Biomedical subjects

Antonio Villalobo

Publications and source records attributed to Antonio Villalobo.

12 recordsLinked to original sources

PubMed2007

Membrane-permeable calmodulin inhibitors (e.g. W-7/W-13) bind to membranes, changing the electrostatic surface potential: dual effect of W-13 on epidermal growth factor receptor activation.

Membrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca2+/CaM) in living cells. If two chemically different inhibitors (e.g. W-7 and trifluoperazine) produce similar effects, investigators often assume the effects are due to CaM inhibition. Zeta potential measurements, however, show that these amphipathic weak bases bind to phospholipid vesicles at the same concentrations as they inhibit Ca2+/CaM; this suggests that they also bind to the inner leaflet of the plasma membrane, reducing its negative electrostatic surface potential. This change will cause electrostatically bound clusters of basic residues on peripheral (e.g. Src and K-Ras4B) and integral (e.g. epidermal growth factor receptor (EGFR)) proteins to translocate from the membrane to the cytoplasm. We measured inhibitor-mediated translocation of a simple basic peptide corresponding to the calmodulin-binding juxtamembrane region of the EGFR on model membranes; W-7/W-13 causes translocation of this peptide from membrane to solution, suggesting that caution must be exercised when interpreting the results obtained with these inhibitors in living cells. We present evidence that they exert dual effects on autophosphorylation of EGFR; W-13 inhibits epidermal growth factor-dependent EGFR autophosphorylation under different experimental conditions, but in the absence of epidermal growth factor, W-13 stimulates autophosphorylation of the receptor in four different cell types. Our interpretation is that the former effect is due to W-13 inhibition of Ca2+/CaM, but the latter results could be due to binding of W-13 to the plasma membrane.

Animals↗

PubMed2006

Nitric oxide and cell proliferation.

Nitric oxide (NO*) has been proposed to be a physiological modulator of cell proliferation, able to promote in most cases cell cycle arrest. In this review I explore the molecular basis of this mechanism of action. The modulatory action of NO* on the intracellular concentration of cGMP and the machinery directly involved in the control of cell cycle progression, including the expression and activity of diverse cyclins and cyclin-dependent kinases, their physiological inhibitors, and the master transcriptional regulator retinoblastoma protein, will be discussed. The role of NO* in proliferation mediated by tyrosine kinase receptors such as the epidermal growth factor receptor and downstream signalling pathways will also be considered. Finally, the involvement of NO* in proliferative processes relevant for normal development will be outlined.

Animals↗

PubMed2005

The adaptor Grb7 is a novel calmodulin-binding protein: functional implications of the interaction of calmodulin with Grb7.

We demonstrate using Ca2+-dependent calmodulin (CaM)-affinity chromatography and overlay with biotinylated CaM that the adaptor proteins growth factor receptor bound (Grb)7 and Grb7V (a naturally occurring variant lacking the Src homology 2 (SH2) domain) are CaM-binding proteins. Deletion of an amphiphilic basic amino-acid sequence (residues 243-256) predicted to form an alpha-helix located in the proximal region of its pleckstrin homology (PH) domain demonstrates the location of the CaM-binding domain. This site is identical in human and rodents Grb7, and shares great homology with similar regions of Grb10 and Grb14, and the Mig10 protein from Caenorhabditis elegans. We show that Grb7 and Grb7V are present in the cytosol and bound to membranes, while the deletion mutants (Grb7Delta and Grb7VDelta) have less capacity to be associated to membranes. Grb7Delta maintains in part the capacity to bind phosphoinositides, and CaM competes for phosphoinositide binding. Activation of ErbB2 by heregulin beta1 decreases the pool of Grb7 associated to membranes. The cell-permeable CaM antagonist W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), but not the CaM-dependent protein kinase II inhibitor KN93, prevents this effect. Highly specific cell-permeable CaM inhibitory peptides decrease the association of Grb7 to membranes. This suggests that CaM regulates the intracellular mobilization of Grb7 in living cells. Direct interaction between enhanced yellow fluorescent protein (EYFP)-Grb7 and enhanced cyan fluorescent protein (ECFP)-CaM chimeras at the plasma membrane of living cells was demonstrated by fluorescence resonance energy transfer (FRET). The FRET signal dramatically decreased in cells loaded with a cell-permeable Ca2+ chelator, and was significantly attenuated when enhanced yellow fluorescent protein-Grb7 chimera (EYFP-Grb7)Delta instead of EYFP-Grb7 was used. Finally, we show that conditioned media from cells transiently transfected with Grb7Delta and Grb7VDelta lost its angiogenic activity, in contrast to those from cells transiently transfected with their wild-type counterparts.

Adenocarcinoma↗

PubMed2005

Gap junction channels reconstituted in two closely apposed lipid bilayers.

