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Phosphorylation of the lymphoid cell kinase p56lck is stimulated by micromolar concentrations of Zn2+
Abstract
In particulate fractions from LSTRA lymphoma cells, tyrosine phosphorylation of the lymphoid specific tyrosine kinase p56lck is elicited by Zn2+ in the absence of other divalent cations. Zn2+ alone also induces autophosphorylation of immunoprecipitated p56lck. The effect of Zn2+ is dose dependent; it is detected at concentrations of Zn2+ as low as 5 microM and reaches a maximum at 100 microM Zn2+. Among other divalent cations tested, Mn2+, and Co2+ to a lesser extent, were also effective. Zn2+ also stimulated p56lck phosphorylation in the presence of Mg2+ ions at physiological concentration, whereas orthovanadate had no effect. These results suggest that Zn2+ activates the autophosphorylation of p56lck; this fact could be related with the stimulating effect of Zn2+ in the activation of T lymphocytes.
... In vitro manipulation of Zn can modulate the activity of several T lymphocyte signal transduction proteins, including 56 lck , phospholipase C␥1 (PLC␥1) 5 and protein kinase C (PKC) (Csermely et al. 1988,Ottolenghi 1965, Pernelle et al. 1991. p56 lck is a lymphoid-specific protein tyrosine kinase that is principally expressed in T lymphocytes (Weil and Viellette 1996). ...
... The interaction of cysteine motifs in p56 lck and CD4 or CD8 does not involve disulfide linkage as demonstrated by co-precipitation experiments under reducing conditions (Turner et al. 1990). Rather, a role for Zn to stabilize the interaction was proposed because micromolar concentrations of Zn (in the absence of other divalent cations) elicits substantial tyrosine phosphorylation of p56 lck compared to other divalent cations (Pernelle et al. 1991). Furthermore, it was demonstrated that Zn stimulates phosphorylation of p56 lck in a dose-dependent manner (Pernelle et al. 1991) and that addition of a Zn chelator disrupts the association of p56 lck with the Zn-finger proteins CD4 or CD8 (Turner et al. 1990). ...
... Rather, a role for Zn to stabilize the interaction was proposed because micromolar concentrations of Zn (in the absence of other divalent cations) elicits substantial tyrosine phosphorylation of p56 lck compared to other divalent cations (Pernelle et al. 1991). Furthermore, it was demonstrated that Zn stimulates phosphorylation of p56 lck in a dose-dependent manner (Pernelle et al. 1991) and that addition of a Zn chelator disrupts the association of p56 lck with the Zn-finger proteins CD4 or CD8 (Turner et al. 1990). Similarly, activities of PLC and PKC are inactivated by chelator treatment and restored by in vitro Zn (Csermely et al. 1988, Ottolenghi 1965). ...
Compromised immune function is common to Zn deficiency, protein and energy malnutrition; however, the causative mechanisms at the molecular level have not been elucidated. The T lymphocyte signal transduction pathway contains several Zn-finger proteins, and it is possible that the in vivo functioning of these proteins could be affected by dietary deficiency of Zn and amino acids. Thus, the objective was to investigate the effects, on expression of the T lymphocyte signal transduction proteins p56lck, phospholipase Cg1 (PLCg1) and protein kinase C (PKCa), of dietary Zn deficiency (ZnDF, , 1 mg Zn/kg diet) and protein-energy malnutrition syndromes (2% protein deficiency (LP), combined Zn and 2% protein deficiency (ZnDF1LP), and diet restriction (DR, body weight equal to ZnDF)) compared with control (C) mice. Indices of nutritional status and splenocyte counts were also determined. Based on serum albumin and liver lipid concentrations, the ZnDF1LP and LP groups had protein-type malnutrition, whereas the ZnDF and DR groups had energy-type malnutrition. For Western immunoblotting of the signal transduction proteins, mouse splenic T lymphocytes were isolated by immunocolumns. The expression of T lymphocyte p56lck was significantly elevated in the ZnDF1LP, ZnDF and DR groups compared to the C group. In contrast, the expression of PLCg1 and PKC was unaffected. There was a significant negative correlation between T lymphocyte p56lck expression and serum Zn (r 52 0.65, P 5 0.0007) or femur Zn (r 52 0.73, P 5 0.0001) concentrations. We propose that elevated T lymphocyte p56lck may contribute to altered thymoctye maturation, apoptosis and lymphopenia in Zn deficiency and protein-energy malnutrition syndromes. J. Nutr. 129: 620-627, 1999.
... In vitro manipulation of Zn can modulate the activity of several T lymphocyte signal transduction proteins, including 56 lck , phospholipase C␥1 (PLC␥1) 5 and protein kinase C (PKC) (Csermely et al. 1988,Ottolenghi 1965, Pernelle et al. 1991. p56 lck is a lymphoid-specific protein tyrosine kinase that is principally expressed in T lymphocytes (Weil and Viellette 1996). ...
... The interaction of cysteine motifs in p56 lck and CD4 or CD8 does not involve disulfide linkage as demonstrated by co-precipitation experiments under reducing conditions (Turner et al. 1990). Rather, a role for Zn to stabilize the interaction was proposed because micromolar concentrations of Zn (in the absence of other divalent cations) elicits substantial tyrosine phosphorylation of p56 lck compared to other divalent cations (Pernelle et al. 1991). Furthermore, it was demonstrated that Zn stimulates phosphorylation of p56 lck in a dose-dependent manner (Pernelle et al. 1991) and that addition of a Zn chelator disrupts the association of p56 lck with the Zn-finger proteins CD4 or CD8 (Turner et al. 1990). ...
... Rather, a role for Zn to stabilize the interaction was proposed because micromolar concentrations of Zn (in the absence of other divalent cations) elicits substantial tyrosine phosphorylation of p56 lck compared to other divalent cations (Pernelle et al. 1991). Furthermore, it was demonstrated that Zn stimulates phosphorylation of p56 lck in a dose-dependent manner (Pernelle et al. 1991) and that addition of a Zn chelator disrupts the association of p56 lck with the Zn-finger proteins CD4 or CD8 (Turner et al. 1990). Similarly, activities of PLC and PKC are inactivated by chelator treatment and restored by in vitro Zn (Csermely et al. 1988, Ottolenghi 1965). ...
