RESUMO
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.
Assuntos
Caseína Quinase I/química , Caseína Quinase I/metabolismo , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Sequência de Aminoácidos , Proteínas Sanguíneas/metabolismo , Caseína Quinase I/genética , Adesão Celular , Movimento Celular , Proteínas do Líquido Cefalorraquidiano/metabolismo , Proteínas da Matriz Extracelular/genética , Técnicas de Inativação de Genes , Ontologia Genética , Humanos , Dados de Sequência Molecular , Fosfoproteínas/análise , Via Secretória , Especificidade por SubstratoRESUMO
CK2 is a constitutively active protein kinase that assuring a constant level of phosphorylation to its numerous substrates supports many of the most important biological functions. Nevertheless, its activity has to be controlled and adjusted in order to cope with the varying needs of a cell, and several examples of a fine-tune regulation of its activity have been described. More importantly, aberrant regulation of this enzyme may have pathological consequences, e.g. in cancer, chronic inflammation, neurodegeneration, and viral infection. Our review aims at summarizing our current knowledge about CK2 regulation. In the first part, we have considered the most important stimuli shown to affect protein kinase CK2 activity/expression. In the second part, we focus on the molecular mechanisms by which CK2 can be regulated, discussing controversial aspects and future perspectives.
Assuntos
Caseína Quinase II/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Transdução de Sinais , Viroses/enzimologia , Animais , Humanos , Inflamação/enzimologiaRESUMO
Protein phosphorylation is the most frequent post-translational modification by which the properties of eukaryotic proteins can be reversibly modified. In humans, over 500 protein kinases generate a huge phosphoproteome including more than 200,000 individual phosphosites, a figure which is still continuously increasing. The in vivo selectivity of protein kinases is the outcome of a multifaceted and finely tuned process where numerous factors play an integrated role. To gain information about the actual contribution to this process of local features that reflect the interaction of the protein targets with the catalytic site of the kinases, the prevalence of the commonest motifs determining the consensus sequence of Ser/Thr-specific kinases has been examined in the whole human phosphoproteome and in the phosphoproteomes generated by a panel of the 47 most pleiotropic protein kinases. Our analysis shows that: (1) most phosphosites do conform to at least one of the motifs considered, with a substantial proportion conforming to two or more of them; (2) some motifs, with special reference to the one recognized by protein kinase CK2 (pS/pT-x-x-E/D) are very promiscuous, being abundantly represented also at the phosphosites of all the other protein kinases considered; (3) by contrast, other phosphorylated motifs, notably pS/pT-P, pS/pT-Q and pS-x-E, are more discriminatory and selective, being nearly absent in the phosphosites that are not attributable to certain categories of kinases. The information provided will prove helpful to make reliable inferences based on the manual inspection of individual phosphosites.
Assuntos
Motivos de Aminoácidos/genética , Fosforilação/genética , Processamento de Proteína Pós-Traducional/genética , Proteoma/genética , Caseína Quinase II/genética , Domínio Catalítico/genética , Humanos , Fosfotransferases/genéticaRESUMO
Viable clones of C2C12 myoblasts where both catalytic subunits of protein kinase CK2 had been knocked out by the CRISPR/Cas9 methodology have recently been generated, thus challenging the concept that CK2 is essential for cell viability. Here we present evidence that these cells are still endowed with a residual "CK2-like" activity that is able to phosphorylate Ser-13 of endogenous CDC37. Searching for a molecular entity accounting for such an activity we have identified a band running slightly ahead of CK2α' on SDS-PAGE. This band is not detectable by in-gel casein kinase assay but it co-immuno-precipitates with the ß-subunit being downregulated by specific CK2α' targeting siRNA treatment. Its size and biochemical properties are consistent with those of CK2α' mutants deleted upstream of Glu-15 generated during the knockout process. This mutant sheds light on the role of the CK2 N-terminal segment as a regulator of activity and stability. Comparable cytotoxic efficacy of two selective and structurally unrelated CK2 inhibitors support the view that survival of CK2α/α'-/- cells relies on this deleted form of CK2α', whose discovery provides novel perspectives about the biological role of CK2.
