonstrate that CK2-phosphorylation of eIF3j at Ser127 promotes the assembly of the eIF3 complex, a crucial step in the activation of the translation initiation machinery. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta 04/2015; 1853(7). DOI:10.1016/j.bbamcr.2015.04.004 · 4.66 Impact Factor
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    ABSTRACT: The deubiquitylating enzyme OTUB1 is present in all tissues and targets many substrates, in both the cytosol and nucleus. We found that casein kinase 2 (CK2) phosphorylated OTUB1 at Ser(16) to promote its nuclear accumulation in cells. Pharmacological inhibition or genetic ablation of CK2 blocked the phosphorylation of OTUB1 at Ser(16), causing its nuclear exclusion in various cell types. Whereas we detected unphosphorylated OTUB1 mainly in the cytosol, we detected Ser(16)-phosphorylated OTUB1 only in the nucleus. In vitro, Ser(16)-phosphorylated OTUB1 and nonphosphorylated OTUB1 exhibited similar catalytic activity, bound K63-linked ubiquitin chains, and interacted with the E2 enzyme UBE2N. CK2-mediated phosphorylation and subsequent nuclear localization of OTUB1 promoted the formation of 53BP1 (p53-binding protein 1) DNA repair foci in the nucleus of osteosarcoma cells exposed to ionizing radiation. Our findings indicate that the activity of CK2 is necessary for the nuclear translocation and subsequent function of OTUB1 in DNA damage repair. Copyright © 2015, American Association for the Advancement of Science.
    Science Signaling 04/2015; 8(372):ra35. DOI:10.1126/scisignal.aaa0441 · 6.28 Impact Factor
  • Cinzia Franchin · Mauro Salvi · Giorgio Arrigoni · Lorenzo A Pinna
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    ABSTRACT: 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 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 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 complex 19S conferring the ability to degrade the ubiquitinated proteins are unaffected. This article is part of a Special Issue entitled:Inhibitors of Protein Kinases. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta 04/2015; DOI:10.1016/j.bbapap.2015.04.002 · 4.66 Impact Factor
  • Luca Cesaro · Lorenzo A. Pinna · Mauro Salvi
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    ABSTRACT: Post-translational modification is the most common mechanism of regulating protein function. If phosphorylation is considered a key event in many signal transduction pathways, other modifications must be considered as well. In particular the side chain of lysine residues is a target of different modifications; notably acetylation, methylation, ubiquitylation, sumoylation, neddylation, etc. Mass spectrometry approaches combining highly sensitive instruments and specific enrichment strategies have enabled the identification of modified sites on a large scale. Here we make a comparative analysis of the most representative lysine modifications (ubiquitylation, acetylation, sumoylation and methylation) identified in the human proteome. This review focuses on conserved amino acids, secondary structures preference, subcellular localization of modified proteins, and signaling pathways where these modifications are implicated. We discuss specific differences and similarities between these modifications, characteristics of the crosstalk among lysine post translational modifications, and single nucleotide polymorphisms that could influence lysine post-translational modifications in humans.
    Current Genomics 03/2015; 16(2). DOI:10.2174/1389202916666150216221038 · 2.34 Impact Factor
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    ABSTRACT: Colorectal cancer (CRC) is the fourth leading cause of cancer related death worldwide due to high apoptotic resistance and metastatic potential. Since mutations as well as deregulation of CK1 isoforms contribute to tumor development and tumor progression, CK1 has become an interesting drug target. In this study we show that CK1 isoforms are differently expressed in colon tumor cell lines and that growth of these cell lines can be inhibited by CK1-specific inhibitors. Furthermore, expression of CK1δ and ε is changed in colorectal tumors compared to normal bowel epithelium, and high CK1ε expression levels significantly correlate with prolonged patients’ survival. In addition to changes in CK1δ and ε expression, mutations within exon 3 of CK1δ were detected in colorectal tumors. These mutations influence ATP binding resulting in changes in kinetic parameters of CK1δ. Overexpression of these mutants in HT29 cells alters their ability to grow anchorage independently. Consistent with these results, these CK1δ mutants lead to differences in proliferation rate and tumor size in xenografts due to changes in gene expression, especially in genes involved in regulation of cell proliferation, cell cycle, and apoptosis. In summary, our results provide evidence that changes in the expression levels of CK1 isoforms in colorectal tumors correlate with patients’ survival. Furthermore, CK1 mutants affect growth and proliferation of tumor cells and induce tumor growth in xenografts, leading to the assumption that CK1 isoforms provide interesting targets for the development of novel effective therapeutic concepts to treat colorectal cancer. This article is protected by copyright. All rights reserved.