Compromised immune function is common to Zn deficiency, protein and energy malnutrition; however, the causative mechanisms at the molecular level have not been elucidated. The T lymphocyte signal transduction pathway contains several Zn-finger proteins, and it is possible that the in vivo functioning of these proteins could be affected by dietary deficiency of Zn and amino acids. Thus, the objective was to investigate the effects, on expression of the T lymphocyte signal transduction proteins p56(lck), phospholipase Cgamma1 (PLCgamma1) and protein kinase C (PKCalpha), of dietary Zn deficiency (ZnDF, < 1 mg Zn/kg diet) and protein-energy malnutrition syndromes [2% protein deficiency (LP), combined Zn and 2% protein deficiency (ZnDF+LP), and diet restriction (DR, body weight equal to ZnDF)] compared with control (C) mice. Indices of nutritional status and splenocyte counts were also determined. Based on serum albumin and liver lipid concentrations, the ZnDF+LP and LP groups had protein-type malnutrition, whereas the ZnDF and DR groups had energy-type malnutrition. For Western immunoblotting of the signal transduction proteins, mouse splenic T lymphocytes were isolated by immunocolumns. The expression of T lymphocyte p56(lck) was significantly elevated in the ZnDF+LP, ZnDF and DR groups compared to the C group. In contrast, the expression of PLCgamma1 and PKC was unaffected. There was a significant negative correlation between T lymphocyte p56(lck) expression and serum Zn (r= -0.65, P = 0.0007) or femur Zn (r = -0.73, P = 0.0001) concentrations. We propose that elevated T lymphocyte p56(lck) may contribute to altered thymoctye maturation, apoptosis and lymphopenia in Zn deficiency and protein-energy malnutrition syndromes.
... The Lck's Zn(II)-induced change of state from monomeric to dimeric one was pointed to be the reason for competition between Zn(II)-induced homodimerization and binding of Pro-rich peptides [142]. Furthermore, Zn(II) supplementation on the murine lymphoma cells with Lck overexpression resulted in an increased phosphorylation and activation of Lck [143]. Besides the importance of Zn(II) in the efficient zinc clasp assembly and Lck's activity, also the membrane anchorage has been shown to play a role. ...
... As far as Lck is a cytoplasmic protein, in the contrary to CD4 coreceptor, its embedding in the inner membrane leaflet is possible through myristoylation of its N-terminus. Literature data support the importance of Lck myristoylation on the CD4-Lck complex formation, but resting cells comprise of freely diffusing fraction in the majority [128,143]. ...
... Co-precipitation experiments under reducing conditions have demonstrated that disulfide bonds do not account for the interaction of the cysteine motifs in p56 lck and CD4 or CD8 (Turner et al. 1990). Instead, it has been proposed that Zn stabilizes this interaction since micromolar concentrations of Zn (in the absence of other divalent cations) results in significant tyrosine phosphorylation of p56 lck compared to other divalent cations (Pernelle et al. 1991). In fact, Zn added in vitro stimulates phosphorylation of p56 lck in a dose dependent manner (Pernelle et al. 1991) and Zn chelators disrupt the association of p56 lck with the Zn-finger proteins CD4 or CD8 (Turner et al. 1990). ...
... Instead, it has been proposed that Zn stabilizes this interaction since micromolar concentrations of Zn (in the absence of other divalent cations) results in significant tyrosine phosphorylation of p56 lck compared to other divalent cations (Pernelle et al. 1991). In fact, Zn added in vitro stimulates phosphorylation of p56 lck in a dose dependent manner (Pernelle et al. 1991) and Zn chelators disrupt the association of p56 lck with the Zn-finger proteins CD4 or CD8 (Turner et al. 1990). Tyrosine phosphorylation of p56 lck leads to activation of other proteins, including phospholipaseγ 1 (PLCγ 1). ...
Impaired immune function in dietary zinc (Zn) deficiency is characterized in part by reduced lymphocyte numbers (lymphopenia) and depressed cell-mediated (T lymphocyte) immune function, however, the causative mechanisms at the molecular level have not been elucidated. This paper will focus on the role of dietary Zn in T lymphocyte signal transduction, and specifically, the early Zn-dependent steps for phosphorylation and the putative Zn-finger proteins or Zn-metalloenzymes that may be part of the molecular mechanism for explaining immune dysfunction in Zn deficiency. One of the major recent findings is that murine splenic T lymphocyte p56lck expression is elevated in dietary Zn deficiency and caloric deficiency. Based on the known functions of p56lck, it is proposed that elevated p56lck may contribute to altered thymocyte maturation, apoptosis, and lymphopenia in dietary Zn deficiency and other malnutrition syndromes.
... Zinc promotes T cell activation through the synthesis of protein kinase C and lymphocyte protein tyrosine kinase (LCK). 16,17 However, T cell activation can be inhibited in the presence of a zinc overdose in culture. 33 Besides, zinc reduces the incidence of infection by increasing the number of CD4 + T cells and CTL, mainly by increasing the production of IL-2 and sIL-2R. ...
T cells are crucial for adaptive immunity to regulate proper immune response and immune homeostasis. T cell development occurs in the thymus and mainly differentiates into CD4⁺ and CD8⁺ T cell subsets. Upon stimulation, naive T cells differentiate into distinct CD4⁺ helper and CD8⁺ cytotoxic T cells, which mediate immunity homeostasis and defend against pathogens or tumours. Trace elements are minimal yet essential components of human body that cannot be overlooked, and they participate in enzyme activation, DNA synthesis, antioxidant defence, hormone production, etc. Moreover, trace elements are particularly involved in immune regulations. Here, we have summarized the roles of eight essential trace elements (iron, zinc, selenium, copper, iodine, chromium, molybdenum, cobalt) in T cell development, activation and differentiation, and immune response, which provides significant insights into developing novel approaches to modulate immunoregulation and immunotherapy.
... Zinc is found mainly in oyster, red meat, poultry, beans, nuts, whole grains, seafoods and dairy products and exerts several important biological function, including development, growth, apoptosis, synthesis of heme, proteins and DNA, wound healing and immune functions (NIH, 2020). It plays a key role in macrophages phagocytosis and pathogens killing, in the production of oxygen radicals by neutrophils, in chemotaxis and in the oxidative stress balance, being an essential cofactor of superoxide dismutase (Dawson 3rd et al., 2014;Ibs & Rink, 2003;Powell, 2000); it is also involved in the maturation and activation of B-and T-cells, in the production of memory T-cells after antigen presentation and in the production and release of proinflammatory cytokines by monocytic cells (Haase et al., 2008;Haase & Rink, 2007;Pernelle, Creuzet, Loeb, & Gacon, 1991). Not surprisingly, low levels of zinc lead to a decrease of INF-γ, IL-2, TNF-α and immunoglobulin G production, a reduced function of NK cells as well as a downregulation of NF-κB and complement activity (Beck, Prasad, Kaplan, Fitzgerald, & Brewer, 1997;Kudrin, 2000). ...
