Genetically engineered calmodulins differentially activate target enzymes

Three mutant calmodulin (CaM) genes together with the normal chicken CaM cDNA have been expressed in bacteria for the purpose of determining structure/function relationships in CaM. The mutant CaM genes were generated by in vitro recombination between a chicken CaM cDNA and a processed pseudogene that encodes a full-length CaM but with 19 amino acid substitutions as compared to authentic vertebrate CaM. The calmodulin-like (CaML) proteins derived from the pseudogene are called CaML19, CaML16, and CaML3 and contain 19, 16, and 3 amino acid substitutions, respectively. CaML3 is functionally identical to CaM by all criteria tested. The functional characteristics of CaML16 and CaML19 are also indistinguishable yet quite different from normal CaM. CaML19 and CaML16 will maximally activate myosin light chain kinase but will only half-maximally activate calcineurin and CaM-dependent multiprotein kinase. In addition, CaML16 and CaML19 do not activate phosphorylase kinase. The differential activation of these enzymes does not result from the loss of Ca2+-binding sites, since CaML16 binds four Ca2+ with affinity similar to CaM or CaM23. It is more likely that the functional characteristics of the mutant proteins result from an altered tertiary structure, since the Ca2+-dependent enhancement of tyrosine fluorescence and limited proteolysis pattern of CaML16 are different from that of CaM. The data demonstrate that the nature of the interaction of CaM with myosin light chain kinase is different from its interaction with calcineurin, CaM-dependent multiprotein kinase, and phosphorylase kinase and may involve different functional domains in CaM.     

PhoP can activate its target genes in a PhoQ-independent manner

The PhoP/PhoQ two-component system controls the extracellular magnesium depletion response in Salmonella enterica. Previous studies have shown that PhoP is unable to up-regulate its target genes in the absence of PhoQ function. In this work, we demonstrate that PhoP overexpression can substitute for PhoQ- and phosphorylation-dependent activation. Either a high concentration of PhoP or activation via phosphorylation stimulates PhoP self-association.     

Calmodulin activation of target enzymes. Consequences of deletions in the central helix

The central helical region of calmodulin (CaM) includes amino acids 65-92 and serves to separate the two pairs of Ca2(+)-binding sites. This region may impart conformational flexibility and also interact with target proteins. The functional effects of deleting two, three, five, or eight amino acids from the central helix were monitored by examining the activation of phosphodiesterase, smooth muscle myosin light chain (MLC) kinase, and Ca2+/CaM-dependent protein kinase II (CaM kinase II). CaMDM(-8), a calmodulin-deletion mutant with 8 amino acids deleted from the middle of the central helix, failed to activate MLC kinase, phosphodiesterase, or CaM kinase II at physiologically significant concentrations of activator but also had altered electrophoretic mobility and tyrosine fluorescence properties suggesting major changes in the structure of this mutant. Deletion of five amino acids (77-81) resulted in an increase in apparent Kact for phosphodiesterase (150-fold), CaM kinase II (25-fold), and MLC kinase (5-fold) relative to CaM. The maximal autophosphorylation activity of CaM kinase II was also diminished 70% with CaMDM(-5). For phosphodiesterase activation, CaMDM(-2) has a 15-fold increase in apparent Kact while CaMDM(-3) had an apparent Kact value only 3-fold higher than native CaM. In contrast, the activation of MLC kinase by the two (79-80)- and three (79-81)-amino acid deletion mutants were indistinguishable from each other or native CaM. CaMDM(-2) and CaMDM(-3) stimulated CaM kinase II autophosphorylation to 85 and 70%, respectively, of native CaM with less than a 2-fold increase in Kact. Therefore, all deletions in the central helix of CaM reduce the efficiency of phosphodiesterase activation as reflected by substantial alterations in Kact. MLC kinase activation, however, is relatively insensitive to small two or three amino acid deletions. CaM kinase II interacts with the central helix deletion mutants in a complex manner with alterations in both the Kact and the maximum activity. The data suggest the central helix of CaM may serve as a flexible tether for MLC kinase (and to a lesser extent CaM kinase II) but that an extended conformation of CaM, as predicted from the crystal structure, may be required for phosphodiesterase activation.     