    International Journal of Cancer 11/2014; 136(12). DOI:10.1002/ijc.29346 · 5.09 Impact Factor
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    ABSTRACT: Polo-like kinase 2 (PLK2) has been recently recognized as the major enzyme responsible for phosphorylation of α-synuclein at S129 in vitro and in vivo, suggesting that this kinase may play a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Moreover PLK2 seems to be implicated in cell division, oncogenesis, and synaptic regulation of the brain. However little is known about the phosphoproteome generated by PLK2 and, consequently the overall impact of PLK2 on cellular signaling. To fill this gap we exploited an approach based on in vitro kinase assay and quantitative phosphoproteomics. A proteome-derived peptide library obtained by digestion of undifferentiated human neuroblastoma cell line was exhaustively dephosphorylated by lambda phosphatase followed by incubation with or without PLK2 recombinant kinase. Stable isotope labeling based quantitative phosphoproteomics was applied to identify the phosphosites generated by PLK2. A total of 98 unique PLK2-dependent phosphosites from 89 proteins were identified by LC-MS/MS. Analysis of the primary structure of the identified phosphosites allowed the detailed definition of the kinase specificity and the compilation of a list of potential PLK2 targets among those retrieved in PhosphositePlus, a curated database of in cell/vivo phosphorylation sites.
    PLoS ONE 10/2014; DOI:10.1371/journal.pone.0111018 · 3.23 Impact Factor
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    ABSTRACT: Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in Cftr(F508del) homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation.
    Autophagy 10/2014; 10(11). DOI:10.4161/15548627.2014.973737 · 11.75 Impact Factor
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    ABSTRACT: CK2 is an extremely pleiotropic Ser/Thr protein kinase, responsible for the generation of a large proportion of the human phosphoproteome and implicated in a wide variety of biological functions. CK2 plays a global role as an anti-apoptotic agent, a property which is believed to partially account for the addiction of many cancer cells to high CK2 levels. To gain information about the CK2 targets whose phosphorylation is primarily implicated in its pro-survival signaling advantage has been taken of quinalizarin (QZ) a cell permeable fairly specific CK2 inhibitor, previously shown to be able to block endogenous CK2 triggering an apoptotic response. HEK-293 T cells either treated or not for 3 hours with 50 μM QZ were exploited to perform a quantitative SILAC phosphoproteomic analysis of phosphosites readily responsive to QZ treatment. Our analysis led to the identification of 4883 phosphosites, belonging to 1693 phosphoproteins. 71 phosphosites (belonging to 47 proteins) underwent a 50% or more decreased occupancy upon QZ treatment. Almost 50% of these fulfilled the typical consensus sequence recognized by CK2 (S/T-x-x-E/D/pS) and in several cases were validated as bona fide substrates of CK2 either based on data in the literature or by performing in vitro phosphorylation experiments with purified proteins. The majority of the remaining phosphosites drastically decreased upon QZ treatment display the pS/T-P motif typical of proline directed protein kinases and a web logo extracted from them differentiates from the web logo extracted from all the proline directed phosphosites quantified during our analysis (1151 altogether). A paradoxical outcome of our study was the detection of 116 phosphosites (belonging to 92 proteins altogether) whose occupancy is substantially increased (50% or more), rather than decreased by QZ treatment: 40% of these display the typical motif recognized by proline directed kinases, while about 25% fulfill the CK2 consensus. Collectively taken our data on one side have led to the disclosure of a subset of CK2 targets which are likely to be implicated in the early steps of CK2 signaling counteracting apoptosis, on the other they provide evidence for the existence of side and off-target effects of the CK2 inhibitor quinalizarin, paving the road toward the detection of other kinases susceptible to this compound. This article is part of a Special Issue entitled: Medical Proteomics.
    Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 09/2014; 1854(6). DOI:10.1016/j.bbapap.2014.09.017 · 2.75 Impact Factor
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    Andrea Venerando · Maria Ruzzene · Lorenzo A Pinna
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    ABSTRACT: The term 'casein kinase' has been widely used for decades to denote protein kinases sharing the ability to readily phosphorylate casein in vitro. These fall into three main classes: two of them, later renamed as protein kinases CK1 (casein kinase 1, also known as CKI) and CK2 (also known as CKII), are pleiotropic members of the kinome functionally unrelated to casein, whereas G-CK, or genuine casein kinase, responsible for the phosphorylation of casein in the Golgi apparatus of the lactating mammary gland, has only been identified recently with Fam20C [family with sequence similarity 20C; also known as DMP-4 (dentin matrix protein-4)], a member of the four-jointed family of atypical protein kinases, being responsible for the phosphorylation of many secreted proteins. In hindsight, therefore, the term 'casein kinase' is misleading in every instance; in the case of CK1 and CK2, it is because casein is not a physiological substrate, and in the case of G-CK/Fam20C/DMP-4, it is because casein is just one out of a plethora of its targets, and a rather marginal one at that. Strikingly, casein kinases altogether, albeit representing a minimal proportion of the whole kinome, appear to be responsible for the generation of up to 40-50% of non-redundant phosphosites currently retrieved in human phosphopeptides database. In the present review, a short historical explanation will be provided accounting for the usage of the same misnomer to denote three unrelated classes of protein kinases, together with an update of our current knowledge of these pleiotropic enzymes, sharing the same misnomer while playing very distinct biological roles.
    Biochemical Journal 06/2014; 460(2):141-56. DOI:10.1042/BJ20140178 · 4.40 Impact Factor
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    ABSTRACT: Akt (also known as PKB) is a survival kinase frequently up-regulated in cancer; three isoforms of Akt exist, and among them Akt1 and Akt2 are the most widely and highly expressed. They share the same structure and activation mechanism and have many overlapping functions; nevertheless isoform-specific roles and substrates have been reported, which are expected to rely on sequence diversities. In particular, a special role in differentiating Akt1 and Akt2 isoforms has been assigned to the linker region, a short segment between the PH and the catalytic domains. We have previously found that a residue in the linker region (Ser129) is directly phosphorylated by protein kinase CK2 in Akt1; the phosphorylation of the homologous residue in Akt2 (Ser131) has never been analyzed. Here we show that Akt2, endogenously or ectopically expressed in different cell lines, is not phosphorylated on Ser131 by CK2, while in vitro recombinant Akt2 is a CK2 substrate. These data support the hypothesis that in vivo a steric hindrance occurs which prevents the access to the CK2 site. Additionally, we have found that Ser129 phosphorylation is involved in the recognition of the Akt1-specific substrate palladin; this observation provides an explanation of why Akt2, lacking Ser131 phosphorylation in the linker region, has a low efficiency in targeting palladin. CK2-dependent phosphorylation is therefore a crucial event which, discriminating between Akt1 and Akt2, can account for different substrate specificity, and, more in general, for fine tuning of Akt activity in the control of isoform-dependent processes.
    Biochimica et Biophysica Acta 04/2014; 1843(9). DOI:10.1016/j.bbamcr.2014.04.020 · 4.66 Impact Factor
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    ABSTRACT: The motif "SYDE", incorporating the protein kinase CK2 consensus sequence (S-x-x-E) has been found to be phosphorylated at both its serine and tyrosine residues in several proteins. Of special interest is the case of cystic fibrosis Transmembrane-conductance Regulator (CFTR), where this motif is close to the residue (F508), whose deletion is the by far commonest cause of cystic fibrosis. Intriguingly, however, CFTR S511 cannot be phosphorylated by CK2 to any appreciable extent. Using a number of peptide substrates encompassing the CFTR "SYDE" site we have recently shown that: (1) failure of CK2 to phosphorylate the S(511)YDE motif is due to the presence of Y512; (2) CK2 readily phosphorylates S511 if Y512 is replaced by a phospho-tyrosine; (3) the Src family protein tyrosine kinase Lyn phosphorylates Y512 in a manner that is enhanced by the deletion of F508. These data, in conjunction with the recent observation that by inhibiting CK2 the degradation of F508delCFTR is reduced, lead us to hypothesize that the hierarchical phosphorylation of the motif SYDE by the concerted action of protein tyrosine kinases and CK2 is one of the mechanisms that cooperate to the premature degradation of F508delCFTR.