Inflammation is a key mechanism of the immune system that can be elicited by several factors, among them several chemical, physical and biological agents. Once stimulated, the inflammatory response activates a series of signaling pathways and a number of immune cells which promote, in a very coordinated manner, the neutralization of the infectious agent. However, if uncontrolled, the inflammatory status may become chronic leading, potentially, to tissue damage and disease onset. Several risk factors are associated with the development of chronic inflammation and, among these factors, diet plays an essential role. In this chapter the effects of some dietary bioactive compounds, including micronutrients, omega-3 fatty acids, nucleotides and polyphenols, on the immunoinflammatory responses in different cellular, animal and human studies have been summarized.
... Various studies revealed zinc's effect on mitogen-activated protein kinases (MAPK), TLR and TCR signaling pathways. Zinc acts at several steps in TCRmediated T cell activation: Zinc directly activates lymphocyte-specific c-src family protein tyrosine kinase (Lck) which is located upstream of all TCR-dependent signaling pathways [69]. Subsequently, Lck is recruited to the TCR signaling complex, where it is autophosphorylated [70]. ...
A functional immune system is essential for healthy life. This is achieved by the coordinate activation and interaction of different immune cells. One should be aware that activation of the immune response is as important as its de-activation when the pathogens are cleared, as otherwise host tissue can be damaged up to life-threatening levels.
Autoimmune diseases (AID) represent a phenomenon of immune cells attacking host cells and tissue. 5 – 8% of the world´s population are currently affected by 80 – 100 AID. In recent years, the incidence has been constantly increasing reaching alarmingly high numbers particularly for type 1 diabetes mellitus, crohn’s disease, rheumatoid arthritis, sjogren’s syndrome and multiple sclerosis. This indicates a higher societal burden of AID for the future. This article provides an overview of general concepts of triggers and underlying mechanisms leading to self-destruction. Lately, several original concepts of disease etiology were revised and there is a variety of hypotheses on triggers, underlying mechanisms and preventive actions.
This article concentrates on the importance of nutrition, especially zinc and vitamin D, for balancing the immune function. Homespun nutritional remedies seem to re-enter today’s therapeutic strategies. Current treatment approaches are largely symptomatic or suppress the immune system. However, recent studies reveal significant benefits of nutrition-related therapeutic approaches including prevention and treatment of established disease, which offers a cost-efficient and trigger-unspecific alternative addressing balancing rather than suppression of the immune system. Zinc and vitamin D are currently the best studied and most promising candidates for therapeutic intervention.
... Dietary zinc can influence highly proliferative immune system, i.e. T-cell proliferation [22][23][24][25]. In the experimental rat models it was found that dietary zinc deficiency could decrease Tcell proliferation by reducing the availability of p56lck protein. ...
Background
Thalassemia is a common hereditary anemia in humans, and beta thalassemia represents a group of recessively inherited hemoglobin disorders first described by Cooley and Lee and characterized by the abnormal synthesis of β-globin chain. The homozygous state results in severe anemia, which needs regular blood transfusion. Although such treatments increase the patient's life span, a variety of complications, including endocrine, metabolic, skeletal, and growth disorders are being observed due to increased iron storage in the body.
Objective
There are some reports emphasizing the role of zinc deficiency and its associated outcomes among thalassemia patients, but none from this part of the world. The aim of this study was to determine the serum zinc levels in children with beta thalassemia major.
Methods
This is a prospective case-control study, which included 35 children between the ages five and 15 years, who were diagnosed as suffering from beta thalassemia major. An equal number of age matched healthy subjects were recruited as controls. The study was carried out at the thalassemia center attached to the Prathima Institute of Medical Sciences (PIMS), Karimnagar, Telangana, India, during the year 2016. Blood samples were collected from both the cases and control subjects and serum zinc activities were analyzed using a semi-automated analyzer. Statistical Package for the Social Sciences (SPSS, Version 15.0) (SPSS Inc., Chicago, USA) was used to calculate the unpaired and independent Student's t-test (p value) to find the significance of the results.
Results
The mean concentrations of serum zinc among the cases and the controls were 39.25 ± 13.45 and 85.31 ± 13.53 (p <0.0001), respectively. Among the cases, 26 (65%) thalassemia patients had zinc concentration below 60 μg/dl, confirming hypozincemia.
Conclusion
This study revealed that hypozincemia was prevalent in beta thalassemia major patients. Further evaluation regarding the role of zinc in the development and progression of thalassemia is recommended.
... In line with that, zinc supplementation was shown to induce p38 phosphorylation [14,66]. This functions by diverse mechanisms, as on the one hand, zinc directly activates the Src kinase Lck, which is located upstream of all TCR-dependent signaling pathways [67]. By binding to two distinct protein interface sites, zinc induces recruitment of Lck to the TCR signaling complex and its activation by autophosphorylation [68,69]. ...
Scope:
Regulatory T cells (Treg) play a pivotal role in immune regulation. For proper immune function, also trace elements like zinc, and anti-inflammatory cytokines including transforming growth factor beta 1 (TGF-β1) and interleukin (IL)-10 are indispensable. Hence, in this study the influence of TGF-β1, IL-10, and zinc supplementation on Treg cells differentiation was investigated.
Methods and results:
A synergistic effect of a combined zinc and TGF-β1 treatment on Foxp3 expression in peripheral blood mononuclear cells (PBMC) and mixed lymphocyte cultures (MLC) was found by per-forming Western blot analysis. Additionally, combined treatment causes elevated Smad 2/3 phosphorylation, which plays an important role in Foxp3 expression. This is due to a TGF-β1-mediated increase of intracellular free zinc measured by zinc probes Fluozin3-AM and ZinPyr-1. Moreover, zinc as well as TGF-β1 treatment caused significantly reduced interferon (IFN)-γ secretion in MLC.
Conclusion:
Combined zinc and TGF-β1 treatment provoked an increased Treg cell induction due to a triggered intracellular zinc signal, which in association with an increased Smad 2/3 activation leads to a boosted Foxp3 expression and resulting in an ameliorated allogeneic reaction in MLC. Thus, zinc can be used as favorable additive, to elevate the induction of Treg cells in adverse immune reactions. This article is protected by copyright. All rights reserved.
... The latter mechanism is thought to facilitate Lck activation by promoting its intermolecular autophosphorylation. Phosphorylation of Lck can be induced by Zn 21 , suggesting that changes of free cellular Zn 21 , which are subject of the next section, might be involved in TCR-signaling via regulation of Lck activation [122]. However, a recent study has demonstrated that at least for the recruitment Lck to the TCRsignaling complex there seems to be no regulatory function for Zn 21 [123]. ...
