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Signaling by calcium ion (Ca2+) plays a prominent role in cell physiology, and these mechanisms are frequently altered in tumor cells. In this review, we consider the interplay of Ca2+ signaling and the functions of the proto-oncogene non-receptor tyrosine kinase c-Src in tumor cells, and the viral oncogenic variant v-Src in transformed cells. Also, other members of the Src-family kinases are considered in this context. The role of Ca2+ in the cell is frequently mediated by Ca2+-binding proteins, where the Ca2+-sensor protein calmodulin (CaM) plays a prominent, essential role in many cellular signaling pathways. Thus, we cover the available information on the role and direct interaction of CaM with c-Src and v-Src in cancerous cells, the phosphorylation of CaM by v-Src/c-Src, and the actions of different CaM-regulated Ser/Thr-protein kinases and the CaM-dependent phosphatase calcineurin on v-Src/c-Src. Finally, we mention some clinical implications of these systems to identify mechanisms that could be targeted for the therapeutic treatment of human cancers. [ABSTRACT FROM AUTHOR]

Overexpression and mutations of the epidermal growth factor receptor (EGFR/ErbB1/HER1) and other tyrosine kinase receptors of the ErbB family (ErbB2/HER2, ErbB3/HER3 and ErbB4/HER4) play an essential role in enhancing the proliferation, the migratory capacity and invasiveness of many tumor cells, leading to cancer progression and increased malignancy. To understand these cellular processes in detail is essential to understand at a molecular level the signaling pathways and regulatory mechanisms controlling these receptors. In this regard, calmodulin (CaM) is a Ca2+-sensor protein that directly interacts with and regulates ErbB receptors, as well as some CaM-dependent kinases that also regulate these receptors, particularly EGFR and ErbB2, adding an additional layer of CaM-dependent regulation to this system. In this short review, an update of recent advances in this area is presented, covering the direct action of Ca2+/CaM on the four ErbB family members mostly in tumor cells and the indirect action of Ca2+/CaM on the receptors via CaM-regulated kinases. It is expected that further understanding of the CaM-dependent mechanisms regulating the ErbB receptors in future studies could identify new therapeutic targets in these systems that could help to control or delay cancer progression. [ABSTRACT FROM AUTHOR]

Calcium (Ca2+) is a universal second messenger that plays a key role in cellular signaling. However, Ca2+ signals are transduced with the help of Ca2+-binding proteins, which serve as sensors, transducers, and elicitors. Among the collection of these Ca2+-binding proteins, calmodulin (CaM) emerged as the prototypical model in eukaryotic cells. This is a small protein that binds four Ca2+ ions and whose functions are multiple, controlling many essential aspects of cell physiology. CaM is universally distributed in eukaryotes, from multicellular organisms, such as human and land plants, to unicellular microorganisms, such as yeasts and ciliates. Here, we review most of the information gathered on CaM in Paramecium, a group of ciliates. We condense the information here by mentioning that mature Paramecium CaM is a 148 amino acid-long protein codified by a single gene, as in other eukaryotic microorganisms. In these ciliates, the protein is notoriously localized and regulates cilia function and can stimulate the activity of some enzymes. When Paramecium CaM is mutated, cells show flawed locomotion and/or exocytosis. We further widen this and additional information in the text, focusing on genomic data. [ABSTRACT FROM AUTHOR]

The epidermal growth factor receptor (EGFR) harbors a calmodulin (CaM)‐binding domain (CaM‐BD) and a CaM‐like domain (CaM‐LD) upstream and downstream, respectively, of the tyrosine kinase (TK) domain. We demonstrate in this paper that deletion of the positively charged CaM‐BD (EGFR/CaM‐BD∆) inactivated the TK activity of the receptor. Moreover, deletion of the negatively charged CaM‐LD (EGFR/CaM‐LD∆), leaving a single negative residue (glutamate), reduced the activity of the receptor. In contrast, substituting the CaM‐LD with a histidine/valine‐rich peptide (EGFR/InvCaM‐LD) caused full inactivation. We also demonstrated using confocal microscopy and flow cytometry that the chimera EGFR‐green fluorescent protein (GFP)/CaM‐BD∆, the EGFR/CaM‐LD∆, and EGFR/InvCaM‐LD mutants all bind tetramethylrhodamine‐labelled EGF. These EGFR mutants were localized at the plasma membrane as the wild‐type receptor does. However, only the EGFR/CaM‐LD∆ and EGFR/InvCaM‐LD mutants appear to undergo ligand‐dependent internalization, while the EGFR‐GFP/CaM‐BD∆ mutant seems to be deficient in this regard. The obtained results and in silico modelling studies of the asymmetric structure of the EGFR kinase dimer support a role of a CaM‐BD/CaM‐LD electrostatic interaction in the allosteric activation of the EGFR TK. [ABSTRACT FROM AUTHOR]

