1,721,004 research outputs found
Dismantling the aberrant signaling network in chronic lymphocytic leukemia: PP2A and SHP-1 as promising targets for drug discovery
Full text linkReversible protein phosphorylation is a fundamental post-translational modification by which virtually all cellular events are regulated, enabling cells to properly respond to intra- and extracellular cues. Protein kinases and protein phosphatases are the principal factors involved in this in this dynamic process and are placed at the different levels of cellular signalling, and, albeit traditionally considered as functionally opposed to one another, not rarely act in an interplay to finely orchestrate and appropriately drive signal transduction. The imbalance of their expression and function affects the cell life and fate, which frequently underlies the onset and progression of a plethora of diseases. B cell chronic lymphocytic leukemia (CLL), the most common leukemia in the Western world, is no exception to this paradigm, many studies having highlighted a crucial role of kinases in the sustained signals from the signalosome downstream of the B cell receptor (BCR), and having led to the development of the promising second-line drugs, but also the blockade of a number of phosphatases underlying pro-survival and anti-apoptotic signals. In this regard, Protein Phosphatase 2A (PP2A) and the Src homology 2 domain-containing phosphatase 1 (SHP-1) exhibit a marked functional inhibition in this disease, which can be properly circumvented by a pharmacological approach, thereby inducing apoptosis of cancer cells. Nintedanib and MP-0766, a drug acting as an angiokinase inhibitor and a new fingolimod analogue devoid of immunosuppressive action, activating respectively SHP-1 and PP2A have enabled for the discovery of a signalling axis that when activated provokes massive cell death, and might provide a new paradigm for the treatment of CLL, which now endorses kinase inhibitors.La fosforilazione proteica è una fondamentale modificazione post-traduzionale che regola virtualmente tutti i processi cellulari, permettendo alla cellula di rispondere a stimoli intra- ed extracellulari. Le protein chinasi e le protein fosfatasi sono i fattori principali coinvolti in questo processo dinamico e si localizzano a diversi livelli del signaling cellulare, e, sebbene tradizionalmente considerate opposte le une alle altre sotto il profilo funzionale, non raramente compartecipano per finemente modulare e opportunamente dirigere la trasduzione del segnale. Uno squilibrio di espressione e/o funzione di questi fattori si riflette sulla vita e il destino della cellula, cosa che frequentemente è alla base dell’insorgenza nocnhé l’evoluzione di un gran numenro di patologie. La leucemia linfatica cronica a cellule B (B Chronic Lymphocytic Leukemia, CLL), la più comune leucemia in occidente, non fa eccezione a tale paradigma e, sebbene la ricerca per lo più si è concentrata sull’anomala attività di diverse protein chinasi con lo sviluppo di promettenti farmaci di seconda linea, sempre più di frequente viene confermata l’ipotesi che la sopravvivenza e la resistenza all’apoptosis delle cellule tumorali dipende anche dalla ridotta espressione o funzionalità delle protein fosfatasi. A questo riguardo, la protein fosfatasi 2A (Protein Phosphatase 2A, PP2A) e la fosfatasi 1 contenente domini Src homology 2 (Src Homology 2 domain-containing phosphatase 1 (SHP-1) in questa patologia si dimostrano funzionalmente inibiti, ma che, quando opportunamente attivate farmacologicamente, inducono morte delle cellule tumorali. Nintedanib, un farmaco che agisce come inibitore 'angiochinasico”, e MP-0766, un nuovo analogo del fingolimod privo di azione immunosoppressiva, si sono dimostrati in grado di attivare SHP-1 e PP2A rispettivamente, permettendo inoltre di individuare un asse di signaling cellulare che provoca la morte di celle cellule leucemiche, potenzialmente rappresentando un nuovo paradigma per il trattamento della CLL, che ad oggi privilegia gli inibitori chinasici
Label-free fluorescence detection of kinase activity using a gold nanoparticle based indicator displacement assay
Full text linkA straightforward indicator-displacement assay (IDA) has been developed for the quantitative analysis of
ATP→ADP conversion. The IDA relies on the use of gold nanoparticles passivated with a monolayer of
thiols terminating with a 1,4,7-triazacyclononane (TACN)·Zn2+ head group. The analytes ATP and ADP
compete to a different extent with a fluorescent probe for binding to the monolayer surface. In the presence
of ATP the fluorescent probe is free in solution, whereas in the presence of ADP the fluorescent
probe is captured by the nanoparticles and its fluorescence is quenched. The linear response of the fluorescence
signal towards different ratios of ATP : ADP permitted the detection of protein kinase activity
simply by adding aliquots of the enzyme solution to the assay solution followed by measurement of the
fluorescent intensity. The assay poses no restrictions on the target kinase nor does it require labeling of
the kinase substrate. The assay was tested on the protein kinases PIM-1 and Src and validated through a
direct comparison with the classical radiometric assay using the [γ-32P]-labeled ATP
The pleiotropic protein kinase CK2 phosphorylates HTLV-1 Tax protein in vitro, targeting its PDZ-binding motif
No full textThe HTLV-1 transactivator Tax is an oncoprotein capable of deregulating the expression of many cellular genes and interfering with signalling pathways. Here we show that Tax-1 is phosphorylated in vitro by the pleiotropic human serine/threonine kinase CK2 at three residues, Ser-336, Ser-344 and Thr-351, close to and within its C-terminal PDZ-binding motif. We also show that the mutation of Thr-351 to aspartate abolishes Tax-1 binding to the scaffold protein hDlg, a tumour suppressor factor, while having no effect on transactivation. These results suggest that CK2, whose constitutive activity is often hijacked by viruses to sustain their vital cycle, could modulate Tax-1 oncogenic interactions