Zinc is known to be an essential trace element that is highly important for all proliferating cells in the human body, especially for the immune system. Free zinc influences several signaling pathways, such as Toll-like receptor 4 or T-cell receptor signaling, by binding reversibly to regulatory sites in signaling proteins, resulting in a change of free zinc concentrations that can affect signal transduction; thus, cellular responses can be altered. Zinc signals have been observed in cells of the innate as well as of the adaptive immune system, that is, neutrophil granulocytes, mast cells, monocytes, dendritic cells, and T cells, mostly in changes of the cytoplasmic zinc concentration. To characterize zinc signals one can distinguish them by the timescale in which they take place. First, zinc signals can occur within a few seconds to minutes, and are therefore called fast zinc signals. Second, a slightly slower type of zinc signal is known and described as “zinc wave.” Third, some zinc signals occur on a timescale significantly longer than the others. In these cases, the signals are typically involved in altered expression of proteins involved in zinc homeostasis.
Zinc signals occurring in different cell types and signaling pathways that are mentioned in this chapter are classified regarding the specific discrimination of fast zinc signal, zinc wave, and late zinc signal.
... The latter mechanism is thought to facilitate Lck activation by promoting its intermolecular autophosphorylation . Phosphorylation of Lck can be induced by Zn 21 , suggesting that changes of free cellular Zn 21 , which are subject of the next section, might be involved in TCR-signaling via regulation of Lck activation [122] ...
... The latter mechanism is thought to facilitate Lck activation by promoting its intermolecular autophosphorylation . Phosphorylation of Lck can be induced by Zn 21 , suggesting that changes of free cellular Zn 21 , which are subject of the next section, might be involved in TCR-signaling via regulation of Lck activation [122]. However, a recent study has demonstrated that at least for the recruitment Lck to the TCRsignaling complex there seems to be no regulatory function for Zn 21 [123]. ...
For more than 50 years, it has been known that zinc deficiency compromises immune function. During this time, knowledge about the biochemistry of zinc has continued to grow, but only recent years have provided in-depth molecular insights into the multiple aspects of zinc as a regulator of immunity. A network based on ZnT and ZIP proteins for transport and metallothionein for storage tightly regulates zinc availability, and virtually all aspects of innate and adaptive immunity are affected by zinc. In vivo, zinc deficiency alters the number and function of neutrophil granulocytes, monocytes, natural killer (NK)-, T-, and B-cells. T cell functions and balance between the different subsets are particularly susceptible to changes in zinc status. This article focuses in particular on the main mechanisms by which zinc ions exert essential functions in the immune system. On the one hand, this includes tightly protein bound zinc ions serving catalytic or structural functions in a multitude of different proteins, in particular enzymes and transcription factors. On the other hand, increasing evidence arises for a regulatory role of free zinc ions in signal transduction, especially in cells of the immune system. Identification of several molecular targets, including phosphatases, phosphodiesterases, caspases, and kinases suggest that zinc ions are a second messenger regulating signal transduction in various kinds of immune cells. © 2013 BioFactors, 2013.
... The cytoplasmic domains of CD4 and CD8 interact with the N-terminal of p56 lck through their cysteine residues as a "zinc clasp"-like structure [82,83]. The pairs of cysteine residues form a zinc-binding site, suggesting that zinc binding is necessary to stimulate p56 lck phosphorylation [84]. It is believed that once CD4 or CD8 stimulates p56 lck , it phosphorylates the CD3 augmenting the TCR signals [85]. ...
Zinc deficiency is one of the leading risk factors for developing disease and yet we do not have a clear understanding of the mechanisms behind the increased susceptibility to infection. This review will examine the interrelationships among the hypothalamus-pituitary-adrenal stress axis, p56(lck), and T-cell maturation in both zinc deficiency and responses during zinc repletion. We will highlight differences between the adult mouse model (wasting malnutrition) and growing rat model (stunting malnutrition) of dietary zinc deficiency and discuss the use of various controls to separate out the effects of zinc deficiency from the associated malnutrition. Elevated serum corticosterone in both zinc deficient and pair-fed rats does not support the hypothesis that zinc deficiency per se leads to corticosterone-induced apoptosis and lymphopenia. In fact, the zinc deficient rat does not have lymphopenia. Thymocytes from zinc deficient mice and rats have elevated levels of p56(lck), a signalling protein with a zinc clasp structure, but this does not appear to affect thymocyte maturation. However, post-thymic T-cell maturation appears to be altered based on the lower proportion of splenic late thymic emigrants in zinc deficient rats. Fewer new T-cells in the periphery could adversely affect the T-cell repertoire and contribute to immunodeficiency in zinc deficiency.
... Moreover, zinc plays a role in T-lymphocyte activation by means of tyrosine kinase p56 lck , an essential protein in the early steps of activation. Zinc stimulates autophosphorylation of tyrosine residues by p56 lck and subsequent phosphorylation of the antigen receptor complex involving CD45 (Pernelle et al., 1991). Reduction of p56 lck activity, such as in ageing (Shankar and Prasad, 1998), could reduce the ability of chemokine receptors on T-cells to bind the CC or CXC families of chemokines. ...
Infections may cause mortality in old age due to damaged immune responses. As zinc is required as a catalyst, structural (zinc fingers) and regulatory ion, it is involved in many biological functions, including immune responses. Low zinc ion bioavailability and impaired cell-mediated immunity are common in ageing and may be restored by physiological supplementation with zinc for 1–2 months, impacting upon morbidity and survival. This article reviews the role of zinc in immune efficacy during ageing, and also describes the main biochemical pathways involved in the role of zinc in resistance to infections in ageing in order to better understand the possible causes of immunosenescence.
... The latter mechanism is thought to facilitate Lck activation by promoting its intermolecular autophosphorylation. Phosphorylation of Lck can be induced by Zn 21 , suggesting that changes of free cellular Zn 21 , which are subject of the next section, might be involved in TCR-signaling via regulation of Lck activation [122]. However, a recent study has demonstrated that at least for the recruitment Lck to the TCRsignaling complex there seems to be no regulatory function for Zn 21 [123]. ...
Zn is an essential trace element for all organisms. In human subjects body growth and development is strictly dependent on Zn. The nervous, reproductive and immune systems are particularly influenced by Zn deficiency, as well as by increased levels of Zn. The relationship between Zn and the immune system is complex, since there are four different types of influence associated with Zn. (1) The dietary intake and the resorption of Zn depends on the composition of the diet and also on age and disease status. (2) Zn is a cofactor in more than 300 enzymes influencing various organ functions having a secondary effect on the immune system. (3) Direct effects of Zn on the production, maturation and function of leucocytes. (4) Zn influences the function of immunostimulants used in the experimental systems. Here we summarize all four types of influence on the immune function. Nutritional aspects of Zn, the physiology of Zn, the influence of Zn on enzymes and cellular functions, direct effects of Zn on leucocytes at the cellular and molecular level, Zn-altered function of immunostimulants and the therapeutic use of Zn will be discussed in detail.
