Utilització de plantes d'Arabidopsis thaliana mutants de CK2 per a l'establiment de línies cel·lulars i per a l'estudi de la implicació de la CK2 en l'estructuració de la cromatina

La proteïna CK2 és una Ser/Thr fosfotransferasa evolutivament conservada. Està formada per dues subunitats diferents, α (catalítica) i β (reguladora), que s’associen en un complex heterotetramèric (α2β2). L’activitat d’aquest enzim està regulada per varis mecanismes que la modulen diferencialment en funció del substrat, entre ells, la localització subcel·lular de les diferents subunitats. A partir de plantes transgèniques que contenen les subunitats de CK2 fusionades a una proteïna fluorescent (GFP o YFP) hem establert línies cel·lulars que permetran analitzar, en treballs futurs, els canvis de localització subcel·lular de les subunitats de CK2 provocats per diferents estímuls. La CK2 intervé en un gran nombre de processos cel·lulars, entre d’altres, metabolisme, transport a nucli, control del cicle cel·lular i reparació del DNA. Amb la finalitat d’estudiar el paper de la CK2 en el desenvolupament de les plantes, membres del grup van generar un mutant dominant negatiu per transformació estable de plantes d’Arabidopsis thaliana. Estudis previs mostraven que la pèrdua d’activitat CK2 en aquestes plantes provocava una alteració de l’activitat mitòtica i un bloqueig del cicle cel·lular. Els resultats obtinguts en aquest treball indiquen que aquests fenotips podrien ser conseqüència de la implicació de la CK2 en processos d’estructuració de la cromatina.

Paper de la quinasa IKKalpha al càncer colorectal

Prèviament havíem identificat la presència d’una forma de l’IKKα de 45kD que s’expressa específicament en el nucli de les cèl.lules de cancer colorectal. A més havíem demostrat que aquesta forma estava fosforilada la qual cosa suggereix que es tracta d’una forma activa d’IKKa. Un dels objectius que ens varem plantejar va ser determinar el mecanisme per el qual es generava aquesta forma de la quinasa. Mitjançant eines bioinformàtiques hem identificat possibles llocs de processament proteolític en la seqüència d’IKKα que podrien ser responsables de generar el fragment de 45kD present en les cèl.lules tumorals. Després, hem demostrat que les proteases identificades in silico són capaces de processar IKKa in vitro, específicament en els llocs predits. S’ha pogut constatar, mitjançant la mutació dels possibles llocs de processament, que només un dels llocs identificats bioinformàticament corresponent a Cathepsin B/L era funcional in vitro, mentre que els altres llocs predits no ho eren. D’igual manera, l’expressió ectòpica de la Cathepsin B o L és capaç de produïr el processament d’IKKα. Pel contrari, la inhibició de l’activitat de la proteasa mitjançant inhibidors específics és capaç de bloquejar el processament d’IKKa en cèl.lules tumorals. Finalment, hem demostrat que els nivells de la Cathepsin B i L, proteases identificades com a responsables de processar IKKa es troben sobre-expressades en la majoria de les mostres humanes de càncer de colon analitzades comparat amb el teixit normal adjacent del mateixos pacients.

Whole genome analysis of p38 SAPK-mediated gene expression upon stress

Background: Cells have the ability to respond and adapt to environmental changes through activation of stress-activated protein kinases (SAPKs). Although p38 SAPK signalling is known to participate in the regulation of gene expression little is known on the molecular mechanisms used by this SAPK to regulate stress-responsive genes and the overall set of genes regulated by p38 in response to different stimuli.Results: Here, we report a whole genome expression analyses on mouse embryonic fibroblasts (MEFs) treated with three different p38 SAPK activating-stimuli, namely osmostress, the cytokine TNFα and the protein synthesis inhibitor anisomycin. We have found that the activation kinetics of p38α SAPK in response to these insults is different and also leads to a complex gene pattern response specific for a given stress with a restricted set of overlapping genes. In addition, we have analysed the contribution of p38α the major p38 family member present in MEFs, to the overall stress-induced transcriptional response by using both a chemical inhibitor (SB203580) and p38α deficient (p38α-/-) MEFs. We show here that p38 SAPK dependency ranged between 60% and 88% depending on the treatments and that there is a very good overlap between the inhibitor treatment and the ko cells. Furthermore, we have found that the dependency of SAPK varies depending on the time the cells are subjected to osmostress. Conclusions: Our genome-wide transcriptional analyses shows a selective response to specific stimuli and a restricted common response of up to 20% of the stress up-regulated early genes that involves an important set of transcription factors, which might be critical for either cell adaptation or preparation for continuous extra-cellular changes. Interestingly, up to 85% of the up-regulated genes are under the transcriptional control of p38 SAPK. Thus, activation of p38 SAPK is critical to elicit the early gene expression program required for cell adaptation to stress.

