1,721,006 research outputs found
Isoform specific phosphorylation of p53 Ser-20 by CK1 is dictated by both a local consensus and a remote docking site
Full text linkProtein kinase CK1 makes up a small independent group of Ser/Thr protein kinases. So far, seven different CK1 isoforms (α, β, γ1, γ2, γ3, δ, and ε) have been characterized in mammals. In addition, alternate splicing generates several variants of their coding genes. While the crystal structure of the δ and γ isoforms have been solved, structural information about CK1α is not yet available. Members of the CK1 family have been implicated in a variety of biological functions including chromosome segregation, spindle formation, circadian rhythm, nuclear import, Wnt pathway and apoptosis. Deregulation of CK1 has been described in several diseases such as neurodegenerative diseases, including Alzheimer’s and Parkinson’s disorders, the familial advanced sleep phase syndrome, hepatitis C, leishmaniasis, and cancer. Recently, silencing of CK1 has been associated with disappearance of metastases formation.
Although the list of substrates phosphorylated by CK1 has been growing in the last decade, features underlying substrate specificity by CK1 are still a matter of debate and investigation. Thus, it is difficult to predict residues phosphorylated by CK1 in its potential substrates and evaluate the actual contribution of CK1 to available phosphoproteome data bases. Early studies mostly performed with artificial substrates revealed that CK1 is a "phosphate directed" protein kinase, able to phosphorylate with high efficiency Ser/Thr residues specified by a pre-phosphorylated side chain (either phospho-Ser or phospho-Thr) at position n-3 whose function is not as efficiently replaced by the negatively charged side chains of glutamic and aspartic acids. This means that the requirement of a priming phosphorylation by another kinase restricted CK1 to exert its activity only in the hierarchical phosphorylation of substrates. However, it soon became clear that CK1 not necessarily required a "primed" substrate, since many of its physiological substrates do not contain phosphorylated residues; rather, it often acts as a priming kinase, its intervention being required to generate the consensus sequence for phosphate directed kinases, with special reference to GSK3. Examining the growing list of non-primed CK1 sites it appears that only a few of these are specified by clusters of acidic residues, shown to be able to effectively replace the individual phosphorylated determinant at position n-3, whereas the majority are to be considered as non-canonical sites whose targeting by CK1 depends on different and somewhat elusive local determinants. Although these atypical local determinants seem to be important for the phosphorylation of full length protein substrates, they are not sufficient alone to confer high affinity to the kinase, as revealed by high Km values of the derived peptide substrates (close to the millimolar range) as compared to those calculated with whole proteins. In the case of β-catenin, the differences between kinetic parameters observed on whole protein with respect to derived peptide used as substrates in vitro were shown to be accounted for by a remote docking site located in the first Armadillo repeat of β-catenin whose removal caused an increase in the Km value of three order of magnitude.
The objective of this thesis has been the characterization of substrate specificity of different CK1 isoforms on two different substrates, p53 and adenomatous polyposis coli protein (APC), which play key roles in signal pathways. The use of both recombinant proteins and synthetic peptides reproducing the primary sequence of the phosphoacceptor segments of these two proteins, allowed us to (i) gain deeper insight into the actual ability of different CK1 isoforms to phosphorylate individual residues in the N-terminus of p53 and identify local determinants on p53 and a conserved remote docking site on protein kinase CK1 which is responsible for high affinity binding of p53 with CK1δ/ε and α but not with the γ isoforms; (ii) demonstrate that APC multiple phosphorylation can occur through a cooperative mechanism involving protein kinase CK1 and GSK3.
Furthermore, in collaboration with Giorgio Cozza (Padua), we have identified through virtual-screening and in vitro enzymology approaches, new powerful protein kinase CK1 inhibitors that show high selectivity for the δ isoform of CK1.