... Zinc deficiency reduces the number of peripheral and thymic T cells, their proliferation in response to phytohemagglutinin, and the functions of T helper (TH) and cytotoxic T cells, but also acts indirectly by reducing the levels of active serum thymulin. On the molecular level, zinc stimulates the autophosphorylation of the protein tyrosine kinase Lck by non-covalent interaction with the cytoplasmic tails of CD4 and CD8, leading to T cell activation [110, 137, 157] . As one result, the delayed-type hypersensitivity reaction is usually reduced in zinc-deficient individuals . ...
Zinc is required for multiple cellular tasks, and especially the immune system depends on a sufficient availability of this essential trace element. During the last decades, many studies attempted to affect the outcome of various diseases by zinc supplementation. These efforts either aimed at supporting immunity by zinc administration or at correcting a loss of zinc secondary to the disease to restore the zinc-dependent functions of the immune system. This review aims to summarize the respective findings and to discuss possible molecular mechanisms by which zinc could influence viral, bacterial, and parasitic infections, autoimmune diseases, and the response to vaccination. Zinc supplementation in diseases such as diarrhea, chronic hepatitis C, shigellosis, leprosy, tuberculosis, pneumonia, acute lower respiratory infection, and leishmaniasis seems beneficial. In contrast, the results for the common cold and malaria are still not conclusive, and zinc was ineffective in most vaccination and rheumatoid arthritis studies. For AIDS and type 1 diabetes, zinc supplementation may even be a risk factor for increased mortality or deterioration of the glucose metabolism, respectively. In these cases, zinc supplementation should be used with care and limited to clearly zinc-deficient individuals.
Significance
While zinc (Zn ²⁺ ) is a vital ion for cell function and human health, little is known about the role it plays in regulating cell signaling. Here, we use fluorescent tools to study the interaction between Zn ²⁺ and cell signaling pathways that play a role in cell growth and proliferation. Importantly, we use small, non-toxic Zn ²⁺ concentrations to ensure that our Zn ²⁺ changes are closer to what cells would experience in the body and not stress inducing. We also demonstrate that these signaling changes are driven by Ras activation, which contradicts one of the major hypotheses in the field. Our sensors shed light on how cells respond to an important micronutrient in real time.
Zn 2+ is a second messenger for signal transduction in immune cells. Zinc signaling is a complex event and in recent years several molecular details of this multifaceted biochemical puzzle have been disentangled. These include the identification of receptors that trigger zinc signals, the role of various elements of zinc homeostasis (transport proteins, zincosomes, and storage proteins), the investigation of zinc signals themselves, the discovery of sensors that translate zinc signals into a biochemical event, and, finally, the cellular targets that are regulated by zinc signals. Taken together, all these pieces of information already allow a much better understanding of the role of the essential trace element zinc in immune cells. Yet, many aspects still remain unclear, and their full elucidation will take our knowledge of immunity and health even further.
Zinc is a key trace metal ion that is important in regulating a wide variety of metabolic, hormonal, immunological, neuronal and epithelial cell functions [1–4] (Chapters 2, 4 and 5). Over 300 enzymic reactions are known to depend on the presence of zinc [3]. These roles of zinc may be considered to have importance for (a) structural components of metalloproteins (e.g. in thymulin, gene-regulatory proteins, steroid receptors, (b) catalytic activity of enzymes (e.g. in various oxido-reductases, hydrolases, ligases, lyases) and (c) co-active functions (e.g. with Cu in superoxide dismutase, or phospholipase C [3].
Zinc is important for virtually all aspects of cellular functions. Two recent developments significantly advanced our understanding of human zinc biology: the recognition that zinc is a constituent of several thousand proteins and the discoveries that cellular zinc homeostasis requires at least three dozen proteins and a high degree of regulation and integration into cellular signaling networks. This chapter focuses on yet another development, namely emerging general and cell-specific functions of zinc ions beyond their roles in established zinc proteins. How zinc is controlled in human cells will be the basis for discussing the functions of zinc ions in cellular regulation and signal transduction, especially in phosphorylation cascades, and their transient interactions with some proteins that are generally not recognized as zinc proteins. Comparative and specific aspects of the zinc biology in plants, single-cell eukaryotes, or microorganisms are beyond the scope of this chapter. New functions of zinc can be summarized under a role of zinc ions as intercellular and intracellular signals, although they embody quite different principles of action. In essence, these functions establish additional biological activities and a much broader role of zinc in biology than previously acknowledged.
Dietary supplementation has traditionally consisted of adding vitamins and/or minerals to correct or prevent a nutritional deficiency. When supplementing the diet with other inflammatory mediators, such as essential fatty acids, there is an adjunctive benefit to the standard therapies used in the control of chronic inflammatory diseases such as Crohn's disease or rheumatoid arthritis. This review focuses on the strategies utilized for therapeutic modulation of the inflammatory cascade through dietary supplementation with specific biomolecules. Examples of how these biomolecules affect local and systemic immune responses to chronic inflammation are examined. In particular, an overview of the literature identifying the potential to modify the host response to chronic periodontitis is provided.
The major tyrosine protein kinase from HL60 (a human non-differentiated promyelocytic cell line) has been purified almost to homogeneity as judged by silver-stained SDS/PAGE. The procedure involved four chromatographic steps: DEAE-Sepharose, casein-agarose, cibacron-blue–agarose and hexyl-agarose. The purification resulted in more than 1000-fold enrichment in angiotensin II phosphorylation activity. A gel-sizing experiment, labeling with [35S]ATP[γs] and autophosphorylation of the enzyme in the presence of [γ-32P]ATP, all led to the identification of a single protein species with a molecular mass of about 40 kDa. Western blot experiments showed that this protein does not belong to the src family and is not related to the abl and fes oncogene products. Phosphorylation of angiotensin II and casein by this 40-kDa human promyelocytic kinase was stimulated by high ionic strength especially from class IA metal salts. The Km for ATP was 2 μM and the Vmax 3.1 nmol · min−1· mg−1 using angiotensin II as a substrate. The kinase requires the presence of either Mn2+ or Mg2+ for full activity and utilizes ATP or dATP but not GTP as phosphate donor. Based on numerous biochemical observations, it was possible to demonstrate that kinase is different from any other tyrosine protein kinases described in the literature. This 40-kDa protein was used as a molecular tool for testing some tyrosine protein kinase inhibitors described in the literature. It is one of the rare tyrosine protein kinases purified from human cancer cells to date.