Lifespan extension by calorie restriction relies on the Sty1 MAP kinase stress pathway

Either calorie restriction, loss of function of the nutrient-dependent PKA or TOR/SCH9 pathways, or activation of stress defences improves longevity in different eukaryotes. However, the molecular links between glucose depletion, nutrient-dependent pathways and stress responses are unknown. Here we show that either calorie restriction or inactivation of nutrient-dependent pathways induces life-span extension in fission yeast, and that such effect is dependent on the activation of the stress-dependent Sty1 MAP kinase. During transition to stationary phase in glucose-limiting conditions, Sty1 becomes activated and triggers a transcriptional stress program, whereas such activation does not occur under glucose-rich conditions. Deletion of the genes coding for the SCH9-homologue Sck2 or the Pka1 kinases, or mutations leading to constitutive activation of the Sty1 stress pathway increase life span under glucose-rich conditions, and importantly such beneficial effects depend ultimately on Sty1. Furthermore, cells lacking Pka1 display enhanced oxygen consumption and Sty1 activation under glucose-rich conditions. We conclude that calorie restriction favours oxidative metabolism, reactive oxygen species production and Sty1 MAP kinase activation, and this stress pathway favours life-span extension.

Activation of Srk1 by the Mitogen-activated Protein Kinase Sty1/Spc1 Precedes Its Dissociation from the Kinase and Signals Its Degradation

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. The Schizosaccharomyces pombe SAPK Sty1/Spc1 orchestrates general changes in gene expression in response to diverse forms of cytotoxic stress. Here we show that Sty1/Spc1 is bound to its target, the Srk1 kinase, when the signaling pathway is inactive. In response to stress, Sty1/Spc1 phosphorylates Srk1 at threonine 463 of the regulatory domain, inducing both activation of Srk1 kinase, which negatively regulates cell cycle progression by inhibiting Cdc25, and dissociation of Srk1 from the SAPK, which leads to Srk1 degradation by the proteasome.

PCAF regulates the stability of the transcriptional regulator and cyclin-dependent kinase inhibitor p27Kip1

P27(Kip1) (p27) is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors. Recently, a new function of p27 as transcriptional regulator has been reported. It has been shown that p27 regulates the expression of target genes mostly involved in splicing, cell cycle, respiration and translation. We report here that p27 directly binds to the transcriptional coactivator PCAF by a region including amino acids 91-120. PCAF associates with p27 through its catalytic domain and acetylates p27 at lysine 100. Our data showed that overexpression of PCAF induces the degradation of p27 whereas in contrast, the knockdown of PCAF stabilizes the protein. A p27 mutant in which K100 was substituted by arginine (p27-K100R) cannot be acetylated by PCAF and has a half-life much higher than that of p27WT. Moreover, p27-K100R remains stable along cell-cycle progression. Ubiquitylation assays and the use of proteasome inhibitors indicate that PCAF induces p27 degradation via proteasome. We also observed that knockdown of skp2 did not affect the PCAF induced degradation of p27. In conclusion, our data suggest that the p27 acetylation by PCAF regulates its stability.

The transcriptional co-activator PCAF regulates cdk2 activity.

Cyclin dependent kinases (cdks) regulate cell cycle progression and transcription. We report here that the transcriptional co-activator PCAF directly interacts with cdk2. This interaction is mainly produced during S and G2/M phases of the cell cycle. As a consequence of this association, PCAF inhibits the activity of cyclin/cdk2 complexes. This effect is specific for cdk2 because PCAF does not inhibit either cyclin D3/cdk6 or cyclin B/cdk1 activities. The inhibition is neither competitive with ATP, nor with the substrate histone H1 suggesting that somehow PCAF disturbs cyclin/cdk2 complexes. We also demonstrate that overexpression of PCAF in the cells inhibits cdk2 activity and arrests cell cycle progression at S and G2/M. This blockade is dependent on cdk2 because it is rescued by the simultaneous overexpression of this kinase. Moreover, we also observed that PCAF acetylates cdk2 at lysine 33. As this lysine is essential for the interaction with ATP, acetylation of this residue inhibits cdk2 activity. Thus, we report here that PCAF inhibits cyclin/cdk2 activity by two different mechanisms: (i) by somehow affecting cyclin/cdk2 interaction and (ii) by acetylating K33 at the catalytic pocket of cdk2. These findings identify a previously unknown mechanism that regulates cdk2 activity.

