Néstor Gómez

Trk1 and Trk2 define the major K(+) transport system in fission yeast.

The trk1(+) gene has been proposed as a component of the K(+) influx system in the fission yeast Schizosaccharomyces pombe. Previous work from our laboratories revealed that trk1 mutants do not show significantly altered content or influx of K(+), although they are more sensitive to Na(+). Genome database searches revealed that S. pombe encodes a putative gene (designated here trk2(+)) that shows significant identity to trk1(+). We have analyzed the characteristics of potassium influx in S. pombe by using trk1 trk2 mutants. Unlike budding yeast, fission yeast displays a biphasic transport kinetics. trk2 mutants do not show altered K(+) transport and exhibit only a slightly reduced Na(+) tolerance. However, trk1 trk2 double mutants fail to grow at low K(+) concentrations and show a dramatic decrease in Rb(+) influx, as a result of loss of the high-affinity transport component. Furthermore, trk1 trk2 cells are very sensitive to Na(+), as would be expected for a strain showing defective potassium transport. When trk1 trk2 cells are maintained in K(+)-free medium, the potassium content remains higher than that of the wild type or trk single mutants. In addition, the trk1 trk2 strain displays increased sensitivity to hygromycin B. These results are consistent with a hyperpolarized state of the plasma membrane. An additional phenotype of cells lacking both Trk components is a failure to grow at acidic pH. In conclusion, the Trk1 and Trk2 proteins define the major K(+) transport system in fission yeast, and in contrast to what is known for budding yeast, the presence of any of these two proteins is sufficient to allow growth at normal potassium levels.

PP1/PP2A phosphatases inhibitors okadaic acid and calyculin A block ERK5 activation by growth factors and oxidative stress

Okadaic acid is an inhibitor of the protein Ser/Thr phosphatases PP1 and PP2A, which blocks the activation of extracellular signal‐regulated protein kinase 5 (ERK5), a member of the MAP kinase family activated by growth factors and several types of stressors. The blocking of ERK5 activation by okadaic acid was observed in HeLa cells exposed to epidermal growth factor and H2O2 as well as in PC12 cells stimulated by nerve growth factor and H2O2. Calyculin A, another PP1 and PP2A inhibitor, behaved similarly although these compounds are not structurally related. This suggests that either PP1 or PP2A or both are necessary for ERK5 activation. Protein kinase C (PKC) acts as a negative regulator of the ERK5 activation pathway, however our data suggest that the effects of PKC and the phosphatase are unrelated.

Apigenin and LY294002 prolong EGF-stimulated ERK1/2 activation in PC12 cells but are unable to induce full differentiation.

In rat pheochromocytoma cell line (PC12) cells, initial epidermal growth factor (EGF)‐stimulated extracellular signal‐regulated protein kinases 1/2 (ERK1/2) phosphorylation was similar to that promoted by nerve growth factor (NGF), but declined rapidly. Pre‐treatment with apigenin or LY294002 sustained EGF‐stimulated ERK1/2 phosphorylation whereas wortmannin partially blocked initial ERK1/2 phosphorylation. Changes in ERK1/2 phosphorylation correlated with alterations in p90 ribosomal S6 kinase activity. Wortmannin, LY294002 and apigenin totally blocked growth factor‐induced protein kinase B phosphorylation. However, none of them potentiated Raf activation, which was in fact decreased by LY290042 and wortmannin. The sustained EGF‐induced ERK1/2 activation promoted by apigenin was not sufficient to commit PC12 cells to differentiate, which was achieved by stimulation with NGF, either alone or in the presence of apigenin.

Determination of monoamine oxidase concentrations in rat liver by inhibitor binding

The concentrations of monoamine oxidase-A and -B have been determined in mitochondria, mitochondrial outer membranes and microsomes from Sprague-Dawley and Wistar rats by determining the binding of tritium-labelled pargyline. Although the amounts of each form present depended on the source and the preparation method, this was paralleled by the specific activity such that the molecular turnover number was found to remain constant. The catalytic constants, kcat/Km, which represents the apparent second-order rate constant for the combination of enzyme and substrate, were about 0.13 and 2.1 sec-1 X microM-1 for 5-hydroxytryptamine and 2-phenethylamine, respectively, regardless of the source. Estimations of the amounts of the two forms by determining the concentrations of the inhibitors clorgyline, (-)-deprenyl, J-508 or pargyline necessary to give complete inhibition were shown to give overestimates of the true values because of the non-specific binding of these inhibitors to sites other than the monoamine oxidase active site.