    Cellular and Molecular Life Sciences CMLS 02/2014; 71(12). DOI:10.1007/s00018-014-1581-8 · 5.81 Impact Factor
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    ABSTRACT: Homeodomain-interacting protein kinase 2 (HIPK2) is a Ser/Thr kinase controlling cell proliferation and survival, whose investigation has been hampered by the lack of specific inhibitors able to dissect its cellular functions. SB203580, a p38 MAP kinase inhibitor, has been used as a tool to inhibit HIPK2 in cells, but here we show that its efficacy as HIPK2 inhibitor is negligible (IC50>40 µM). In contrast by altering the scaffold of the promiscuous CK2 inhibitor TBI a new class of HIPK2 inhibitors has been generated. One of these, TBID, displays toward HIPK2 unprecedented efficacy (IC50 = 0.33 µM) and selectivity (Gini coefficient 0.592 out of a panel of 76 kinases). The two other members of the HIPK family, HIPK1 and HIPK3, are also inhibited by TBID albeit less efficiently than HIPK2. The mode of action of TBID is competitive with respect to ATP, consistent with modelling. We also provide evidence that TBID is cell permeable by showing that HIPK2 activity is reduced in cells treated with TBID, although with an IC50 two orders of magnitude higher (about 50 µM) than in vitro.
    PLoS ONE 02/2014; 9(2):e89176. DOI:10.1371/journal.pone.0089176 · 3.23 Impact Factor
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    ABSTRACT: Protein kinase CK2 is a pleiotropic serine/threonine kinase responsible for the generation of a substantial proportion of the human phosphoproteome. CK2 is generally found as a tetramer with two catalytic, α and α´ and two non catalytic β subunits. CK2α C-terminal tail phosphorylation is regulated during the mitotic events and the absence of these phosphosites in α´ suggests an isoform specialization. We used a proteomic approach to identify proteins specifically phosphorylated by a CK2α phosphomimetic mutant, CK2αT344ET360ES362ES370E (CK2α4E), in human neuroblastoma SKNBE cellular extract. One of these proteins is Lysine-Specific Demethylase 1 (LSD1 or KDM1A), an important player of the epigenetic machinery. LSD1 is a FAD-dependent amine oxidase and promotes demethylation of lysine 4 and lysine 9 of mono- and di-methylated histone H3. We found that LSD1 is a new substrate and an interacting partner of protein kinase CK2. Three CK2 phosphosites, (Ser131, Ser137 and Ser166) in the N-terminal region of LSD1 have been identified. This domain is found in all chordates but not in more ancient organisms and it is not essential for LSD1 catalytic event while it could modulate the interaction with CK2 and with other partners in gene repressing and activating complexes. Our data support the view that the phosphorylation of the N-terminal domain by CK2 may represent a mechanism for regulating histone methylation, disclosing a new role for protein kinase CK2 in epigenetics.
    Biochimica et Biophysica Acta 01/2014; 1844(4). DOI:10.1016/j.bbapap.2014.01.014 · 4.66 Impact Factor

Publication Stats

14k Citations
1,696.24 Total Impact Points


  • 1997–2015
    • INO - Istituto Nazionale di Ottica
      Florens, Tuscany, Italy
  • 1965–2015
    • University of Padova
      • • Department of Biomedical Sciences - DSB
      • • Interdepartmental Research Centre for Innovative Biotechnologies CRIBI
      • • Department of Medicine DIMED
      Padua, Veneto, Italy
  • 2013
    • University-Hospital of Padova
      Padua, Veneto, Italy
  • 2002–2012
    • Venetian Institute of Molecular Medicine
      Padua, Veneto, Italy
  • 2007
    • The Neurosciences Institute
      La Jolla, California, United States
  • 2006
    • University of Florence
      Florens, Tuscany, Italy
  • 2001–2006
    • It-Robotics
      Vicenza, Veneto, Italy
  • 2005
    • University of Zurich
      Zürich, Zurich, Switzerland
  • 2003
    • Autonomous University of Barcelona
      • Department of Biochemistry and Molecular Biology
      Cerdanyola del Vallès, Catalonia, Spain
  • 1968–1999
    • National Research Council
      Roma, Latium, Italy
  • 1998
    • University of Cologne
      Köln, North Rhine-Westphalia, Germany
  • 1996
    • University of Melbourne
      • School of Chemistry
      Melbourne, Victoria, Australia
  • 1995
    • University of Minnesota Duluth
      • Laboratory Medicine and Pathology
      Duluth, Minnesota, United States
  • 1993–1995
    • Universität des Saarlandes
      • Fachbereich Medizinische Biochemie und Molekularbiologie
      Saarbrücken, Saarland, Germany
  • 1994
    • Universitätsklinikum des Saarlandes
      Homburg, Saarland, Germany
  • 1991
    • University of Glasgow
      • Division of Biochemistry
      Glasgow, SCT, United Kingdom
  • 1985–1986
    • University of Zagreb
      Zagrabia, Grad Zagreb, Croatia
  • 1967
    • Johns Hopkins Medicine
      • Department of Biological Chemistry
      Baltimore, Maryland, United States