The appearance of modular proteins is a widespread phenomenon during the evolution of proteins. The combinatorial arrangement of different functional and/or structural domains within a single polypeptide chain yields a wide variety of activities and regulatory properties to the modular proteins. In this review, we will discuss proteins, that in addition to their catalytic, transport, structure, localization or adaptor functions, also have segments resembling the helix-loop-helix EF-hand motifs found in Ca2+-binding proteins, such as calmodulin (CaM). These segments are denoted CaM-like domains (CaM-LDs) and play a regulatory role, making these CaM-like proteins sensitive to Ca2+ transients within the cell, and hence are able to transduce the Ca2+ signal leading to specific cellular responses. Importantly, this arrangement allows to this group of proteins direct regulation independent of other Ca2+-sensitive sensor/transducer proteins, such as CaM. In addition, this review also covers CaM-binding proteins, in which their CaM-binding site (CBS), in the absence of CaM, is proposed to interact with other segments of the same protein denoted CaM-like binding site (CLBS). CLBS are important regulatory motifs, acting either by keeping these CaM-binding proteins inactive in the absence of CaM, enhancing the stability of protein complexes and/or facilitating their dimerization via CBS/CLBS interaction. The existence of proteins containing CaM-LDs or CLBSs substantially adds to the enormous versatility and complexity of Ca2+/CaM signaling. [ABSTRACT FROM AUTHOR]

Calmodulin (CaM) is a versatile Ca2+ -sensor/transducer protein that modulates hundreds of enzymes, channels, transport systems, transcription factors, adaptors and other struc- tural proteins, controlling in this manner multiple cellular functions. In addition to its cap- acity to regulate target proteins in a Ca2+ -dependent and Ca2+ -independent manner, the posttranslational phosphorylation of CaM by diverse Ser/Thr- and Tyr-protein kinases has been recognized as an important additional manner to regulate this protein by fi ne-tuning its functionality. In this review, we shall cover developments done in recent years in which phospho-CaM has been implicated in signalling pathways that are relevant for the onset and progression of diverse pathophysiological processes. These include diverse systems playing a major role in carcinogenesis and tumour development, prion-induced encephal- opathies and brain hypoxia, melatonin-regulated neuroendocrine disorders, hypertension, and heavy metal-induced cell toxicity. [ABSTRACT FROM AUTHOR]

We identified the Grb7 family members, Grb10 and Grb14, as Ca2+-dependent CaM-binding proteins using Ca2+-dependent CaM-affinity chromatography as we previously did with Grb7. The potential CaM-binding sites were identified and experimentally tested using fluorescent-labeled peptides corresponding to these sites. The apparent affinity constant of these peptides for CaM, and the minimum number of calcium ions bound to CaM that are required for effective binding to these peptides were also determined. We prepared deletion mutants of the three adaptor proteins lacking the identified sites and determined that they lost or strongly diminished their CaM-binding capacity following the sequence Grb7 > > Grb14 > Grb10. More than one CaM-binding site and/or accessory CaM-binding sites appear to exist in Grb10 and Grb14, as compared to a single one present in Grb7. [ABSTRACT FROM AUTHOR]