Ionizing radiation can activate the insertion of mitochondrial DNA fragments in the nuclear genome

In analytical review is considered the possibility of the insertion of mitochondrial DNA (mtDNA) fragments into the nuclear genome of cells, exposed ionizing radiation (IR). Many studies show that integration fragment mtDNA in nuclear genome, as well as its fastening as NUMT-pseudogenes, proceed at ancient periods of the evolutions not only, but also at more late periods. The number of the investigations shows that under influence endogenous reactive oxygen species, chemical agent, UV-light and IR mtDNA is damaged with greater frequency, than nucleus DNA. Furthermore, the repair systems in mitochondria are low efficiency. In irradiated by IR cells mtDNA fragments can transition from the mitochondria to the cytoplasm. The binding of mtDNA fragment to a complex with proteins provides them the protection from nuclease destroying. Possibly, at such safe condition they and are carried to nucleus. At inductions of DNA double-strand breaks (under the action of IR and activated their reparation) mtDNA fragments may be inserted to nuclear genome. Such integration of mtDNA to nuclear genome, with shaping NUMT-pseudogenes de novo, may be proceed in irradiated cells in the course of the reparations DNA double-strand breaks by the nonhomologous end-joining pathway. These insertions of mtDNA can cardinally change the structure of nuclear genomes in area of their introduction and render the essential influence upon the realization of genetic information. Available information in literature also allows to suppose that integration mtDNA in nuclear genome can proceed and at raised genomic instability observed in cells at post radiation period. It in equal extent pertains and to malignant cells with raised by instability mitochondrial and nuclear genomes. As the most efficient agent, initiating insertion fragment mtDNA in nuclear genome, is considered ionizing radiation.     

Antimicrotubule agents can activate different apoptotic pathways

The effect of agents (taxol, vincristine, and nocodazole) disturbing the microtubule network in MCF-7 human breast carcinoma cells has been examined. The aim of the study was to determine the subtypes of mitotic catastrophe and the dependence of cell death on the status of protein p53. Antimicrotubule agents can not only induce mitotic catastrophe, that is, cell death during mitosis and the death of micronucleated cells, but also activate apoptosis in interphase cells. We assume that the G1 checkpoint activation in this case occurs as a result of microtubule disruption. Apoptosis can be activated in a p53-independent manner in K-mitotic cells and after the complete disruption of the microtubule network.     

Calmodulin is a potent target for new hypothalamic neuropeptides

Recently, five glycopeptides with coronaro-constrictory properties were isolated from bovine hypothalamus [(1988) Neurochemistry (USSR) 7, 519-524]. Calmodulin has been recognized in our laboratory as a target protein for the neuropeptides isolated from hypothalamus. The results of indirect enzyme-linked immunosorbent assay have shown that the new hypothalamic neuropeptides antagonize with the monospecific anti-calmodulin antibody for calmodulin binding although they are not fragments of calmodulin. The inhibitory potency of the peptides is dependent on their concentration and the length of the polypeptide chain. Four out of five peptides are effective in nM concentration range. Ca2+ stimulates the binding of peptides to calmodulin; however, immunocomplex can be formed in the absence of Ca2+ as well. The effects of trifluoperazine and peptides on the calmodulin/antibody interaction are not additive, suggesting the cooperativity between the binding sites on calmodulin. Under physiological conditions the presence of the peptides could produce distinct conformers of calmodulin which may exhibit altered potency for stimulation/inhibition of target enzymes.     

Heparin can activate a receptor tyrosine kinase.

Heparin, a densely sulfated glycosaminoglycan produced by mast cells, is best known for its inhibitory effects on the blood coagulation system. Heparin or heparan sulfate proteoglycans are also essential cofactors for the interaction of fibroblast growth factors (FGFs) with their receptor tyrosine kinases (FGFRs). Here we show that heparin is a growth factor-independent activating ligand for FGFR-4. Heparin stimulates FGFR-4 autophosphorylation on transfected myoblasts, fibroblasts and lymphoid cells, and is most potent on cells lacking surface heparan proteoglycan. Two functional analogs of heparin, fucoidan and dextran sulfate, are also activators of FGFR-4, while neither heparin nor its analogs can stimulate FGFR-1 in the absence of FGF. A mutation in the FGFR-4 ectodomain which impairs receptor activation by FGFs does not interfere with activation by heparin, demonstrating that receptor domains required for heparin or FGF activation are not identical. Heparin activation of FGFR-4 or of a chimeric receptor bearing FGFR-4 ectodomain and FGFR-1 cytodomain triggers downstream tyrosine phosphorylation of several signaling proteins, and induces proliferation of cells bearing the chimeric receptor. Consistent with these findings, a soluble FGFR-4 ectodomain has strong FGF-independent affinity for immobilized heparin resin, while soluble FGFR-1 requires FGF for stable heparin interaction. Heparin activation of FGFR-4 is the first example of a mammalian polysaccharide serving as a signaling ligand.     

Modified DNA fragments activate NaeI cleavage of refractory DNA sites.