Alternative ERK5 regulation by phosphorylation during the cell cycle

ERK5 is a member of the mitogen-activated protein kinase (MAPK) family that, after stimulation, is activated selectively by dual phosphorylation in the TEY motif by MAPK kinase 5 (MEK5). ERK5 plays an important role in regulating cell proliferation, survival, differentiation and stress response. Moreover, it is involved in G2/M progression and timely mitotic entry. ERK5 is phosphorylated during mitosis, but the molecular mechanism by which it is regulated during this phase is still unclear. Here we show that although ERK5 is phosphorylated in mitosis, this does not occur on the activation motif (TEY), but at its C-terminal half. We have identified five sites of ERK5 phosphorylation in mitosis, two of them unknown. Furthermore, we demonstrate that ERK5 phosphorylation in mitosis is not MEK5-dependent, but rather, cyclin-dependent kinase (CDK)-dependent. Using a mutagenesis approach, we analysed the importance of the phosphorylated residues in ERK5 function; our evidence show that phosphorylation in mitosis of the residues identified inhibits ERK5 activity and regulates ERK5 shuttling from cytoplasm to the nucleus. These results reveal a previously unreported form of ERK5 regulation by phosphorylation and establish a link between CDK and ERK5 pathways during mitosis, which could be crucial for the correct progression of the cell cycle.

A comparative study of some kinetic and molecular properties of microsomal and mitochondrial monoamine oxidase.

This experimental work tries to characterize the monoamine oxidase of microsomal origin through its kinetic and molecular properties, and to establish a comparative study with the enzyme present in rat liver mitochondria. The temperature effect upon this catalytic activity was examined and similar behaviour of MAO A and MAO B between both cellular fractions was found. The study of the pH dependence of initial velocity showed similar results both in mitochondria and in microsomes. The FAD cofactor is covalently attached to the MAO of microsomal origin. The FAD containing subunits corresponding to MAO A and MAO B, previous binding of the enzyme with [3H]pargyline and posterior SDS electrophoresis and fluorography, showed molecular weights of 65,900 and 62,400, respectively, in both cellular fractions. The inhibition curves with clorgyline, deprenyl, semicarbazide and KCN, measuring the remaining activity towards 1 microM of benzylamine, indicated that in mitochondria 5% of the total activity is due to the presence of SSAO activity whereas in microsomes this activity represents about 20%. From all these results it appears that mitochondrial and microsomal MAO are related enzymes, although further structural studies are necessary to confirm their possible identity.

Interaction of PDK1 with Phosphoinositides Is Essential for Neuronal Differentiation but Dispensable for Neuronal Survival

ABSTRACT 3-Phosphoinositide-dependent protein kinase 1 (PDK1) operates in cells in response to phosphoinositide 3-kinase activation and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] production by activating a number of AGC kinases, including protein kinase B (PKB)/Akt. Both PDK1 and PKB contain pleckstrin homology (PH) domains that interact with the PtdIns(3,4,5)P3 second messenger. Disrupting the interaction of the PDK1 PH domain with phosphoinositides by expressing the PDK1 K465E knock-in mutation resulted in mice with reduced PKB activation. We explored the physiological consequences of this biochemical lesion in the central nervous system. The PDK1 knock-in mice displayed a reduced brain size due to a reduction in neuronal cell size rather than cell number. Reduced BDNF-induced phosphorylation of PKB at Thr308, the PDK1 site, was observed in the mutant neurons, which was not rate limiting for the phosphorylation of those PKB substrates governing neuronal survival and apoptosis, such as FOXO1 or glycogen synthase kinase 3 (GSK3). Accordingly, the integrity of the PDK1 PH domain was not essential to support the survival of different embryonic neuronal populations analyzed. In contrast, PKB-mediated phosphorylation of PRAS40 and TSC2, allowing optimal mTORC1 activation and brain-specific kinase (BRSK) protein synthesis, was markedly reduced in the mutant mice, leading to impaired neuronal growth and differentiation.