Zinc is an essential nutrient with remarkable importance for immunity, in particular for T-cell function. This is, at least in part, based on an involvement of zinc ions in immune cell signal transduction; dynamic changes of the intracellular free zinc concentration have recently been recognized as signaling events. Because the molecular targets of zinc signals remain incompletely understood, we investigated the impact of elevated intracellular free zinc on mitogen-activated protein kinase (MAPK) activity and MAPK-dependent cytokine production in human T-cells. p38 was activated by treatment with zinc and the ionophore pyrithione, whereas ERK1/2 and c-Jun N-terminal kinases were unaffected. In contrast, after T-cell receptor stimulation with antibodies against CD3, ERK1/2-phosphorylation was selectively suppressed by intracellular zinc. Mechanisms that had been shown to mediate zinc-effects in other cells, such as activation of the Src kinase Lck, inhibition of the protein tyrosine phosphatase CD45 or MAPK phosphatases and cyclic nucleotide/protein kinase A signaling were not involved. This indicates that the differential impact of zinc on the MAPK families in T-cells is mediated by mechanisms that differ from the ones observed in other cell types. Further investigation of the activation of p38 by zinc demonstrated that this MAPK is responsible for the zinc-mediated activation of CREB and mRNA expression of the Th1 cytokines interferon-gamma and interleukin-2. In conclusion, regulation of MAPK activity contributes to the impact of zinc on T-cell function.
Zink ist für die zellulären Funktionen von T-Zellen essentiell. Ein Zink-Mangel beeinträchtigt u.a. T-Zellfunktionen. Zum Beispiel ist das Th1/Th2 Gleichgewicht gestört, was zu einer reduzierten Produktion von Th1-Cytokinen wie IFN-gamma und IL-2 führt. Eine Zink-Supplementierung in vivo führt zur Wiederherstellung der Immunfunktionen und zu einem Ausgleich zwischen Th1- und Th2-Zellen. Im Rahmen der vorliegenden Doktorarbeit sollte die Rolle von Zink in der Signaltransduktion von T-Zellen charakterisiert werden. Dabei konnte anhand von Phosphorylierungsnachweisen in primären T-Zellen und der humanen T-Zelllinie Jurkat festgestellt werden, dass die gängige Aktivierung über den TCR und eine Stimulation mit Zink in Kombination mit dem Ionophor Pyrithion zu einer unterschiedlichen Aktivierung der Kinasen Lck, p38, JNK2 und ERK1/2 führt. Die Untersuchungen zeigen, dass TCR-stimulierte T-Zellen intrazellulär Zink freisetzen, welches selbst keinen Einfluss auf die Signalweiterleitung in die Zelle hat. Werden die Zellen allerdings mit Zink/Pyrithion stimuliert, werden die Kinasen Lck und p38 aktiviert, während die TCR-stimulierte ERK1/2-Phosphorylierung inhibiert wird. Um diesen differentiellen Zink-Effekt aufzuklären, wurden im Anschluss verschiedene, in anderen Zelltypen bereits bekannte Wirkmechanismen von Zink hinsichtlich ihrer Gültigkeit in T-Zellen untersucht. Die Lck wird durch Zink in ihrer Aktivität auf unterschiedlichen Ebenen beeinflusst. In dieser Arbeit konnte gezeigt werden, dass die Aktivierung nach Zink/Pyrithion Stimulation zusätzlich abhängig ist von der Proteintyrosinphosphatase CD45. Des Weiteren ist der beobachtete Zink-Effekt unabhängig von CD3 und stellt damit ein intrazelluläres Ereignis dar. Eine Inhibition von MAPK Phosphatasen durch Zink als mögliche Erklärung für die p38-Aktivierung ist auszuschließen. Eine Zink-induzierte Phosphorylierung der p38 durch die alternative Aktivierung konnte durch Inhibitorstudien und den Einsatz von defizienten Zelllinien ebenfalls ausgeschlossen werden. Auch die Beteiligung des PKA-Signalweges konnte unter Verwendung verschiedener Aktivatoren und Inhibitoren ausgeschlossen werden. Nach Überprüfung unterschiedlicher Signalwege muss festgehalten werden, dass der molekulare Mechanismus des beobachteten Zink-Effektes in T-Zellen von den gängigen Mechanismen in anderen Zellen abweicht. Trotz unvollständiger Aufklärung des Mechanismus konnte ein Zusammenhang zwischen der Induktion von Th1-Antworten und der Aktivierung der p38/CREB Kaskade nachgewiesen werden. Durch die vorliegende Arbeit wird erstmals der Einfluss von Zink auf Th1-Zellen mit der Aktivierung der MAPK p38 in Zusammenhang gebracht. Die weitere Aufklärung des molekularen Mechanismus könnte Einfluss auf die Behandlung T-Zell vermittelter Erkrankungen haben und demnach neue Therapieperspektiven eröffnen. Zinc is an essential trace element with a variety of cellular functions in all organ systems. Disturbance of the cellular zinc availability by zinc deficiency leads to multiple disorders. Predominantly the cell-mediated immune response is influenced by zinc deprivation, and here in particular T-cell mediated functions. For example, the Th1/Th2 balance is disturbed leading to a reduced production of Th1 cytokines such as IFN-gamma and IL-2. Zinc supplementation in vivo causes reconstitution of Th1-mediated responses. To achieve a broad picture about the role of zinc in T-cell signal transduction, phosphorylation of several effector kinases in primary human T-cells and in the human T-cell line Jurkat was investigated. CD3-mediated activation and stimulation by zinc and the ionophore pyrithione led to differential activation of kinase Lck, MAPK p38, JNK1/2 and ERK1/2. Experiments showed that CD3-stimulated T-cells release intracellular zinc which has no effect on signal transduction itself. However, if cells were stimulated with zinc and pyrithione, the kinases Lck and p38 were activated, while CD3-induced ERK1/2 phosphorylation was reduced. To elucidate this differential zinc effect, several known zinc-mediated signalling mechanisms were investigated for their validity in T-cells. Lck is influenced by zinc in its activity at different levels. Experiments with Lck-negative cells showed that activation after stimulation with zinc and pyrithione additionally depends on protein tyrosine phosphatase CD45. Furthermore the observed zinc effect is independent from CD3 signalling and represents an intracellular event. MAPK phosphatase inhibition by zinc could be excluded as one possible explanation for p38 activation. Zinc induced phosphorylation of p38 by alternative activation mediated by Lck and ZAP70 could be also eliminated by inhibitor studies and the use of Lck and ZAP70 deficient cell lines. Further on, the involvement of the PKA signalling pathway was no explanation for the observed differential zinc effect, confirmed by the use of different inhibitors and activators. After verification of several signalling pathways, the molecular pathway of the observed zinc effect in T-cells seems to differ from established pathways in other cells. Although the mechanism could not be elucidated, it could be demonstrated that there is a connection between the induction of Th1 answers and the activation of p38/CREB cascade. For the first time the impact of zinc on Th1 cells could be linked to p38 activation. Further work on the molecular mechanism could have an influence on the treatment of T-cell mediated diseases and could open up new therapy perspectives.