Endonuclease NaeI cleaves DNA using a two-site mechanism. The DNA-binding sites are nonidentical: they recognize different families of flanking sequences. A unique NaeI site that is resistant to cleavage resides in M13 double-stranded DNA. NaeI can be activated to cleave this site by small DNA fragments containing one or more NaeI sites. These activators are not practical for genetic engineering because unphosphorylated activators that are consumed during the cleavage of substrate give ends that may interfere with subsequent ligations. We show that a DNA fragment containing phosphorothioate linkages at the NaeI scissile bonds (S-activator) is not cleaved by NaeI, even though this S-activator binds to the substrate site. The S-activator activates NaeI to cleave M13 DNA under conditions that completely exhaust unsubstituted activator. These results demonstrate that activation is not coupled to cleavage of activator, that NaeI reverts to its inactive state soon after dissociation of the EA complex, and that S-activator makes for a nondepletable activator during prolonged incubations.     

Normal but not altered mucins activate neutrophils

Interactions between leucocytes and their surroundings are mediated through oligosaccharide epitopes, some of which are also expressed on ocular mucins. Neutrophils represent the majority of immune cells in the proinflammatory environment of the ocular surface during sleep. We have tested whether changes in mucin glycosylation, as occur in dry eyes, influence the phenotype and activation of neutrophils. Peripheral blood leucocytes were circulated over equal concentration mats of ocular surface mucins purified from normal volunteers and dry-eye patients, and in sequence over normal and pathological mucins in all combinations. Non-adherent cells were tagged with monoclonal fluorescent antibodies to leucocyte determinants and analysed by flow cytometry. Oxidative burst, assessed with dihydrorhodamine, was followed in cells and supernatant. At a speed similar to that of leucocyte traffic in the retina, normal mucins caused a decrease in neutrophil cathepsin G fluorescence, a decrease that was not observed with mucins from patients with Meibomian gland disease or Sjögren syndrome. No effect was detected at a higher flow. Supernatant and cells collected after circulation over normal mucin showed increased rhodamine fluorescence, indicative of oxidative burst. Fluorescence could also be observed in intact cells adherent to dry-eye mucins. Non-adherent cells could be activated with phorbol 12-myristate 13-acetate after flow over any mucin or combination of mucins. Differences in neutrophil activation after exposure to normal and pathological mucins highlight reciprocal influences at the interface between local and systemic immunity.     

Etoposide and adriamycin but not genistein can activate the checkpoint kinase Chk2 independently of ATM/ATR.

We have investigated the effects of three unrelated topoisomerase 2 inhibitors, genistein, adriamycin, and etoposide, on phosphorylation/activation of the checkpoint kinase Chk2 in normal or ATM-deficient (ATM-) human fibroblasts and in cells overexpressing a catalytically inactive ATR kinase. We demonstrate that genistein activates Chk2 in a strictly ATM-dependent manner, whereas etoposide and adriamycin can trigger Chk2 activation in long-term cultures of ATM- cells. Moreover, these two latter genotoxic compounds were found to activate Chk2 in fibroblasts expressing the dominant negative form of ATR. We also report a significant decrease in the accumulation in G2-phase of ATM- cells when genistein did not activate Chk2. In conclusion, our results strongly support that activation of Chk2 could be dependent on the type and/or extent of DNA damage and under the control of either an ATM-dependent or an ATM and, maybe, an ATR-independent pathway.     

Calmodulin in action: diversity in target recognition and activation mechanisms

Recent structural studies on calmodulin complexes with anthrax adenylyl cyclase and rat Ca2+-activated K+ channel have uncovered unexpected ways by which calmodulin interacts with target proteins.     

DNA-binding domain mutations in SMAD genes yield dominant-negative proteins or a neomorphic protein that can activate WG target genes in Drosophila

Mutations in SMAD tumor suppressor genes are involved in approximately 140,000 new cancers in the USA each year. At this time, how the absence of a functional SMAD protein leads to a tumor is unknown. However, clinical and biochemical studies suggest that all SMAD mutations are loss-of-function mutations. One prediction of this hypothesis is that all SMAD mutations cause tumors via a single mechanism. To test this hypothesis, we expressed five tumor-derived alleles of human SMAD genes and five mutant alleles of Drosophila SMAD genes in flies. We found that all of the DNA-binding domain mutations conferred gain-of-function activity, thereby falsifying the hypothesis. Furthermore, two types of gain-of-function mutation were identified - dominant negative and neomorphic. In numerous assays, the neomorphic allele SMAD4(100T) appears to be capable of activating the expression of WG target genes. These results imply that SMAD4(100T) may induce tumor formation by a fundamentally different mechanism from other SMAD mutations, perhaps via the ectopic expression of WNT target genes - an oncogenic mechanism associated with mutations in Adenomatous Polyposis Coli. Our results are likely to have clinical implications, because gain-of-function mutations may cause tumors when heterozygous, and the life expectancy of individuals with SMAD4(100T) is likely to be different from those with other SMAD mutations. From a larger perspective, our study shows that the genetic characterization of missense mutations, particularly in modular proteins, requires experimental verification.     