Intercellular communication mediated by gap junction channels plays an important role in many cellular processes. In contrast to other channels, gap junction channels span two plasma membranes resulting in an intracellular location for both ends of the junctional pore and the regulatory sites for channel gating. This configuration presents unique challenges for detailed experimental studies of junctional channel physiology and ligand-activation in situ. Availability of an appropriate model system would significantly facilitate future studies of gap junction channel function and structure. Here we show that the double-membrane channel can be reconstituted in pairs of closely apposed lipid bilayers, as experienced in cells. We have trapped the calcium-sensitive dye, arsenazo III (AIII), partially calcium-saturated (AIII-Ca), in one population of connexin32 reconstituted-liposomes, and EGTA in a second one. In such mixtures, the interaction of EGTA with AIII-Ca was measured by a large color shift from blue to red (decreased absorbance at 652 nm). The exchange of these compounds through gap junctions was proportional to these decrements. Results indicate that these connexon-mediated interliposomal channels are functional and are inhibited by the addition of alpha-glycyrrhetinic acid and by flufenamic acid, two gap junction communication inhibitors. Future use of this model system has the potential to improve our understanding of the permeability and modulation of junctional channels in its native intercellular assembly.

Arsenicals↗

PubMed2005

Characterisation of tyrosine-phosphorylation-defective calmodulin mutants.

Using site-directed mutagenesis, we have produced three calmodulin (CaM) mutants in which one or the two tyrosine residues of native CaM were substituted by phenylalanine. The three variants, denoted CaM(Y99F), CaM(Y138F), and CaM(Y99F/Y138F), were highly expressed in transformed Escherichia coli BL21(DE3)pLysS and purified in high yield. The three CaM mutants were able to activate the cyclic nucleotide phosphodiesterase and the plasma membrane Ca(2+)-ATPase, and present the characteristic Ca(2+)-induced electrophoretic mobility shift of native CaM. CaM(Y138F) and CaM(Y99F/Y138F), however, showed a slightly higher electrophoretic mobility than CaM(Y99F) or wild type CaM. The molar extinction coefficient of native CaM at 276 nm decreases 50% in CaM(Y99F) and CaM(Y138F), while the 276nm peak disappears in CaM(Y99F/Y138F). Terbium fluorescence studies with the different CaM mutants indicate that Y99 (but not Y138) closely interacts with Ca(2+) in the III Ca(2+)-binding domain. The epidermal growth factor receptor (EGFR) and the non-receptor tyrosine kinase c-Src phosphorylate CaM(Y99F) and CaM(Y138F) at a lesser extent than wild type CaM, while they fail to phosphorylate CaM(Y99F/Y138F) as expected. All resulting phospho-(Y)CaM species present the characteristic Ca(2+)-induced electrophoretic mobility shift observed in non-phosphorylated CaM. Quantitative analysis of the different phospho-(Y)CaM species suggests that the relative phosphorylation of Y99 and Y138 in wild type CaM by both the EGFR and c-Src is different than the respective phosphorylation of either Y99 in CaM(Y138F) or Y138 in CaM(Y99F).

Amino Acid Substitution↗

PubMed2004

The ErbB2/Neu/HER2 receptor is a new calmodulin-binding protein.

We have demonstrated previously that the EGFR (epidermal growth factor receptor) is a calmodulin (CaM)-binding protein. To establish whether or not the related receptor ErbB2/Neu/HER2 also binds CaM, we used human breast adenocarcinoma SK-BR-3 cells, because these cells overexpress this receptor thus facilitating the detection of this interaction. In the present paper, we show that ErbB2 could be pulled-down using CaM-agarose beads in a Ca2+-dependent manner, as detected by Western blot analysis using an anti-ErbB2 antibody. ErbB2 was also isolated by Ca2+-dependent CaM-affinity chromatography. We also demonstrate using an overlay technique with biotinylated CaM that CaM binds directly to the immunoprecipitated ErbB2. The binding of biotinylated CaM to ErbB2 depends strictly on the presence of Ca2+, since it was prevented by the presence of EGTA. Moreover, the addition of an excess of free CaM prevents the binding of its biotinylated form, demonstrating that this was a specific process. We excluded any interference with the EGFR, as SK-BR-3 cells express considerably lower levels of this receptor, and no detectable EGFR signal was observed by Western blot analysis in the immunoprecipitated ErbB2 preparations used to perform the overlay assays with biotinylated CaM. We also demonstrate that treating living cells with W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide], a cell-permeant CaM antagonist, down-regulates ErbB2 phosphorylation, and show that W7 does not interfere non-specifically with the activity of ErbB tyrosine kinases. We also show that W7 inhibits the phosphorylation (activation) of both ERK1/2 (extracellular-signal-regulated kinases 1 and 2) and Akt/PKB (protein kinase B), in accordance with the inhibition observed in ErbB2 phosphorylation. In contrast, W7 treatment increased the phosphorylation (activation) of CREB (cAMP-response-element-binding protein) and ATF1 (activating transcription factor-1), two Ca2+-sensitive transcription factors that operate downstream of these ErbB2 signalling pathways, most likely because of the absence of calcineurin activity. We conclude that ErbB2 is a new CaM-binding protein, and that CaM plays a role in the regulation of this receptor and its downstream signalling pathways in vivo.