Metal-mediated protein oligomerization is an emerging mode of protein-protein interaction. The C-terminal cytosolic domains of T-cell coreceptors CD4 and CD8alpha form zinc-bridged heterodimers with the N-terminal region of the kinase Lck, with each protein contributing two cysteinate ligands to the complex. Using size exclusion chromatography, (1)H NMR, and UV/visible absorption spectroscopy with cobalt(II) as a spectroscopic probe, we demonstrate that small peptides derived from these regions form metal-bridged heterodimers but also homodimers, in contrast to previous reports. The Lck-CD4 and Lck-CD8alpha cobalt(II)-bridged heterodimer complexes are more stable than the corresponding (Lck)(2)cobalt(II) complex by factors of 11 +/- 4 and 22 +/- 9, respectively. These studies were aided by the discovery that cobalt(II) complexes with a cobalt(II)(-Cys-X-X-Cys-)(-Cys-X-Cys-) chromophore show unusual optical spectra with one component of the visible d-d ((4)A(2)-to-(4)T(1)(P)) transition red-shifted and well separated from the other components. These results provide insights into the basis of specificity of metal-bridged complex formation and on the potential biological significance of metal-bridged homodimers in T-cells.
The trace element zinc is a crucial cofactor for many proteins involved in cellular processes like differentiation, proliferation and apoptosis. Zinc homeostasis is tightly regulated and disturbance of this homeostasis due to genetic defects, zinc deficiency, or supplementation influences the development and the progression of various infectious and autoimmune diseases. The immune system is strongly impaired during zinc deficiency, predominantly the cell-mediated response by T-lymphocytes. During zinc deprivation T-lymphocyte development, polarization into effector cells, and function are impaired. This leads to reduced T-cell numbers, a decreased ratio of type 1 to type 2 T-helper cells with reduced production of T-helper type 1 cytokines like interferon-gamma, and compromised T-cell mediated immune defense. Accordingly, disturbed zinc homeostasis increases the risk for infections, and zinc supplementation restores normal immune function. Furthermore, several disorders, like mycobacterial infections, asthma, diabetes, and rheumatoid arthritis are accompanied by decreased zinc levels and in some cases disease progression can be affected by zinc supplementation. On the molecular level, apoptosis of T-cell precursors is influenced by zinc via the Bcl-2/Bax ratio, and zinc ions inhibit caspases-3, -6, -7, and -8. In mature T-cells, zinc interacts with kinases involved in T-cell activation, like protein kinase C and the lymphocyte protein tyrosine kinase (Lck), while higher zinc concentrations are inhibitory, reducing the activities of the interleukin-1 receptor-associated kinase (IRAK) and calcineurin. Taken together, zinc homeostasis influences T-lymphocytes via several molecular targets, leading to a modulation of T-cell-dependent immune responses.
Recent years have brought a paradigm shift for the role of the essential trace element zinc in immunity. Although its function as a structural component of many enzymes has been known for decades, current experimental evidence points to an additional function of the concentration of free or loosely bound zinc ions as an intracellular signal. The activity of virtually all immune cells is modulated by zinc in vitro and in vivo. In this review, we discuss the interactions of zinc with major signaling pathways that regulate immune cell activity, and the implications of zinc deficiency or supplementation on zinc signaling as the molecular basis for an effect of zinc on immune cell function.
Zinc cations at concentrations of 0.2 mM and greater catalyzed specific phosphorylation, by ATP, of two membrane-associated proteins from rat hippocampus. These proteins, corresponding to molecular weights of 60 and 49 kDa, were phosphorylated primarily at tyrosine residues. The 60-kDa protein was identified as p60c-src by immunoprecipitation using two different p60src-specific monoclonal antibodies. The 49-kDa protein co-immunoprecipitated with p60c-src. Cyanogen bromide cleavage of p60c-src and the 49-kDa protein phosphorylated in the presence of Zn2+ gave different patterns of phosphopeptides. It is suggested that tyrosine phosphorylation of p60c-src and the p60c-src-associated 49-kDa protein may be a way of zinc participation in hippocampal neurotransmission.
The levels of intracellular free Ca(II) and Zn(II) during dexamethasone (dex)-induced apoptosis in CEM cell lines were determined by 19F nuclear magnetic resonance (NMR), using the fluorinated intracellular chelator 1,2-bis-(2- amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5-FBAPTA). The effects of these divalent metal ions on growth rate and DNA degradation were evaluated. Measurements were done on one dex-sensitive (CEM-C7) and three different dex-resistant variants (CEM-C1, CEM-4R4, and CEM-ICR27). Dex caused a continuous increase in the Ca(II) level in dex-sensitive CEM-C7 cells, while in CEM-C1 cells dex caused an initial increase in the Ca(II) level which in approximately 36 h was restored to its normal value. The intracellular Ca(II) level in CEM-4R4 cells was not significantly affected by dex, while that of CEM-ICR27 cells decreased after dex incubation. Only the dex-sensitive CEM-C7 cells showed dex-induced DNA degradation. An intracellular free Zn(II) level of approximately 1 nM was measured for the dex-resistant CEM-C1 cells. No detectable level of intracellular Zn(II) was found in the other cell lines. Incubation with < 100 microM Zn(II) did not inhibit dex-induced apoptosis in CEM-C7 cells (e.g., DNA degradation). Treatment with approximately 250 microM Zn(II) caused significant decrease in growth rate in all cell lines and prevented dex-induced DNA degradation in CEM-C7 cells. A calibrated amount of Ca(II) ionophore (A23187), used to increase Ca(II) concentrations up to the dex-induced levels, did not induce DNA degradation in CEM-C7 or CEM-C1 cells. While elevation of intracellular Ca(II) by itself is not sufficient to initiate apoptosis in CEM-C7 cells, the results reported here suggest that Ca(II) is involved in the killing mechanism as a secondary factor. The combination of dex and ionophore caused significant DNA degradation in CEM-C1 cells, which normally showed resistance to each compound individually. The combination of dex and the Zn(II) chelator phenanthroline also caused extensive DNA degradation in the normally dex-resistant CEM-C1 cells, suggesting that Zn(II) plays a role in the dex resistance of these cells.
Zinc is known to play a central role in the immune system, and zinc-deficient persons experience increased susceptibility to a variety of pathogens. The immunologic mechanisms whereby zinc modulates increased susceptibility to infection have been studied for several decades. It is clear that zinc affects multiple aspects of the immune system, from the barrier of the skin to gene regulation within lymphocytes. Zinc is crucial for normal development and function of cells mediating nonspecific immunity such as neutrophils and natural killer cells. Zinc deficiency also affects development of acquired immunity by preventing both the outgrowth and certain functions of T lymphocytes such as activation, Th1 cytokine production, and B lymphocyte help. Likewise, B lymphocyte development and antibody production, particularly immunoglobulin G, is compromised. The macrophage, a pivotal cell in many immunologic functions, is adversely affected by zinc deficiency, which can dysregulate intracellular killing, cytokine production, and phagocytosis. The effects of zinc on these key immunologic mediators is rooted in the myriad roles for zinc in basic cellular functions such as DNA replication, RNA transcription, cell division, and cell activation. Apoptosis is potentiated by zinc deficiency. Zinc also functions as an antioxidant and can stabilize membranes. This review explores these aspects of zinc biology of the immune system and attempts to provide a biological basis for the altered host resistance to infections observed during zinc deficiency and supplementation.