The last part of this thesis concerns the study of p13, a small accessory protein of Human T-cell Leukemia Virus type 1. The chemical synthesis of full length p13 allowed us to characterize structurally and functionally this protein. Unpublished data on possible role of phosphorylation in the regulation of p13 function are also reported.La proteina chinasi CK1 rappresenta un piccolo ed indipendente gruppo delle Ser/Thr-proteine chinasi. Ad ora, nei mammiferi sono state caratterizzate sette diverse isoforme di CK1 (α, β, γ1, γ2, γ3, δ, and ε). Inoltre, lo splicing alternativo dei geni che le codificano genera diverse varianti delle isoforme di CK1. Se da un lato le strutture cristallografiche delle isoforme δ e γ sono state risolte, non sono invece ancora disponibili informazioni strutturali dell’isoforma α. I membri di questa famiglia di chinasi partecipano a diverse funzioni biologiche tra cui si possono annoverare la segregazione cromosomica, la formazione del fuso mitotico, il ritmo circadiano, il trasporto nel nucleo, la via del segnale Wnt e l’apoptosi. Una regolazione alterata di CK1 è stata associata a diverse malattie, quali malattie neurodegenerative, tra cui le sindromi di Alzheimer e di Parkinson, la sindrome ereditaria delle fasi del sonno, l’epatite di tipo C, la leishmaniosi ed infine il cancro. Recentemente, il silenziamento trascrizionale di CK1 è stato associato alla scomparsa della formazione di metastasi.
Sebbene la lista di substrati fosforilati da CK1 sia aumentata nell’ultimo decennio, le caratteristiche che contraddistinguono la specificità di substrato di CK1 sono ancora oggetto di discussione e studio. Di conseguenza, è difficile predire siti bersaglio di CK1 nei suoi potenziali substrati e valutare l’effettivo contributo di CK1 al fosfoproteoma oggi disponibile. I primi studi, per la maggior parte condotti su substrati artificiali, classificarono CK1 come una protein chinasi “fosfato-diretta” data la sua capacità di fosforilare efficacemente residui di serina o treonina caratterizzati da un residuo pre-fosforilato (fosfo-serina o fosfo-treonina) in posizione n-3, la cui funzione non viene efficacemente mimata dalle catene laterali cariche negativamente di un residuo di acido glutammico o aspartico. Questo significa che la necessità di una fosforilazione che faccia da “priming” da parte di un’altra chinasi, relegava CK1 ad esercitare la propria attività solo all’interno di meccanismi gerarchici di fosforilazione di substrati. Tuttavia, presto divenne chiaro che CK1 non necessariamente richiede un substrato pre-fosforilato, dal momento che molti dei suoi substrati fisiologici non contengono residui fosforilati, ma all’opposto svolge la funzione di chinasi “priming”, essendo necessario il suo intervento per generare sequenze consenso per altre chinasi fosfato-dirette, in particolare per GSK3. Analizzando la crescente lista di siti CK1 non pre-fosforilati, appare chiaro che solo alcuni sono caratterizzati da sequenze di residui acidi, che si sono rivelati capaci di sostituire efficacemente il singolo residuo pre-fosforilato in posizione n-3, mentre la maggior parte di essi sono da considerarsi siti non canonici la cui fosforilazione da parte di CK1 dipende da determinanti locali diversi e in alcuni casi sfuggenti. Sebbene questi determinanti locali atipici sembrano essere importanti per la fosforilazione di substrati proteici, non sono tuttavia sufficienti da soli a conferire una alta affinità per la chinasi, come dimostrano alti valori di Km ottenuti con substrati peptidici che riproducono porzioni della proteina (nell’intervallo del millimolare) se comparati con quelli ottenuti con la proteina intera. Nel caso della β-catenina, le differenze tra i parametri cinetici osservati con la proteina intera e quelli ottenuti con peptidi che riproducono parte di essa usati come substrati in esperimenti in vitro, si sono dimostrate riconducibili a siti di legame remoti situati nel primo Armadillo repeat di β-catenina, la rimozione dei quali causa un aumento di tre ordini di grandezza del valore di Km.
L’obbiettivo di questa tesi è stato quello di caratterizzare la specificità di substrato della diverse isoforme di CK1 prendendo come esempio due substrati di CK1, p53 e la proteina adenomatous polyposis coli (APC), che rivestono un ruolo chiave nelle vie del segnale cellulare. L’utilizzo di proteine ricombinanti e di peptidi sintetici che riproducono la sequenza primaria della porzione fosforilabile di questi due substrati proteici, ci ha permesso di (i) ottenere maggiori informazioni sulla reale capacità delle diverse isoforme di CK1 di fosforilare singoli residui nella porzione N-terminale di p53 e di identificare sia i determinanti locali di fosforilazione su p53 sia un sito di legame legame remoto conservato sulla protein chinasi CK1 responsabile dell’alta affinità di legame di p53 con CK1δ/ε e α ma non con le isoforme γ; (ii) dimostrare che la fosforilazione multipla di APC può avvenire attraverso un meccanismo cooperativo che coinvolge le protein chinasi CK1 e GSK3.