Inhibition of Mitogen-Activated Protein Kinase Erk1/2 Promotes Protein Degradation of ATP Binding Cassette Transporters A1 and G1 in CHO and in HuH7 cells.

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.

TrkB and TrkC signaling are required for maturation and synaptogenesis of hippocampal connections

Recent studies have suggested a role for neurotrophins in the growth and refinement of neural connections, in dendritic growth, and in activity-dependent adult plasticity. To unravel the role of endogenous neurotrophins in the development of neural connections in the CNS, we studied the ontogeny of hippocampal afferents intrkB (¿/¿) and trkC (¿/¿) mice. Injections of lipophilic tracers in the entorhinal cortex and hippocampus of newborn mutant mice showed that the ingrowth of entorhinal and commissural/associational afferents to the hippocampus was not affected by these mutations. Similarly, injections of biocytin in postnatal mutant mice (P10¿P16) did not reveal major differences in the topographic patterns of hippocampal connections. In contrast, quantification of biocytin-filled axons showed that commissural and entorhinal afferents have a reduced number of axon collaterals (21¿49%) and decreased densities of axonal varicosities (8¿17%) in both trkB (¿/¿) and trkC (¿/¿) mice. In addition, electron microscopic analyses showed thattrkB (¿/¿) and trkC (¿/¿) mice have lower densities of synaptic contacts and important structural alterations of presynaptic boutons, such as decreased density of synaptic vesicles. Finally, immunocytochemical studies revealed a reduced expression of the synaptic-associated proteins responsible for synaptic vesicle exocytosis and neurotransmitter release (v-SNAREs and t-SNAREs), especially in trkB (¿/¿) mice. We conclude that neither trkB nor trkC genes are essential for the ingrowth or layer-specific targeting of hippocampal connections, although the lack of these receptors results in reduced axonal arborization and synaptic density, which indicates a role for TrkB and TrkC receptors in the developmental regulation of synaptic inputs in the CNS in vivo. The data also suggest that the genes encoding for synaptic proteins may be targets of TrkB and TrkC signaling pathways.

Lipid binding by the unique and SH3 domains of c-Src suggests a new regulation mechanism

c-Src is a non-receptor tyrosine kinase involved in numerous signal transduction pathways. The kinase,SH3 and SH2 domains of c-Src are attached to the membrane-anchoring SH4 domain through the flexible Unique domain. Here we show intra- and intermolecular interactions involving the Unique and SH3 domains suggesting the presence of a previously unrecognized additional regulation layer in c-Src. We have characterized lipid binding by the Unique and SH3 domains, their intramolecular interaction and its allosteric modulation by a SH3-binding peptide or by Calcium-loaded calmodulin binding to the Unique domain. We also show reduced lipid binding following phosphorylation at conserved sites of the Unique domain. Finally, we show that injection of full-length c-Src with mutations that abolish lipid binding by the Unique domain causes a strong in vivo phenotype distinct from that of wild-type c-Src in a Xenopus oocyte model system, confirming the functional role of the Unique domain in c-Src regulation.

Long-lived States in an intrinsically disordered protein domain

Long-lived states (LLS) are relaxation-favoured eigenstates of J-coupled magnetic nuclei. LLS were measured, along with classical 1H and 15 N relaxation rate constants, in aminoacids of the N-terminal Unique domain of the c-Src kinase (USrc), which is disordered in vitro under physiological conditions. The relaxation rates of LLS are a probe for motions and interactions in biomolecules. LLS of the aliphatic protons of glycines, with lifetimes ca. four times longer than their spin-lattice relaxation times, are reported for the first time in an intrinsically disordered protein domain (IDP). LLS relaxation experiments were integrated with 2D spectroscopy methods, further adapting them for studies on proteins.