Assuntos
Caseína Quinase II/química , Caseína Quinase II/metabolismo , Domínio Catalítico , Deleção de Sequência , Sequência de Aminoácidos , Animais , Caseína Quinase II/deficiência , Linhagem Celular , Sobrevivência Celular , Camundongos Knockout , Peptídeos/metabolismo , Fosforilação , Fosfosserina/metabolismo , Estabilidade Proteica , Especificidade por SubstratoRESUMO
A short biographical sketch of Professor David Shugar, the "father of the IPK conferences," is presented, focusing on the growing interest of this eminent scientist for protein kinases and his farsighted perception of the extraordinary therapeutic potential of protein kinase inhibitors, after his discovery in 1986 that 5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole effects are mediated by inhibition of protein kinase CK2. This led David Shugar to conceive the idea of organizing a periodic international conference on protein kinase inhibitors ("IPK conference"). The first conference was held in 1998 and the 10th one under the auspices of International Union of Biochemistry and Molecular Biology in September 2019. David Shugar died at the age of 100 in 2015, shortly after having organized the eight IPK conference.
Assuntos
Inibidores de Proteínas Quinases , Proteínas Quinases , Congressos como Assunto , História do Século XX , História do Século XXI , Humanos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/históriaRESUMO
HSJ1 (DNAJB2), a member of the DNAJ family of molecular chaperones, is a key player in neuronal proteostasis maintenance. It binds ubiquitylated proteins through its Ubiquitin Interacting Motifs (UIMs) and facilitates their delivery to the proteasome for degradation. Mutations in the DNAJB2 gene lead to inherited neuropathies such as Charcot-Marie-Tooth type-2, distal hereditary motor neuropathies, spinal muscular atrophy with parkinsonism and the later stages can resemble amyotrophic lateral sclerosis. HSJ1 overexpression can reduce aggregation of neurodegeneration-associated proteins in vitro and in vivo; however, the regulation of HSJ1 function is little understood. Here we show that CK2, a ubiquitous and constitutively active protein kinase, phosphorylates HSJ1 within its second UIM, at the dominant site Ser250 and the hierarchical site Ser247. A phospho-HSJ1 specific antibody confirmed phosphorylation of endogenous HSJ1a and HSJ1b. A tandem approach of phospho-site mutation and treatment with CK2 specific inhibitors demonstrated that phosphorylation at these sites is accompanied by a reduced ability of HSJ1 to bind ubiquitylated clients and to exert its chaperone activity. Our results disclose a novel interplay between ubiquitin- and phosphorylation-dependent signalling, and represent the first report of a regulatory mechanism for UIM-dependent function. They also suggest that CK2 inhibitors could release the full neuroprotective potential of HSJ1, and deserve future interest as therapeutic strategies for neurodegenerative disease.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Proteínas de Choque Térmico HSP40/genética , Chaperonas Moleculares/genética , Atrofia Muscular Espinal/genética , Neurônios/metabolismo , Sequência de Aminoácidos , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Doença de Charcot-Marie-Tooth/fisiopatologia , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Atrofia Muscular Espinal/fisiopatologia , Mutação , Neurônios/patologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Domínios Proteicos/genética , Dobramento de Proteína , Ubiquitina/genéticaRESUMO
CK2 denotes a ubiquitous and pleiotropic protein kinase whose holoenzyme is composed of two catalytic (α and/or α') and two regulatory ß subunits. The CK2 consensus sequence, S/T-x-x-D/E/pS/pT is present in numerous phosphosites, but it is not clear how many of these are really generated by CK2. To gain information about this issue, advantage has been taken of C2C12 cells entirely deprived of both CK2 catalytic subunits by the CRISPR/Cas9 methodology. A comparative SILAC phosphoproteomics analysis reveals that, although about 30% of the quantified phosphosites do conform to the CK2 consensus, only one-third of these are substantially reduced in the CK2α/α'(-/-) cells, consistent with their generation by CK2. A parallel study with C2C12 cells deprived of the regulatory ß subunit discloses a role of this subunit in determining CK2 targeting. We also find that phosphosites notoriously generated by CK2 are not fully abrogated in CK2α/α'(-/-) cells, while some phosphosites unrelated to CK2 are significantly altered. Collectively taken our data allow to conclude that the phosphoproteome generated by CK2 is not as ample and rigidly pre-determined as it was believed before. They also show that the lack of CK2 promotes phosphoproteomics perturbations attributable to kinases other than CK2.