The activity of calmodulin (CaM) is modulated not only by oscillations in the cytosolic concentration of free Ca2+, but also by its phosphorylation status. In the present study, the role of tyrosine-phosphorylated CaM [P-(Tyr)-CaM] on the regulation of the epidermal growth factor receptor (EGFR) has been examined using in vitro assay systems. We show that phosphorylation of CaM by rat liver solubilized EGFR leads to a dramatic increase in the subsequent phosphorylation of poly-L-(Glu:Tyr) (PGT) by the receptor in the presence of ligand, both in the absence and in the presence of Ca2+. This occurred in contrast with assays where P-(Tyr)-CaM accumulation was prevented by the presence of Ca2+, absence of a basic cofactor required for CaM phosphorylation and/or absence of CaM itself. Moreover, an antibody against CaM, which inhibits its phosphorylation, prevented the extra ligand-dependent EGFR activation. Addition of purified P-(Tyr)-CaM, phosphorylated by recombinant c-Src (cellular sarcoma kinase) and free of non-phosphorylated CaM, obtained by affinity-chromatography using an immobilized antiphospho-(Tyr)-antibody, also increased the ligand-dependent tyrosine kinase activity of the isolated EGFR toward PGT. Also a CaM(Y99D/Y138D) mutant mimicked the effect of P-(Tyr)-CaM on ligand-dependent EGFR activation. Finally, we demonstrate that P-(Tyr)-CaM binds to the same site (645R-R-R-H-I-V-R-KR-T-L-R-R-L-L-Q660) as non-phosphorylated CaM, located at the cytosolic juxtamembrane region of the EGFR. These results show that P-(Tyr)-CaM is an activator of the EGFR and suggest that it could contribute to the CaM-mediated ligand-dependent activation of the receptor that we previously reported in living cells. [ABSTRACT FROM AUTHOR]

Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca2+ signal generated in cells by different effectors, where the Ca2+-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca2+-dependent and Ca2+-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca2+/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca2+-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca2+/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca2+ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred. [ABSTRACT FROM AUTHOR]

Calmodulin (CaM) phosphorylated at different serine/threonine and tyrosine residues is known to exert differential regulatory effects on a variety of CaM-binding enzymes as compared to non-phosphorylated CaM. In this report we describe the preparation and characterization of a series of phospho-(Y)-mimetic CaM mutants in which either one or the two tyrosine residues present in CaM (Y99 and Y138) were substituted to aspartic acid or glutamic acid. It was expected that the negative charge of the respective carboxyl group of these amino acids mimics the negative charge of phosphate and reproduce the effects that distinct phospho-(Y)-CaM species may have on target proteins. We describe some physicochemical properties of these CaM mutants as compared to wild type CaM, after their expression in Escherichia coli and purification to homogeneity, including: i) changes in their electrophoretic mobility in the absence and presence of Ca2+; ii) ultraviolet (UV) light absorption spectra, far- and near-UV circular dichroism data; iii) thermal stability in the absence and presence of Ca2+; and iv) Tb3+-emitted fluorescence upon tyrosine excitation. We also describe some biochemical properties of these CaM mutants, such as their differential phosphorylation by the tyrosine kinase c-Src, and their action as compared to wild type CaM, on the activity of two CaM-dependent enzymes: cyclic nucleotide phosphodiesterase 1 (PDE1) and endothelial nitric oxide synthase (eNOS) assayed in vitro. [ABSTRACT FROM AUTHOR]

This study analyzes whether the release of nitric oxide (NO) and thromboxane A2 (TXA2) depends on the time lapsed since gonadal function is lost, and their correlation with the proliferation of vascular smooth muscle cells (VSMC) mediated by the epidermal growth factor receptor (EGFR). For this purpose, aortic and mesenteric artery segments from control and 6-weeks or 5-months orchidectomized rats were used to measure NO and TXA2 release. The results showed that the basal and acetylcholine (ACh)-induced NO release were decreased 6 weeks post-orchidectomy both in aorta and mesenteric artery, but were recovered 5 months thereafter up to levels similar to those found in arteries from control rats. The basal and ACh-induced TXA2 release increased in aorta and mesenteric artery 6 weeks post-orchidectomy, and was maintained at high levels 5 months thereafter. Since we previously observed that orchidectomy, which decreased testosterone level, enlarged the muscular layer of mesenteric arteries, the effect of testosterone on VSMC proliferation was analyzed. The results showed that treatment of cultured VSMC with testosterone downregulated mitogenic signaling pathways initiated by the ligand-dependent activation of the EGFR. In contrast, the EGFR pathways were constitutively active in mesenteric arteries of long-term orchidectomized rats. Thus, the exposure of mesenteric arteries from control rats to epidermal growth factor (EGF) induced the activation of EGFR signaling pathways. However, the addition of EGF to arteries from orchidectomized rats failed to induce a further activation of these pathways. In conclusion, this study shows that the release of NO depends on the time lapsed since the gonadal function is lost, while the release of TXA2 is already increased after short periods post-orchidectomy. The alterations in these signaling molecules could contribute to the constitutive activation of the EGFR and its downstream signaling pathways after long period post-orchidectomy enhancing the proliferation of the vascular muscular layer. [ABSTRACT FROM AUTHOR]