3' Flanking Region↗

PubMed2004

Endogenous calmodulin interacts with the epidermal growth factor receptor in living cells.

We have previously shown that exogenous calmodulin (CaM) binds to the epidermal growth factor receptor (EGFR) at its cytosolic juxtamembrane region inhibiting its tyrosine kinase activity. We demonstrate in this report that endogenous CaM binds to EGFR in intact cells as CaM co-immunoprecipitates with EGF-activated and non-activated receptors. We also show in living cells that cell-permeable CaM inhibitors prevent the full transphosphorylation of wild type EGFR but not the transphosphorylation of an insertional EGFR mutant in which the CaM-binding domain was divided into two parts. Overall these results suggest that CaM interacts with EGFR in vivo.

Binding Sites↗

PubMed2004

Molecular analysis of the erbB gene family calmodulin-binding and calmodulin-like domains in astrocytic gliomas.

Primarily involved in cell proliferation and differentiation processes, the plasma membrane-bound ErbB tyrosine kinase receptor family is formed by four members: erbB1/EGFR, erbB2/HER2/Neu, erbB3/HER3 and erbB4/HER4. Calmodulin (CaM) is a Ca2+-binding protein involved in the regulation of multiple intracellular processes that binds directly to EGFR in the presence of Ca2+, inhibiting its tyrosine kinase activity. Two main regions in the receptor have been implicated in this relationship: the calmodulin-binding domain (CaM-BD) and the calmodulin-like domain (CaM-LD); their sequences are highly conserved in other members of this family of receptors. The presence of mutations, amplification and/or overexpression and genomic rearrangement of these domains was investigated for all four erbB family genes in a series of 89 glial tumors, including 44 WHO grade IV glioblastomas, 21 WHO grade III anaplastic astrocytomas, and 24 WHO grade II astrocytomas. Gene alterations were only found in the regions of interest in EGFR. One glioblastoma showed an in frame tandem duplication of the intracellular region including CaM-LD (exons 18-25). CaM-BD gene overdose was evidenced in 18 tumors that showed EGFR amplification in other domains. Over-expression of CaM-BD and CaM-LD was detected in 6 and 17 cases, respectively, of the 19 tumors in which this study was performed. The other three genes coding for the ErbB receptors did not present point mutations, or rearrangements, and only a very low amplification rate was found for erbB2 (1 case) and erbB3 (4 cases). No overexpression of erbB2, erbB3 or erbB4 was detected. These findings suggest that EGFR is the main erbB gene family member non-randomly involved in malignant glioma development, and that the two domains under study, due to their high conservation and wide separation in the EGFR sequence, are good marker regions for evaluating EGFR/erbB1 gene amplification, as well as for analysing the presence of transcripts corresponding to truncated cytosolic forms of the receptor in these tumors.

Astrocytoma↗

PubMed2003

Nitric oxide-induced epidermal growth factor-dependent phosphorylations in A431 tumour cells.

Nitric oxide (NO*) strongly inhibits the proliferation of human A431 tumour cells. It also inhibits tyrosine phosphorylation of a 170-kDa band corresponding to the epidermal growth factor receptor (EGFR) and induces the phosphorylation at tyrosine residue(s) of a 58-kDa protein which we have denoted NOIPP-58 (nitric oxide-induced 58-kDa phosphoprotein). The NO*-induced phosphorylation of NOIPP-58 is strictly dependent on the presence of EGF. Phosphorylation of NOIPP-58 and inhibition of the phosphorylation of the band corresponding to EGFR are both cGMP-independent processes. We also demonstrate that the p38 mitogen-activated protein kinase (p38MAPK) pathway is activated by NO* in the absence and presence of EGF, whereas the activity of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and the c-Jun N-terminal kinase 1/2 (JNK1/2) pathways are not significantly affected or are slightly decreased, respectively, on addition of this agent. Moreover, we show that the p38MAPK inhibitor, SB202190, induces rapid vanadate/peroxovanadate-sensitive dephosphorylation of prephosphorylated EGFR and NOIPP-58. We propose that the dephosphorylation of both NOIPP-58 and EGFR are mediated by a p38MAPK-controlled phosphotyrosine-protein phosphatase (PYPP). Activation of the p38MAPK pathway during nitrosative stress probably prevents the operation of this PYPP, allowing NOIPP-58, and in part EGFR, to remain phosphorylated and therefore capable of generating signalling events.