Assuntos
Caseína Quinase II/metabolismo , Fosfopeptídeos/metabolismo , Animais , Caseína Quinase II/genética , Linhagem Celular , Deleção de Genes , Técnicas de Inativação de Genes , Camundongos , Fosfopeptídeos/análise , Fosforilação , Proteômica/métodosRESUMO
Protein kinase CK2 (CK2) is a highly conserved and ubiquitous kinase is involved in crucial biological processes, including proliferation, migration, and differentiation. CK2 holoenzyme is a tetramer composed by two catalytically active (α/α') and two regulatory (ß) subunits and exerts its function on a broad range of targets. In the brain, it regulates different steps of neurodevelopment, such as neural differentiation, neuritogenesis, and synaptic plasticity. Interestingly, CK2 mutations have been recently linked to neurodevelopmental disorders; however, the functional requirements of the individual CK2 subunits in neurodevelopment have not been yet investigated. Here, we disclose the role of CK2 on the migration and adhesion properties of GN11 cells, an established model of mouse immortalized neurons, by different in vitro experimental approaches. Specifically, the cellular requirement of this kinase has been assessed pharmacologically and genetically by exploiting CK2 inhibitors and by generating subunit-specific CK2 knockout GN11 cells (with a CRISPR/Cas9-based approach). We show that CK2α' subunit has a primary role in increasing cell adhesion and reducing migration properties of GN11 cells by activating the Akt-GSK3ß axis, whereas CK2α subunit is dispensable. Further, the knockout of the CK2ß regulatory subunits counteracts cell migration, inducing dramatic alterations in the cytoskeleton not observed in CK2α' knockout cells. Collectively taken, our data support the view that the individual subunits of CK2 play different roles in cell migration and adhesion properties of GN11 cells, supporting independent roles of the different subunits in these processes.
Assuntos
Caseína Quinase II/genética , Neurônios/citologia , Animais , Caseína Quinase II/metabolismo , Adesão Celular , Linhagem Celular , Movimento Celular , Técnicas de Silenciamento de Genes , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos , Mutação , Neurônios/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
Protein kinases constitute one of the largest gene families and control many aspects of cellular life. In retrospect, the first indication for their existence was reported 130 years ago when the secreted protein, casein, was shown to contain phosphate. Despite its identification as the first phosphoprotein, the responsible kinase has remained obscure. This conundrum was solved with the discovery of a novel family of atypical protein kinases that are secreted and appear to phosphorylate numerous extracellular proteins, including casein. Fam20C, the archetypical member, phosphorylates secreted proteins within Ser-x-Glu/pSer motifs. This discovery has solved a 130-year-old mystery and has shed light on several human disorders of biomineralization.
Assuntos
Proteínas Quinases/metabolismo , Animais , Humanos , Proteínas Quinases/genéticaRESUMO
A SILAC analysis performed on HEK-293T cells either treated or not for 3h 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 subunit 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.
Assuntos
Antraquinonas/farmacologia , Caseína Quinase II/metabolismo , Fosfopeptídeos/biossíntese , Inibidores de Proteínas Quinases/farmacologia , Caseína Quinase II/antagonistas & inibidores , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Fosfopeptídeos/metabolismo , ProteômicaRESUMO
Protein kinase CK2 is a tetrameric enzyme composed of two catalytic (α/α') and two regulatory (ß) 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 ß 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. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.
Assuntos
Peptídeos Penetradores de Células/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Peptídeos Penetradores de Células/química , Humanos , Proteínas E7 de Papillomavirus/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Proteínas Recombinantes de Fusão/química , Transdução de Sinais/efeitos dos fármacos , Especificidade por SubstratoRESUMO
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 Mn2+ concentration (>1 mM), and is negligible if Mn2+ is replaced by physiological Mg2+ concentrations. Here we show that sphingosine (but not its biological precursor ceramide) not only stimulates several-fold Fam20C activity in the presence of Mn2+, but also confers a comparable activity to Fam20C assayed with Mg2+. Activation by sphingosine is evident using a variety of substrates, and is accounted for by both higher Vmax and decreased KmATP, as judged from kinetics run with the ß(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.
Assuntos
Caseína Quinase I/genética , Proteínas da Matriz Extracelular/genética , Esclerose Múltipla/genética , Esfingosina/biossíntese , Sequência de Aminoácidos , Caseína Quinase I/química , Proteínas da Matriz Extracelular/química , Ácidos Graxos Monoinsaturados/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Esclerose Múltipla/enzimologia , Fosforilação , Esfingosina/antagonistas & inibidores , Esfingosina/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
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.
