Francisco Centeno

p38γ regulates the localisation of SAP97 in the cytoskeleton by modulating its interaction with GKAP

Activation of the p38 MAP kinase pathways is crucial for the adaptation of mammalian cells to changes in the osmolarity of the environment. Here we identify SAP97/hDlg, the mammalian homologue of the Drosophila tumour suppressor Dlg, as a physiological substrate for the p38γ MAP kinase (SAPK3/p38γ) isoform. SAP97/hDlg is a scaffold protein that forms multiprotein complexes with a variety of proteins and is targeted to the cytoskeleton by its association with the protein guanylate kinase‐associated protein (GKAP). The SAPK3/p38γ‐catalysed phosphorylation of SAP97/hDlg triggers its dissociation from GKAP and therefore releases it from the cytoskeleton. This is likely to regulate the integrity of intercellular–junctional complexes, and cell shape and volume in response to osmotic stress.

Resveratrol‐induced apoptosis in MCF‐7 human breast cancer cells involves a caspase‐independent mechanism with downregulation of Bcl‐2 and NF‐κB

Resveratrol (RES), a chemopreventive molecule, inhibits the proliferation of tumor cells of different etiologies. We previously showed that RES alters the cell cycle and induces apoptosis in MCF‐7 breast tumor cells by interfering with the estrogen receptor (ERaα)–dependent phosphoinositide 3‐kinase (PI3K) pathway. Here, we analyzed signaling downstream of PI3K, to understand the mechanisms of RES‐induced apoptosis. Apoptotic death by RES in MCF‐7 was mediated by Bcl‐2 downregulation since overexpression of this protein abolished apoptosis. Decreased Bcl‐2 levels were not related to cytochrome c release, activation of caspases 3/8 or poly(ADP‐ribose) polymerase proteolysis. However, RES decreased mitochondrial membrane potential and increased reactive oxygen species and nitric oxide production. NF‐κB, a regulator of Bcl‐2 expression, and calpain protease activity, a regulator of NF‐κB, were both inhibited by RES. The patterns for NF‐κB and calpain activities followed that of PI3K and were inhibited by LY294002. NF‐κB inhibition coincided with diminished MMP‐9 activity and cell migration. These data suggest that RES‐induced apoptosis in MCF‐7 could involve an oxidative, caspase‐independent mechanism, whereby inhibition of PI3K signaling converges to Bcl‐2 through NF‐κB and calpain protease activity. Therefore, Bcl‐2 and NF‐κB could be considered potential targets for the chemopreventive activity of RES in estrogen‐responsive tumor cells.

Lithium blocks the PKB and GSK3 dephosphorylation induced by ceramide through protein phosphatase-2A.

The biochemical mechanism of apoptosis induced by ceramide remains still unclear, although it has been reported that dephosphorylation of PKB at Ser-473 may be a key event. In this article, we show that C(2)-ceramide (N-acetyl-sphingosine) induces the dephosphorylation of both protein kinase B (PKB) and glycogen synthase kinase-3 (GSK3) in cerebellar granule cells (CGC). We also show that lithium protects against the apoptosis induced by C(2)-ceramide by blocking the dephosphorylation of both kinases. Since lithium inhibits in vivo the observed protein phosphatase-2A (PP2A) activation induced by ceramide, we hypothesise that the neuroprotective action of lithium may be due to the inhibition of the PP2A activation by apoptotic stimuli.

Lithium inhibits caspase 3 activation and dephosphorylation of PKB and GSK3 induced by K+ deprivation in cerebellar granule cells.

Lithium protects cerebellar granule cells from apoptosis induced by low potassium, and also from other apoptotic stimuli. However, the precise mechanism by which this occurs is not understood. When cerebellar granule cells were switched to low potassium medium, the activation of caspase 3 was detected within 6 h, suggesting a role of caspase 3 in mediating apoptosis under conditions of low potassium. In the same conditions, lithium (5 mm) inhibited the activation of caspase 3 induced by low potassium. As lithium did not inhibit caspase 3 activity in vitro, these results suggest that this ion inhibits an upstream component that is required for caspase 3 activation. Lithium is known to inhibit a kinase termed glycogen sythase kinase 3 (GSK3), which is implicated in the survival pathway of phosphatidylinositol 3‐kinase/protein kinase B (PI3K/PKB). Here we demonstrate that low potassium in the absence of lithium induces the dephosphorylation, and therefore the activation, of GSK3. However, when lithium was present, GSK3 remained phosphorylated at the same level as observed under conditions of high potassium. Low potassium induced the dephosphorylation and inactivation of PKB, whereas when lithium was present PKB was not dephosphorylated. Our results allow us to propose a new hypothesis about the action mechanism of lithium, this ion could inhibit a serine‐threonine phosphatase induced by potassium deprivation.

Stress- and mitogen-induced phosphorylation of the synapse-associated protein SAP90/PSD-95 by activation of SAPK3/p38gamma and ERK1/ERK2.