Our success in producing an active epidermal growth factor receptor (EGFR) tyrosine kinase in Escherichia coli encouraged us to express the full-length receptor in the same host. Despite its large size, we were successful at producing the full-length EGFR protein fused to glutathione S-transferase (GST) that was detected by Western blot analysis. Moreover, we obtained a majoritarian truncated GST-EGFR form detectable by gel electrophoresis and Western blot. This truncated protein was purified and confirmed by MALDI-TOF/TOF analysis to belong to the N-terminal extracellular region of the EGFR fused to GST. Northern blot analysis showed two transcripts suggesting the occurrence of a transcriptional arrest. [ABSTRACT FROM AUTHOR]

Development of neovasculature is a necessary requirement for tumour growth and it provides additional opportunities for therapeutic intervention. However, current antiangiogenic therapies have limited efficacy, mostly because of the development of resistance. Hence, characterization of new antiangiogenic molecular targets is of considerable clinical interest. We report that a calmodulin-binding domain (CaM-BD) deletion mutant of the growth factor receptor bound protein 7 (Grb7) (denoted Grb7Δ) impairs tumour growth and associated angiogenesis in vivo. We implanted glioma C6 cells in rat brains transfected with an enhanced yellow fluorescent protein (EYFP) chimera of Grb7∆, its EYFP-Grb7 wild type counterpart, and EYFP alone. We systematically followed intracerebral growth of the tumours, their cellularity and the functional performance of tumour-associated microvasculature using magnetic resonance imaging, including anatomical T1W and T2W images and functional diffusion and perfusion parameters. Tumours grown from implanted C6 cells expressing EYFP-Grb7Δ developed slower, became smaller and presented lower apparent cellularity than those derived from cells expressing EYFP-Grb7 and EYFP. Vascular perfusion measurements within tumours derived from EYFP-Grb7∆-expressing cells showed significantly lower cerebral blood flow (CBF), cerebral blood volume (CBV) and mean transit time (MTT) values. These findings were independently validated by histological and immunohistochemical techniques. Taken together, these findings confirm that the CaM-BD of Grb7 plays an important role in tumour growth and associated angiogenesis in vivo, thus identifying this region of the protein as a novel target for antiangiogenic treatment. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: We describe in this report the presence of a nuclear localization signal (NLS) overlapping the calmodulin-binding domain (CaM-BD) of the growth factor receptor bound protein 7 (Grb7). We show that deletion of the CaM-BD of Grb7 prevents its nuclear localization, and that its Src homology 2 (SH2) domain might participate as well in the translocation process. Also, treating cells with the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) enhances the presence of Grb7 in the nucleus. We propose that CaM inhibits the translocation of Grb7 to the nucleus after binding to its CaM-BD and therefore occluding its overlapping NLS. [Copyright &y& Elsevier]

In this review, we first describe the mechanisms by which the epidermal growth factor receptor generates a Ca2+ signal and, subsequently, we compile the available experimental evidence regarding the role that the Ca2+/calmodulin complex, formed after the rise in cytosolic free Ca2+ concentration, exerts on the receptor. We focus not only on the indirect action that Ca2+/calmodulin exerts on the epidermal growth factor receptor, as a result of the activation of distinct calmodulin-dependent kinases, but also, and more extensively, on the direct interaction of Ca2+/calmodulin with the receptor. We also describe several mechanistic models that could account for the Ca2+/calmodulin-mediated regulation of epidermal growth factor receptor activity. The control exerted by calmodulin on distinct epidermal growth factor receptor-mediated cellular functions is also discussed. Finally, the phosphorylation of this Ca2+ sensor by the epidermal growth factor receptor is highlighted. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 α-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 (Grb7Δ and Grb7VΔ) have less capacity to be associated to membranes. Grb7Δ maintains in part the capacity to bind phosphoinositides, and CaM competes for phosphoinositide binding. Activation of ErbB2 by heregulin β1 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)Δ instead of EYFP-Grb7 was used. Finally, we show that conditioned media from cells transiently transfected with Grb7Δ and Grb7VΔ lost its angiogenic activity, in contrast to those from cells transiently transfected with their wild-type counterparts.Oncogene (2005) 24, 4206–4219. doi:10.1038/sj.onc.1208591 Published online 4 April 2005 [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

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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. [ABSTRACT FROM AUTHOR]

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 thephosphorylation 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 Srcfamilykinases, 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 willemphasize 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. [ABSTRACT FROM AUTHOR]