Characterization of monoamine oxidase activity present in human granulocytes and lymphocytes

The characterization of monoamine oxidase (MAO) activity in lymphocytes and granulocytes was studied by using cells prepared from human blood. The specific activities of the enzyme towards beta-phenylethylamine (PEA), benzylamine (Bz), tyramine (TYR) and 5-hydroxytryptamine (5-HT) were found to be 5-times higher in lymphocytes than in granulocytes. The absence of the semicarbazide-sensitive amine oxidase (SSAO) was confirmed by the lack of effect of semicarbazide on the benzylamine oxidation. The presence of MAO-B was corroborated by the inhibition of PEA oxidation with nanomolar deprenyl concentrations and by inhibition of TYR oxidation with high clorgyline concentrations, as well as by the simple sigmoid curve obtained in both cases. These results, together with the substrate preferences, suggest that the MAO activity of human granulocytes and lymphocytes is predominantly of the B form. For each fraction the kinetic constants were determined towards PEA, TYR and Bz as substrates. The Km values were similar for both cellular samples, whereas the Vmax values were higher in lymphocytes than in granulocytes. MAO-B was titrated with [3H]pargyline in order to find out the number of active sites. The corresponding molecular concentration, Kcat values and turnover number showed the presence of related enzymes in human granulocytes and lymphocytes.

Canonical and Kinase Activity-Independent Mechanisms for Extracellular Signal-Regulated Kinase 5 (ERK5) Nuclear Translocation Require Dissociation of Hsp90 from the ERK5-Cdc37 Complex

ABSTRACT The mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase 5 (ERK5) plays a crucial role in cell proliferation, regulating gene transcription. ERK5 has a unique C-terminal tail which contains a transcriptional activation domain, and activates transcription by phosphorylating transcription factors and acting itself as a transcriptional coactivator. However, the molecular mechanisms that regulate its nucleocytoplasmatic traffic are unknown. We have used tandem affinity purification to identify proteins that interact with ERK5. We show that ERK5 interacts with the Hsp90-Cdc37 chaperone in resting cells, and that inhibition of Hsp90 or Cdc37 results in ERK5 ubiquitylation and proteasomal degradation. Interestingly, activation of cellular ERK5 induces Hsp90 dissociation from the ERK5-Cdc37 complex, leading to ERK5 nuclear translocation and activation of transcription, by a mechanism which requires the autophosphorylation at its C-terminal tail. Consequently, active ERK5 is no longer sensitive to Hsp90 or Cdc37 inhibitors. Cdc37 overexpression also induces Hsp90 dissociation and the nuclear translocation of a kinase-inactive form of ERK5 which retains transcriptional activity. This is the first example showing that ERK5 transcriptional activity does not require kinase activity. Since Cdc37 cooperates with ERK5 to promote cell proliferation, Cdc37 overexpression (as happens in some cancers) might represent a new, noncanonical mechanism by which ERK5 regulates tumor proliferation.

Structural determinants for ERK5 (MAPK7) and leucine rich repeat kinase 2 activities of benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-ones

The benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-one core was discovered as a novel ERK5 (also known as MAPK7 and BMK1) inhibitor scaffold, previously. Further structure–activity relationship studies of this scaffold led to the discovery of ERK5-IN-1 (26) as the most selective and potent ERK5 inhibitor reported to date. 26 potently inhibits ERK5 biochemically with an IC50 of 0.162 ± 0.006 μM and in cells with a cellular EC50 for inhibiting epidermal growth factor induced ERK5 autophosphorylation of 0.09 ± 0.03 μM. Furthermore, 26 displays excellent selectivity over other kinases with a KINOMEscan selectivity score (S10) of 0.007, and exhibits exceptional bioavailability (F%) of 90% in mice. 26 will serve as a valuable tool compound to investigate the ERK5 signaling pathway and as a starting point for developing an ERK5 directed therapeutic agent.