Carcinoma, Squamous Cell↗

PubMed2002

Evidence for the direct interaction between calmodulin and the human epidermal growth factor receptor.

Previous work from our laboratory has demonstrated that the Ca(2+)-calmodulin complex inhibits the intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGFR), and that the receptor can be isolated by Ca(2+)-dependent calmodulin-affinity chromatography [San José, Bengurija, Geller and Villalobo (1992) J. Biol. Chem. 267, 15237-15245]. Moreover, we have demonstrated that the cytosolic juxtamembrane region of the human receptor (residues 645-660) binds calmodulin in a Ca(2+)-dependent manner when this segment forms part of a recombinant fusion protein [Martijn-Nieto and Villalobo (1998) Biochemistry 37, 227-236]. However, demonstration of the direct interaction between calmodulin and the whole receptor has remained elusive. In this work, we show that calmodulin, in the presence of Ca(2+), forms part of a high-molecular-mass complex built upon covalent cross-linkage of the human EGFR immunoprecipitated from two cell lines overexpressing this receptor. Although several calmodulin-binding proteins co-immunoprecipitated with the EGFR, suggesting that they interact with the receptor, we demonstrated using overlay techniques that biotinylated calmodulin binds directly to the receptor in a Ca(2+)-dependent manner without the mediation of any adaptor calmodulin-binding protein. Calmodulin binds to the EGFR with an apparent dissociation constant (K'(d)) of approx. 0.2-0.3 microM. Treatment of cells with epidermal growth factor, or with inhibitors of protein kinase C and calmodulin-dependent protein kinase II, or treatment of the immunoprecipitated receptor with alkaline phosphatase, does not significantly affect the binding of biotinylated calmodulin to the receptor.

Alkaline Phosphatase↗

PubMed2002

Phosphorylation of calmodulin. Functional implications.

Calmodulin (CaM) is phosphorylated in vitro and in vivo by multiple protein-serine/threonine and protein-tyrosine kinases. Casein kinase II and myosin light-chain kinase are two of the well established protein-serine/threonine kinases implicated in this process. On the other hand, within the protein-tyrosine kinases involved in the phosphorylation of CaM are receptors with tyrosine kinase activity, such as the insulin receptor and the epidermal growth factor receptor, and nonreceptor protein-tyrosine kinases, such as several members of the Src family kinases, Janus kinase 2, and p38Syk. The phosphorylation of CaM brings important physiological consequences for the cell as the diverse phosphocalmodulin species have differential actions as compared to nonphosphorylated CaM when acting on different CaM-dependent systems. In this review we will summarize the progress made on this topic as the first report on phosphorylation of CaM was published almost two decades ago. We will emphasize the description of the phosphorylation events mediated by the different protein kinases not only in the test tube but in intact cells, the phosphorylation-mediated changes of CaM activity, its action on CaM-dependent systems, and the functional repercussion of these phosphorylation processes in the physiology of the cell.

Animals↗

PubMed2002

Antiproliferative effect of nitric oxide on epidermal growth factor-responsive human neuroblastoma cells.

Addition of nitric oxide (NO) donors to NB69 neuroblastoma cells produced a cGMP-independent decrease in cell proliferation, without affecting cell viability or apoptosis. The potency of short half-life NO donors was higher when cell proliferation was stimulated by epidermal growth factor (EGF), as compared with cultures exposed to fetal calf serum (FCS). Immunoprecipitation and western blot analysis of the EGF receptor (EGFR) revealed a significant reduction of its EGF-induced tyrosine phosphorylation in cells treated with the NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO). When total cell lysates were subjected to western blotting, we observed that DEA-NO also reduced tyrosine phosphorylation in EGF-activated phosphoproteins, but not in those proteins whose tyrosine phosphorylation was evident in the absence of EGF. The effect of NO on EGFR transphosphorylation was concentration-dependent and transient, with a total recovery observed between 1.5 and 3 h after addition of DEA-NO to the cells. When cells were incubated for 15 min with DEA-NO and then washed, the EGFR transphosphorylation returned to control levels immediately, indicating that the interaction of NO with the receptor molecule was fully reversible. NB69 cells expressed both the neuronal and the inducible isoforms of NO synthase (NOS) when cultured in the presence of FCS; under this condition, the NOS inhibitor, N(omega)-nitro-L-arginine methyl ester, produced a small but significant increase in cell proliferation. The results suggest that NO is an endogenous antimitotic agent and that its interaction with EGFR contributes to cytostasis in NB69 cells.

Apoptosis↗