The epidermal growth factor (EGF) receptor purified by calmodulin-affinity chromatography from solubilized rat liver plasma membranes phosphorylates connexin32 in gap junction plaques isolated from the same origin. Phosphorylation of connexin32 was stimulated by EGF and mainly occurs at tyrosine residue(s), although phosphorylation of serine and threonine residues was also detected. The kinetics parameters for the phosphorylation of connexin32 parallel those for the transphosphorylation of the EGF receptor. m-Calpain proteolyzes phosphoconnexin32, and its major 26 kDa proteolytic fragment only contains phosphotyrosine residue(s). Calmodulin binds to connexin32 in the absence of calcium and prevents in great extent its phosphorylation by the EGF receptor tyrosine kinase. [ABSTRACT FROM AUTHOR]

Ehrlich ascites tumor cells incorporate [methyl-3H]thymidine into DNA independently of exogenous growth factors or fetal calf serum. Using an acid/ethanol extraction procedure we have obtained from these tumor cells a fraction that induces both the proliferation and the formation of cell foci by Swiss 3T3 mouse fibroblasts in the presence of insulin; inhibits the proliferation of Mv1Lu mink lung epithelial cells; and stimulates the growth of NRK rat kidney fibroblasts in soft-agar in the presence of epidermal growth factor. An antibody against transforming growth factor-β (TGFβ) prevents both the tumor extract-induced proliferation of Swiss 3T3 fibroblasts and the tumor extract-induced proliferative arrest of Mv1Lu cells. The tumor cells secrete a TGFβ-like activity to the extracellular medium in a partially-activated form. However, authentic TGFβ does not affect their proliferation, and no TGFβ receptors were detected using [125I]TGFβ as a ligand. Therefore, the absence of TGFβ receptors with ligand-binding capacity in these tumor cells may bypass the negative control that this factor exerts on the proliferation of their normal cell counterparts. [ABSTRACT FROM AUTHOR]

Biosignalling via lectins may involve modulation of protein kinase activities. This aspect of the biological action of mammalian and plant lectins has been investigated for their effect on the activity of the isolated epidermal growth factor receptor (EGFR). The constitutive tyrosine kinase activity of the epidermal growth factor receptor from rat liver, isolated by calmodulin-affinity chromatography, was activated by concanavalin A (ConA), and wheat germ agglutinin (WGA) to a similar extent as the measured enhancement induced by EGF. In contrast, two mannose-specific lectins, the mannan-binding protein (MBP) and serum amyloid P component (SAP), isolated from human serum, have inhibitory effects, both in the absence and presence of EGF. The differential effects of these lectins were tested using as phosphorylatable substrates a co-polymer of glutamic acid-tyrosine, as well as calmodulin. However, two galactoside-specific lectins, the laminin-binding β-galactoside-binding 14 kDa lectin, isolated from bovine heart (14K-BHL), and the α/β-galactoside-binding lectin, isolated from mistletoe ( Viscum album L.) leaves (VAA), do not inhibit the EGFR tyrosine kinase activity. The sugar dependence of the lectin-mediated action was studied by inhibition assays. Mannose and a mannose-containing neoglycoprotein prevent the activating effect of ConA, and N-acetyl-D-glucosamine partially prevents the activation produced by WGA. However, mannose and mannose-containing neoglycoprotein were ineffective to reduce the inhibitory effect of MBP or SAP. Although the response to binding of ConA and WGA was different to that of MBP or SAP with respect to the tyrosine kinase activity of the EGFR, it should be noted that the four lectins inhibited the binding of [I]EGF to its receptor with similar efficiency. [ABSTRACT FROM AUTHOR]