SAPK3 (stress-activated protein kinase-3, also known as p38gamma) is a member of the mitogen-activated protein kinase family; it phosphorylates substrates in response to cellular stress, and has been shown to bind through its C-terminal sequence to the PDZ domain of alpha1-syntrophin. In the present study, we show that SAP90 [(synapse-associated protein 90; also known as PSD-95 (postsynaptic density-95)] is a novel physiological substrate for both SAPK3/p38gamma and the ERK (extracellular-signal-regulated protein kinase). SAPK3/p38gamma binds preferentially to the third PDZ domain of SAP90 and phosphorylates residues Thr287 and Ser290 in vitro, and Ser290 in cells in response to cellular stresses. Phosphorylation of SAP90 is dependent on the binding of SAPK3/p38gamma to the PDZ domain of SAP90. It is not blocked by SB 203580, which inhibits SAPK2a/p38alpha and SAPK2b/p38beta but not SAPK3/p38gamma, or by the ERK pathway inhibitor PD 184352. However, phosphorylation is abolished when cells are treated with a cell-permeant Tat fusion peptide that disrupts the interaction of SAPK3/p38gamma with SAP90. ERK2 also phosphorylates SAP90 at Thr287 and Ser290 in vitro, but this does not require PDZ-dependent binding. SAP90 also becomes phosphorylated in response to mitogens, and this phosphorylation is prevented by pretreatment of the cells with PD 184352, but not with SB 203580. In neurons, SAP90 and SAPK3/p38gamma co-localize and they are co-immunoprecipitated from brain synaptic junctional preparations. These results demonstrate that SAP90 is a novel binding partner for SAPK3/p38gamma, a first physiological substrate described for SAPK3/p38gamma and a novel substrate for ERK1/ERK2, and that phosphorylation of SAP90 may play a role in regulating protein-protein interactions at the synapse in response to adverse stress- or mitogen-related stimuli.

Partial lithium-associated protection against apoptosis induced by C2-ceramide in cerebellar granule neurons

PRIMARY cultures of cerebellar granule neurons, maintained in a serum-containing medium, underwent apoptosis when exposed to C2-ceramide, as assessed by mitochondrial reduction of MTT and intranucleosomal DNA fragmentation. After an 8h exposure to 50 μM C2-ceramide, cell viability decreased by 25–40%. Addition of lithium together with C2-ceramide resulted in a partial protection of apoptosis, which was maximal ab mM lithium (37% protection). When lithium was added h before the apoptotic stimulus the neuroprotective effect of the ion was clearly increased (66% protection). This effect was not due to intracellular inositol depletion or inhibition of NMDA receptors. Our data broaden the nature of apoptotic insults being reversed by lithium, stressing the neuroprotective effects of the ion.

Mechanisms of MPP+ incorporation into cerebellar granule cells

Exposure of cerebellar granule cells to 1-methyl-4-phenylpiridinium (MPP(+)) results in cell death. We have studied the implication of various membrane transporter systems on MPP(+) neurotoxicity, including the dopamine transporter system (DAT) and cationic amino acid transporters (CAT). We have showed a partial protection against MPP(+) toxicity when the dopamine transporter is inhibited by 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]4-(3-phenylpropyl)piperazinedihydrochloride (GBR-12909). However, almost full protection is only achieved by the simultaneous addition of GBR-12909 and cationic amino acids. These results suggest two ways system of MPP(+) entrance into cerebellar granule cells: the DAT with high activity and the CAT with low activity. We also demonstrated that 5,7-dichlorokynurenic acid (MK-801) failed to protect against MPP(+) exposure, evidencing that N-methyl-D-aspartate (NMDA) receptor is not involved in the MPP(+)-induced cell death.

Characterization of renal damage in canine leptospirosis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting of the urinary proteins.

Canine leptospirosis is a zoonotic disease that can cause interstitial nephritis. As a consequence of the renal damage, proteinuria may occur. To determine the urine protein pattern in the disease we performed sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on the urine from 10 dogs with leptospirosis and 20 healthy dogs. Western blotting analysis of the urine samples with antibodies against canine IgG and IgA was also performed to identify these immunoglobulins in the urine. Urine electrophoresis showed three new bands in the dogs suffering from leptospirosis. Only two of the dogs with leptospirosis showed detectable concentrations of IgG and IgA in urine, while a third animal showed IgG alone. The study showed a 36.7% increase in the excretion of low molecular weight proteins in dogs with leptospirosis but almost no change in the high molecular weight protein pattern. These results, together with the low number of animal with detectable concentrations of IgG and IgA in the urine, support the view that canine leptospirosis is characterized by interstitial nephritis.

Implication of Akt, ERK1/2 and alternative p38MAPK signalling pathways in human colon cancer cell apoptosis induced by green tea EGCG.

We investigated apoptosis induced by the green tea component the epigallocatechin-3-gallate (EGCG) and the pathways underlying its activity in a colon cancer cell line. A complete understanding of the mechanism(s) and molecules targeted by green tea polyphenols could be useful in developing novel therapeutic approaches for cancer treatment. EGCG, which is the major polyphenol in green tea, has cytotoxic effects and induced cell death in HT-29 cell death. In this study, we evaluated the effect EGCG on mitogen-activated protein kinase (MAPK) and Akt pathways. EGCG treatment increased phospho-ERK1/2, -JNK1/2 and -p38α, -p38γ and -p38δ, as well as phospho-Akt levels. Using a combination of kinase inhibitors, we found that EGCG-induced cell death is partially blocked by inhibiting Akt, ERK1/2 or alternative p38MAPK activity. Our data suggest that these kinase pathways are involved in the anti-cancer effects of EGCG and indicate potential use of this compound as chemotherapeutic agent for colon cancer treatment.

Glu-256 is a main structural determinant for oligomerisation of human arginase I.

One determinant that could play a role in the quaternary structure of human arginase is the pair of salt links between the strictly conserved residues R255 from one monomer and E256 from every adjacent subunit. In this work, the ionic interaction between monomers was disrupted by expressing a human arginase where Glu‐256 had been substituted by Gln. Biochemical analyses of the mutant protein showed that: (i) it shares the wild‐type kinetic parameters of the arginine substrate; (ii) E256Q arginase behaves as a monomer by gel filtration; (iii) it is drastically inactivated by dialysis in the presence of EDTA, an inhibitory effect which is reversed by addition of Mn2+; and (iv) the mutant enzyme loses thermal stability. The lack of oligomerisation for E256Q arginase and the conservation of E256 throughout evolution of the protein family suggest that this residue is involved in the quaternary structure of arginases.