Contribution of S6K1/MAPK signaling pathways in the response to oxidative stress: activation of RSK and MSK by hydrogen peroxide

Cells respond to different kind of stress through the coordinated activation of signaling pathways such as MAPK or p53. To find which molecular mechanisms are involved, we need to understand their cell adaptation. The ribosomal protein, S6 kinase 1 (S6K1), is a common downstream target of signaling by hormonal or nutritional stress. Here, we investigated the initial contribution of S6K1/MAPK signaling pathways in the cell response to oxidative stress produced by hydrogen peroxide (H2O2). To analyze S6K1 activation, we used the commercial anti-phospho-Thr389-S6K1 antibody most frequently mentioned in the bibliography. We found that this antibody detected an 80-90 kDa protein that was rapidly phosphorylated in response to H2O2 in several human cells. Unexpectedly, this phosphorylation was insensitive to both mTOR and PI3K inhibitors, and knock-down experiments showed that this protein was not S6K1. RSK and MSK proteins were candidate targets of this phosphorylation. We demonstrated that H2O2 stimulated phosphorylation of RSK and MSK kinases at residues that are homologous to Thr389 in S6K1. This phosphorylation required the activity of either p38 or ERK MAP kinases. Kinase assays showed activation of RSK and MSK by H2O2. Experiments with mouse embryonic fibroblasts from p38 animals" knockout confirmed these observations. Altogether, these findings show that the S6K1 signaling pathway is not activated under these conditions, clarify previous observations probably misinterpreted by non-specific detection of proteins RSK and MSK by the anti-phospho-Thr389-S6K1 antibody, and demonstrate the specific activation of MAPK signaling pathways through ERK/p38/RSK/MSK by H2O2.

Metabolic alterations and increased liver mTOR expression precede the development of autoimmune disease in a murine model of lupus erythematosus

Although metabolic syndrome (MS) and systemic lupus erythematosus (SLE) are often associated, a common link has not been identified. Using the BWF1 mouse, which develops MS and SLE, we sought a molecular connection to explain the prevalence of these two diseases in the same individuals. We determined SLE- markers (plasma anti-ds-DNA antibodies, splenic regulatory T cells (Tregs) and cytokines, proteinuria and renal histology) and MS-markers (plasma glucose, non-esterified fatty acids, triglycerides, insulin and leptin, liver triglycerides, visceral adipose tissue, liver and adipose tissue expression of 86 insulin signaling-related genes) in 8-, 16-, 24-, and 36-week old BWF1 and control New-Zealand-White female mice. Up to week 16, BWF1 mice showed MS-markers (hyperleptinemia, hyperinsulinemia, fatty liver and visceral adipose tissue) that disappeared at week 36, when plasma anti-dsDNA antibodies, lupus nephritis and a pro-autoimmune cytokine profile were detected. BWF1 mice had hyperleptinemia and high splenic Tregs till week 16, thereby pointing to leptin resistance, as confirmed by the lack of increased liver P-Tyr-STAT-3. Hyperinsulinemia was associated with a down-regulation of insulin related-genes only in adipose tissue, whereas expression of liver mammalian target of rapamicyn (mTOR) was increased. Although leptin resistance presented early in BWF1 mice can slow-down the progression of autoimmunity, our results suggest that sustained insulin stimulation of organs, such as liver and probably kidneys, facilitates the over-expression and activity of mTOR and the development of SLE.

Gleevec, an Abl family inhibitor, produces a profound change in cell shape and migration (in press)

The issue of how contractility and adhesion are related to cell shape and migration pattern remains largely unresolved. In this paper we report that Gleevec (Imatinib), an Abl family kinase inhibitor, produces a profound change in the shape and migration of rat bladder tumor cells (NBTII) plated on collagen-coated substrates. Cells treated with Gleevec adopt a highly spread D-shape and migrate more rapidly with greater persistence. Accompanying this more spread state is an increase in integrin-mediated adhesion coupled with increases in the size and number of discrete adhesions. In addition, both total internal reflection fluorescence microscopy (TIRFM) and interference reflection microscopy (IRM) revealed a band of small punctate adhesions with rapid turnover near the cell leading margin. These changes led to an increase in global cell-substrate adhesion strength, as assessed by laminar flow experiments. Gleevec-treated cells have greater RhoA activity which, via myosin activation, led to an increase in the magnitude of total traction force applied to the substrate. These chemical and physical alterations upon Gleevec treatment produce the dramatic change in morphology and migration that is observed.