Calpain I purified from human erythrocyte cytosol activates both the ATP hydrolytic activity and the ATP-dependent Ca2+ transport function of the Ca2(+)-translocating ATPase solubilized and purified from the plasma membrane of human erythrocytes and reconstituted into phosphatidylcholine vesicles. Following partial proteolysis of the enzyme by calpain I, both the initial rates of calcium ion uptake and ATP hydrolysis were increased to near maximal levels similar to those obtained upon addition of calmodulin. The proteolytic activation resulted in the loss of further stimulation of the rates of Ca2+ translocation or ATP hydrolysis by calmodulin as well as an increase of the affinity of the enzyme for calcium ion. However, the mechanistic Ca2+/ATP stoichiometric ratio was not affected by the proteolytic treatment of the reconstituted Ca2(+)-translocating ATPase. The proteolytic activation of the ATP hydrolytic activity of the reconstituted enzyme could be largely prevented by calmodulin. Different patterns of proteolysis were obtained in the absence or in the presence of calmodulin during calpain treatment: the 136-kDa enzyme was transformed mainly into a 124-kDa active ATPase fragment in the absence of calmodulin, whereas a 127-kDa active ATPase fragment was formed in the presence of calmodulin. This study shows that calpain I irreversibly activates the Ca2+ translocation function of the Ca2(+)-ATPase in reconstituted proteoliposomes by producing a calmodulin-independent active enzyme fragment, while calmodulin antagonizes this activating effect by protecting the calmodulin-binding domain against proteolytic cleavage by calpain. [ABSTRACT FROM AUTHOR]

Human erythrocyte Ca2+-translocating ATPase was solubilized from calmodulin-depleted membranes using the detergent Triton X-100, and subsequently purified by calmodulin-affinity chromatography. The purified enzyme was reconstituted in artificial phospholipid vesicles using a cholate-dialysis method and various phospholipids. The reconstituted enzyme was able to translocate Ca2+ inside the vesicles, both in the absence and in the presence of the Ca2+-chelating agent, oxalate, inside the vesicles. The tightness of coupling between ATP hydrolysis and cation translocation was investigated by the use of different ionophoretic compounds. The efficiency of Ca2+ translocation was measured by the ability of the ionophores to stimulate ATP hydrolytic activity of the reconstituted enzyme. It was found that the maximum stimulation of the ATP hydrolytic activity was induced by the electroneutral Ca2+/2H+ ionophore A23187 (9 to 10-fold). A Ca2+ ionophore unable to translocate H+, CYCLEX-2E, was less efficient in stimulating the activity of the reconstituted enzyme (two- to threefold). However, the combined addition of CYCLEX-2E plus protonophores further increased the ATP hydrolytic activity (around fourfold), whereas, the protonophores did not further stimulate ATP hydrolysis in the presence of A23187. Furthermore, in the absence of Ca2+ ionophore, the electroneutral K+(Na+)/H+ ionophoretic exchanger, monensin, stimulated the rate of ATP hydrolysis in the reconstituted enzyme two- or threefold, respectively. These results suggest that the Ca2+-ATPase not only translocates Ca2+ but also H+ in the opposite direction. [ABSTRACT FROM AUTHOR]

Discusses the phosphorylation of calmodulin by plasma-membrane-associated protein kinase(s). Obtaining of the maximum levels of calmodulin phosphorylation; Phosphoamino acid analysis; Identity of endogenous phosphocalmodulin.

An epidermal-growth-factor(EGF)-receptor preparation isolated by calmodulin-affinity chromatography from rat liver plasma membranes is able to phosphorylate calmodulin. Calmodulin phosphorylation was enhanced 3 - 8 fold by EGF, was dependent on the presence of a polycation or basic protein and was inhibited by micromolar concentrations of Ca2. Phosphate incorporation into calmodulin occurs predominantly on tyrosine residues. Partial proteolysis of phosphocalmodulin by thrombin identifies Tyr99, located in the third calcium-binding domain of calmodulin, as the phosphorylated residue. Stoichiometric measurements show a 32P/calmodulin molar ration of approximately 1 when optimal phosphorylation conditions are used. [ABSTRACT FROM AUTHOR]

Calcium in mammalian neurons is essential for developmental processes, neurotransmitter release, apoptosis, and signal transduction. Incorrectly processed Ca2+ signal is well-known to trigger a cascade of events leading to altered response to variety of stimuli and persistent accumulation of pathological changes at the molecular level. To counterbalance potentially detrimental consequences of Ca2+, neurons are equipped with sophisticated mechanisms that function to keep its concentration in a tightly regulated range. Calcium pumps belonging to the P-type family of ATPases: plasma membrane Ca2+-ATPase (PMCA), sarco/endoplasmic Ca2+-ATPase (SERCA) and secretory pathway Ca2+-ATPase (SPCA) are considered efficient line of defense against abnormal Ca2+ rises. However, their role is not limited only to Ca2+ transport, as they present tissue-specific functionality and unique sensitive to the regulation by the main calcium signal decoding protein—calmodulin (CaM). Based on the available literature, in this review we analyze the contribution of these three types of Ca2+-ATPases to neuropathology, with a special emphasis on mental diseases. [ABSTRACT FROM AUTHOR]