2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole: a novel powerful and selective inhibitor of protein kinase CK2
No full textProtein kinase CK2 is a highly pleiotropic enzyme whose high constitutive activity is suspected to be instrumental to the enhancement of the tumour phenotype and to the propagation of infectious diseases. Here we describe a novel compound, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), which is superior to the commonly used specific CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) in several respects. DMAT displays the lowest K(i) value ever reported for a CK2 inhibitor (40 nM); it is cell permeable and its efficacy on cultured cells, both in terms of endogenous CK2 inhibition and induction of apoptosis, is several fold higher than that of TBB. The selectivity of DMAT assayed on a panel of >30 protein kinases is comparable to that of TBB, with the additional advantage of being ineffective on protein kinase CK1 up to 200 microM. These properties make DMAT the first choice CK2 inhibitor for in vivo studies available to date
Viral proteins and Src family kinases: Mechanisms of pathogenicity from a “liaison dangereuse”
No full textpCMB treatment reveals the essential role of cysteinyl residues in conferring functional competence to the regulatory subunit of protein kinase CK2
No full textTo assess the functional role of the four conserved cysteinyl residues in the regulatory beta-subunit of protein kinase CK2, the effect of pCMB and other reagents of sulfhydryl groups has been investigated. The pCMB-treated beta-subunit has lost its ability to form either homodimers or regular alpha(2)beta(2) heterotetramers with the catalytic subunit. It also fails to increase catalytic activity toward peptide substrates and to mediate the stimulatory effect of polylysine. The pCMB-treated beta-subunit, however, is still able to prevent calmodulin phosphorylation and to physically interact with the alpha-subunit to form inactive complexes whose sedimentation coefficient is lower than that of CK2 holoenzyme. These inactive complexes upon treatment with reducing agents like DTT are converted into a fully active heterotetrameric holoenzyme
Cooperative modulation of protein kinase CK2 by separate domains of its regulatory beta-subunit
No full textProtein kinase CK2 ("casein kinase 2") holoenzyme is composed of two catalytic (alpha and/or alpha') and two regulatory beta-subunits. A truncated form of the beta-subunit lacking its C-terminal region (betaDelta171-215) has lost the ability to stably associate with the catalytic subunits and to display a number of properties which are mediated by structural elements still present in its sequence, notably down-regulation of catalytic activity, autophosphorylation, and responsiveness to polycationic effectors. All these functions are restored by simultaneous addition of a synthetic peptide reproducing the deleted fragment, beta170-215, which is able to associate with the catalytic subunits and to stimulate catalytic activity. This peptide includes a segment displaying significant sequence similarity with a region of cyclin A which interacts with the PSTAIRE motif of CDK2 eliciting its catalytic activity. A peptide reproducing this sequence (beta181-203), but not its derivative in which three nonpolar side chains have been replaced by polar ones, interacts with the alpha-subunit and stimulates its catalytic activity; it also partially restores the ability of truncated betaDelta171-215 to autophosphorylate. These data disclose the essential role of a structural module located between residues 181 and 203 in conferring regulatory properties to the beta-subunit of CK2
Subverted signalling by protein kinase CK2 in Delta F508 CFTR expressing cells
No full textCystic Fibrosis (CF) is almost invariably caused by mutations
occurring in the Cystic Fibrosis Transmembrane Regulator
(CFTR), a protein involved in chloride transport across the cell
membrane. The commonest mutation (accounting for 70% or
more of all CF cases) is the deletion of phenylalanine 508
(DF508) in the nucleotide binding domain-1 (NBD1) of CFTR,
which leads to premature degradation of the nascent protein with
consequent alterations of diverse cellular functions. We recently
showed that protein kinase CK2, which unlike the other protein
kinases has no recognised factors controlling its activity, is susceptible
to allosteric modulation in vitro by peptides reproducing
the sequence encompassing the F508 deletion of CFTR. Consequently
the targeting of several substrates by CK2 is deeply
altered by these DF peptides [1,2]. This prompted us to hypothesize
that in DF508 CFTR expressing cells signalling by CK2
might be subverted, rendering CK2 a new target for therapies
aimed at treating CF.
Lysates of cell lines expressing either wild type or DF508 CFTR
incubated with 32P-GTP in the presence or absence of selective
CK2 inhibitors exhibit significant alterations in the phosphorylation
of CK2 protein substrates. By MS analysis, a number of
proteins whose phosphorylation by CK2 is altered in DF508 cells,
have been identified and some of these have been recognized as
implicated in the process of maturation, stabilization and degradation
of CFTR. Treatment of cells with selective CK2 inhibitors
affects the protein level of CFTR, both wild type and DF508, disclosing
the possibility that modulation of CK2-dependent phosphorylation
may represent a tool for the regulation of expression
and maturation of CFTR, especially in a DF508 CFTR background.
References:
1. Pagano M. A. et al. Biochemistry 2008; 47, 7925–7936.
2. Pagano M. A. et al. Biochem J 2010; 426, 19–29
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