Inoltre, in collaborazione con Giorgio Cozza (Padova), attraverso tecniche di virtual-screening e saggi biochimici abbiamo identificato dei nuovi inibitori di CK1 che mostrano una alta selettività per l’isoforma δ di CK1.
L’ultima parte di questa tesi riguarda lo studio di p13, una piccola proteina accessoria del virus HTLV-1. La sintesi chimica di tutta la sequenza aminoacidica di p13 ci ha permesso di caratterizzare questa proteina sia dal punto di vista strutturale che funzionale. Sono inoltre riportati alcuni dati non ancora pubblicati riguardanti il possibile ruolo della fosforilazione nella regolazione della funzione di p13
Pyrvinium pamoate does not activate protein kinase CK1, but promotes Akt/PKB down-regulation and GSK3 activation
No full textThe Selectivity of CK2 Inhibitor Quinalizarin: A Reevaluation
Full text linkMany polyphenolic compounds have been reported to inhibit protein kinases, with special reference to CK2, a pleiotropic serine/threonine kinase, implicated in neoplasia, neurodegenerative disease, and viral infections. In general however these compounds are not endowed with stringent selectivity. Among them quinalizarin (1,2,5,8-tetrahydroxyanthraquinone) turned out to be particularly potent (Ki = 0.058 μM) and quite selective as judged by profiling it on a small panel of 70 protein kinases. Here, by profiling quinalizarin on a larger panel of 140 kinases we reach the conclusion that quinalizarin is one of the most selective inhibitors of CK2, superior to the first-in-class CK2 inhibitor, CX-4945, now in clinical trials for the treatment of cancer. Moreover here we show that quinalizarin is able to discriminate between the isolated CK2 catalytic subunit (CK2α) and CK2 holoenzyme (CK2α2 β2), consistent with in silico and in vitro analyses
Phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) serine-511 by the combined action of tyrosine kinases and CK2: the implication of tyrosine-512 and phenylalanine-508
No full textThe cystic fibrosis transmembrane conductance regulator (CFTR) harbors, close to Phe-508, whose deletion is the commonest cause of cystic fibrosis, a conserved potential CK2 phospho-acceptor site (Ser511), which however is not susceptible to phosphorylation by CK2. To shed light on this apparent paradox, a series of systematically substituted peptides encompassing Ser511 were assayed for their ability to be phosphorylated. The main outcomes of our study are the following: (a) Tyr512 plays a prominent role as a negative determinant as its replacement by Ala restores Ser511 phosphorylation by CK2; (b) an even more pronounced phosphorylation of Ser511 is promoted if Tyr512 is replaced by phospho-tyrosine instead of alanine; (c) Tyr512 and, to a lesser extent, Tyr515 are readily phosphorylated by Lyn, a protein tyrosine kinase of the Src family, in a manner which is enhanced by the concomitant Phe508 deletion. Collectively taken, our data, in conjunction with the notion that Tyr515 is phosphorylated in vivo, disclose the possibility that CFTR Ser511 can be phosphorylated by the combined action of tyrosine kinases and CK2 and disclose a new mechanism of hierarchical phosphorylation where the role of the priming kinase is that of removing negative determinant(s)
A "SYDE" effect of hierarchical phosphorylation: possible relevance to the cystic fibrosis basic defect.
No full textThe 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 S511YDE 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
From phosphoproteins to phosphoproteomes: A historical account
No full textThe first phosphoprotein (casein) was discovered in 1883, yet the enzyme responsible for its phosphorylation was identified only 130 years later, in 2012. In the intervening time, especially in the last decades of the 1900s, it became evident that, far from being an oddity, phosphorylation affects the majority of eukaryotic proteins during their lifespan, and that this reaction is catalysed by the members of a large family of protein kinases, susceptible to a variety of stimuli controlling nearly every aspect of life and death. The aim of this review is to present a historical account of the main steps of this spectacular revolution, which transformed our conception of a biochemical reaction originally held as a sporadic curiosity into the master mechanism governing cell regulation, and, if it is perturbed, causing cell dysregulation
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