Calmodulin (CaM) is the principal Ca2+ sensor protein in all eukaryotic cells, that upon binding to target proteins transduces signals encoded by global or subcellular-specific changes of Ca2+ concentration within the cell. The Ca2+/CaM complex as well as Ca2+-free CaM modulate the activity of a vast number of enzymes, channels, signaling, adaptor and structural proteins, and hence the functionality of implicated signaling pathways, which control multiple cellular functions. A basic and important cellular function controlled by CaM in various ways is cell motility. Here we discuss the role of CaM-dependent systems involved in cell migration, tumor cell invasiveness, and metastasis development. Emphasis is given to phosphorylation/dephosphorylation events catalyzed by myosin light-chain kinase, CaM-dependent kinase-II, as well as other CaM-dependent kinases, and the CaM-dependent phosphatase calcineurin. In addition, the role of the CaM-regulated small GTPases Rac1 and Cdc42 (cell division cycle protein 42) as well as CaM-binding adaptor/scaffold proteins such as Grb7 (growth factor receptor bound protein 7), IQGAP (IQ motif containing GTPase activating protein) and AKAP12 (A kinase anchoring protein 12) will be reviewed. CaM-regulated mechanisms in cancer cells responsible for their greater migratory capacity compared to non-malignant cells, invasion of adjacent normal tissues and their systemic dissemination will be discussed, including closely linked processes such as the epithelial–mesenchymal transition and the activation of metalloproteases. This review covers as well the role of CaM in establishing metastatic foci in distant organs. Finally, the use of CaM antagonists and other blocking techniques to downregulate CaM-dependent systems aimed at preventing cancer cell invasiveness and metastasis development will be outlined. [ABSTRACT FROM AUTHOR]

Overexpression and mutations of ErbB receptors are frequently associated with the high proliferation rate of some tumor cells and the poor prognosis of patients with different types of cancer. The development of new potential therapeutic strategies able to curtail the growth of tumor cells is a highly pursued goal in basic research and in clinicel applications. The inhibition of the proliferation of tumor cells could be mediated by directly or indirectly targeting ErbB receptors. Hence, in this minireview three experimental strategies to attain the arrest of cell proliferation by targeting ErbB receptors with glycobiological tools are explored. This includes: the delivery of cytotoxic lectins to the cell interior using ErbB receptors as carriers, bioengineering tumor cells to modify the normal glycosylation pattern and function of ErbB receptors, and the use of natural occurring inhibitory glycoligands for ErbB receptors. [ABSTRACT FROM AUTHOR]

The posttranslational N- and O-glycosylation of proteins and the recognition of their glycosylated moieties by endogenous lectins play important physiological roles in animal cells. These processes are ubiquitous in the animal kingdom, and have been observed from the lowest nematode to man. After a lectin specifically recognizes a glycoligand, the cellular machinery transfers that information to their different targets to attain specific cellular responses through a network of signaling pathways. In this short assay, the involvement of lectins in signaling processes, the modification of the binding activity of lectins by precise signaling systems, and the possible role of lectins recognizing O-glycosylated intracellular proteins as recruitment sites to initiate new signaling events, will be discussed. [ABSTRACT FROM AUTHOR]

Focuses on the problems in cellular signaling. Relay of communication; Transmission of orders between the multiple cell components; Distinct domains of a given protein.

The epidermal growth factor receptor (EGFR) is a plasma membrane tyrosine kinase that belongs, together with other three related members, to the ErbB family of receptors. These receptors share a large group of polypeptide ligands, that induce homodimerization or heterodimerization among themselves, and the activation of their intrinsic tyrosine kinases followed by their transphosphorylation. The active phosphorylated receptors generate a series of signaling events conducted to initiate cell proliferation, differentiation and/or antiapoptotic responses. In this short review it will be analysed some major signaling pathways initiated by EGFR activation, the regulatory mechanisms controlling its tyrosine kinase activity, some of which are mediated by the calcium signal generated by the own receptor, and its downregulation and internalisation. Moreover, the cross-talk between the EGFR and other ErbB family members, and between the EGFR and other non-related receptors, will be described. Finally, the importance of this receptor in embryogenesis and in proliferative processes in adult individuals will be analysed by the description of some genetic alterations that result in the occurrence of important foetal malformations and the generation of cancerous tumors. [ABSTRACT FROM AUTHOR]