Apoptotic cell fragments locally activate tingible body macrophages in the germinal center

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The caspase-generated fragments of PKR cooperate to activate full-length PKR and inhibit translation

We have studied the involvement of receptor interacting protein kinase-1 (RIP1) and dsRNA-activated protein kinase (PKR) in external dsRNA-induced apoptotic and necrotic cell death in Jurkat T cell lymphoma. Our results suggest that RIP1 plays an imported role in dsRNA-induced apoptosis and necrosis. We demonstrated that contrary to necrosis, protein synthesis is inhibited in apoptosis. Here, we show that phosphorylation of translation initiation factor 2-alpha (eukaryotic initiation factor 2-alpha (eIF2-alpha)) and its kinase, PKR, occur in dsRNA-induced apoptosis but not in necrosis. These events are caspase-dependent and coincide with the appearance of the caspase-mediated PKR fragments, N-terminal domain (ND) and kinase domain (KD). Our immunoprecipitation experiments demonstrated that both fragments could independently co-precipitate with full-length PKR. Expression of PKR-KD leads to PKR and eIF2-alpha phosphorylation and inhibits protein translation, whereas that of PKR-ND does not. Co-expression of PKR-ND and PKR-KD promotes their interaction with PKR, PKR and eIF2-alpha phosphorylation and suppresses protein translation better than PKR-KD alone. Our findings suggest a caspase-dependent mode of activation of PKR in apoptosis in which the PKR-KD fragment interacts with and activates intact PKR. PKR-ND facilitates the interaction of PKR-KD with full-length PKR and thus the activation of the kinase and amplifies the translation inhibitory signal.     

Paracoccus pantotrophus NapC can reductively activate cytochrome cd1 nitrite reductase

The oxidized "as isolated" form of Paracoccus pantotrophus cytochrome cd1 nitrite reductase has a bis-histidinyl coordinated c heme and a histidine/tyrosine coordinated d1 heme. This form of the enzyme has previously been shown to be kinetically incompetent. Upon reduction, the coordination of both hemes changes and the enzyme is kinetically activated. Here, we show that P. pantotrophus NapC, a tetraheme c-type cytochrome belonging to a large family of such proteins, is capable of reducing, and hence activating, "as isolated" cytochrome cd1. NapC is the first protein from P. pantotrophus identified as being capable of this activation step and, given the periplasmic co-location and co-expression of the two proteins, is a strong candidate to be a physiological activation partner.     

Guanine nucleotides can activate the insulin-stimulated phosphodiesterase in liver plasma membranes

The insulin-stimulated cyclic AMP phosphodiesterase from liver plasma membranes is shown to be activated upon incubation with guanine nucleotides in the presence of ATP. The non-hydrolysable analogue of ATP, adenylyl imidodiphosphate failed to substitute for ATP in achieving activation. GTP, its non-hydrolysable analogues p[NH]ppG and GTP-gamma-S, as well as GDP, all elicited activation. It is suggested that guanine nucleotides, and probably insulin, exert their effect on this enzyme through a distinct species of guanine nucleotide regulatory protein.     

Glutathione-dependent enzymes alone can produce paraquat resistance

HL60 cells exposed to increasing paraquat concentrations were screened for clones without increased superoxide dismutase activities in an effort to examine cytotoxic events occurring after superoxide production. The resulting resistance to paraquat was not associated with alterations in paraquat uptake, catalase, or NADPH-P450 reductase activity, but to alterations in glutathione-dependent enzyme activities. While increases in glutathione-dependent enzymes upon exposure to paraquat have been reported, the increases were considered a secondary response to increases in superoxide dismutase activities. Our results demonstrate that glutathione-dependent enzymes alone provide protection against paraquat toxicity, and their increase upon exposure to paraquat can be independent of the response of superoxide dismutases. This supports a previous finding that cells resistant to Adriamycin, based on elevated glutathione peroxidase and transferase activities are also cross-resistant to paraquat. Unlike this previous report, the increase in glutathione peroxidase was not a persistent genetic event, as activities returned to normal upon removal of paraquat. An isolated increase in glutathione peroxidase without accompanying increases in superoxide dismutases was a rare event, as nearly all clones examined after exposure to paraquat had increased superoxide dismutase.