Assuntos
Caseína Quinase II/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Fosfosserina/metabolismo , Biossíntese de Proteínas , Caseína Quinase II/antagonistas & inibidores , Inativação Gênica/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Mutação/genética , Naftiridinas/farmacologia , Fenazinas , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Especificidade por Substrato/efeitos dos fármacosRESUMO
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 of three or four glutamic or aspartic acid residues, a new family of bisubstrate 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 bisubstrate 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 α-helixC that recognize the acidic determinant at position n+3. Compared with the parent inhibitor (K137), K137-E4 is severalfold more potent (IC50 25 compared with 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 the cell and to inhibit endogenous CK2, its pro-apoptotic efficacy being negligible compared with 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. On the basis of the cellular responses to K137-E4, we conclude that ecto-CK2 is implicated in cell motility, whereas its contribution to the pro-survival role of CK2 is negligible.
Assuntos
Benzimidazóis/química , Inibidores de Proteínas Quinases/química , Proteínas Quinases/química , Estrutura Secundária de Proteína/efeitos dos fármacos , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Benzimidazóis/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Cinética , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/metabolismoRESUMO
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 specificities, and, more in general, for fine tuning of Akt activity in the control of isoform-dependent processes.
Assuntos
Caseína Quinase II/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Dissulfetos/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Fosfosserina/metabolismo , Desnaturação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Recombinantes/metabolismoRESUMO
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 ß 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.
Assuntos
Caseína Quinase II/metabolismo , Epigênese Genética , Histona Desmetilases/metabolismo , Histonas/metabolismo , Neurônios/metabolismo , Subunidades Proteicas/metabolismo , Sequência de Aminoácidos , Animais , Caseína Quinase II/genética , Linhagem Celular Tumoral , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Histona Desmetilases/genética , Histonas/genética , Humanos , Lisina/genética , Lisina/metabolismo , Metilação , Dados de Sequência Molecular , Mutação , Neurônios/citologia , Fosforilação , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/genética , Serina/metabolismo , Transdução de SinaisRESUMO
Colorectal cancer (CRC) is the fourth leading cause of cancer related death worldwide due to high apoptotic resistance and metastatic potential. Because 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 É is changed in colorectal tumors compared to normal bowel epithelium, and high CK1É expression levels significantly correlate with prolonged patients' survival. In addition to changes in CK1δ and É 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.
Assuntos
Caseína Quinase 1 épsilon/genética , Caseína Quinase Idelta/genética , Neoplasias Colorretais/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Idoso , Animais , Western Blotting , Caseína Quinase 1 épsilon/metabolismo , Caseína Quinase Idelta/metabolismo , Adesão Celular/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Perfilação da Expressão Gênica , Células HT29 , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Carga Tumoral/genéticaRESUMO
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.
RESUMO
It has been proposed that dual inhibitors of protein kinases CK2 and PIM-1 are tools particularly valuable to induce apoptosis of cancer cells, a property, however, implying cell permeability, which is lacking in the case of selective CK2/PIM-1 inhibitors developed so far. To fill this gap, we have derivatized the scaffold of the promiscuous CK2 inhibitor TBI with a deoxyribose moiety, generating TDB, a selective, cell-permeable inhibitor of CK2 and PIM-1. Here, we shed light on the structural features underlying the potency and narrow selectivity of TDB by exploiting a number of TDB analogs and by solving the 3D structure of the TDB/CK2 complex at 1.25 Å resolution, one of the highest reported so far for this kinase. We also show that the cytotoxic efficacy of TDB is almost entirely due to apoptosis, is accompanied by parallel inhibition of cellular CK2 and PIM-1, and is superior to both those observed combining individual inhibitors of CK2 and PIM-1 and by treating cells with the CK2 inhibitor CX4945. These data, in conjunction with the observations that cancer cells are more susceptible than non-cancer cells to TDB and that such a sensitivity is maintained in a multi-drug resistance background, highlight the pharmacological potential of this compound.
Assuntos
Benzimidazóis/química , Benzimidazóis/farmacologia , Caseína Quinase II/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Benzimidazóis/farmacocinética , Caseína Quinase II/química , Caseína Quinase II/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Proliferação de Células/efeitos dos fármacos , Halogenação , Humanos , Cinética , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Proto-Oncogênicas c-pim-1/química , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Relação Estrutura-AtividadeRESUMO
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.