The possible effects of zinc in the modulation of the activity of glycolytic enzymes phosphofructokinase and pyruvate kinase through tyrosine kinase-mediated signal transduction in isolated digestive gland cells from mussels (Mytilus galloprovincialis Lam.) were investigated. Addition of micromolar concentrations of zinc resulted in both time- and concentration-dependent stimulation of glycolytic enzyme activities similar to those previously observed with insulin; however, zinc pretreatment prevented the glycolytic effect of insulin in mussel cells. The insulin-like effect of zinc was mediated by increased tyrosine phosphorylation of multiple proteins, as demonstrated by Western blotting with antiphosphotyrosine antibodies. The pattern of zinc-induced phosphorylation resembled that induced by insulin. Moreover, both zinc and insulin induced activation of mitogen activated protein kinases (MAPKs); however, whereas zinc gave a clear effect on the stress-activated p-38 MAPK, insulin activated extracellular-activated MAPK (ERK2) and inhibited p-38. The results demonstrate that zinc can act as a physiological regulator of tyrosine kinase-mediated cell signaling in mussel digestive gland cells, in particular at the level of MAPK activation. Activation of p-38 by zinc may be a key step in prevention of the glycolytic effect of insulin in mussel cells. These data underline the importance of cross talk between different MAPKs in determination of the response to extracellular stimuli in marine invertebrate cells.
Inhibition of NK cell cytotoxicity by killer cell Ig-like receptors (KIR) depends on phosphorylation of cytoplasmic tyrosines in KIR, which recruit tyrosine phosphatase Src homology protein tyrosine phosphatase 1. It is not clear how KIR, whose function lies downstream of a tyrosine kinase, succeeds in blocking proximal NK cell activation signals upon binding HLA class I on target cells. Here we show that mixing NK cells with insect cells expressing HLA-C was sufficient to induce clustering of KIR, and phosphorylation of KIR and SHP-1. Transient phosphorylation of KIR was detected in the presence of pervanadate, an inhibitor of protein tyrosine phosphatases, at suboptimal concentration. Phosphorylation of KIR was specifically induced by ligand binding because it was detected only when HLA-C was loaded with a peptide that permits KIR binding. KIR phosphorylation was not dependent on ICAM-1-mediated adhesion and was not blocked by inhibition of actin polymerization, but required Zn(2+). Fluorescence resonance energy transfer between HLA-C molecules revealed close molecular interactions induced by KIR binding. These results demonstrate tight clustering of KIR and rapid KIR phosphorylation induced simply by binding to HLA-C. The unique property of KIR to become phosphorylated in the absence of adhesion and of actin cytoskeleton rearrangement explains how KIR can efficiently block early activation signals during NK-target cell contacts.
The divalent cation-binding properties of the human insulin receptor tyrosine kinase domain were examined kinetically and by electron paramagnetic resonance and circular dichroic spectroscopy. The protein-tyrosine kinase activity of the purified cytoplasmic domain can be activated nearly 10-fold by 3 mM Mn2+ in the presence or absence of 5 mM Mg2+. Electron paramagnetic resonance spectra of the purified, acid-denatured kinase domain and assays of EDTA-treated kinase show that the purified protein does not possess residual, tightly bound Mn2+. Electron paramagnetic resonance spectroscopy was used to directly measure the binding constant of the kinase domain for Mn2+. The results indicate that the recombinant cytoplasmic domain of the human insulin receptor does not bind Mn2+ tightly in the absence or presence of MgATP (Kd greater than 0.8 mM). Furthermore, the enzyme does not show a strong preference for MnATP binding when both MgATP and MnATP are present. The far-ultraviolet circular dichroic spectrum of this domain is characterized by a negative maximum at 207 nm. In the presence of Mn2+, but not Mg2+, changes in the mean residue-weight ellipticity at 207 nm occur that are consistent with a decrease in alpha-helical content. The addition of ATP to Mn2(+)-bound protein does not further perturb the spectrum. We conclude that Mn2+ ions, although they bind weakly, induce an activating conformational change in the secondary structure of the human insulin receptor cytoplasmic domain. Activation by Mn2+ is unlikely to be significant in intact cells, but it may mimic the action of a physiological activator.
In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested (Parker, P.J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M.D., and Ullrich, A. (1986) Science 233, 853-859). In the present report, we demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes, and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn2+, while Fe2+ and Mn2+ are only partially counteractive. Our results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca2+, phorbol ester, or antigen.
cis-Fatty acids such as oleic acid or linoleic acid have been previously shown to induce full activation of protein kinase C in the absence of Ca2+ and phospholipids (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193; Murakami, K., Chan, S.Y., and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429). In this study, we have investigated the effects of various metal ions on protein kinase C activity without the interference of Ca2+ since cis-fatty acid requires no Ca2+ for protein kinase C activation. Here we report a specific interaction of Zn2+ with protein kinase C in either a positive or negative cooperative fashion in concert with Ca2+. At low concentrations (approximately 5 microM) of Ca2+, Zn2+ enhances protein kinase C activity induced by both oleic acid and phosphatidylserine/diolein. In contrast, Zn2+ inhibits the activity at higher concentrations (over 50 microM) of Ca2+. In the absence of Ca2+, Zn2+ shows no effect on protein kinase C activity. Our results suggest that Zn2+ does not recognize or interact with protein kinase C in the absence of Ca2+, that protein kinase C possesses high and low affinity Ca2+-binding sites, and that at least one Zn2+-binding site exists which is distinct from Ca2+-binding sites.
The autophosphorylation reaction responsible for conversion of insulin receptor (from human placenta) to an active tyrosyl-protein kinase was shown to be inhibited by Zn2+ and other divalent metal ions. The order of inhibitory potency was found to be Cu2+ greater than Zn2+, Cd2+ greater than Co2+, Ni2+. Autophosphorylation of insulin receptor was almost completely blocked by 10 microM Zn2+. Zn2+, however, did not appear to affect the binding of insulin to its receptor. Histidine, a chelator of Zn2+, protected against the inhibitory effects of Zn2+. The failure of histidine to regenerate the competence of the Zn2+-inhibited receptor to undergo autophosphorylation suggested that the inhibition by Zn2+ was irreversible. In addition to inhibiting autophosphorylation, Zn2+ inhibited the tyrosyl-protein kinase activity of highly purified phosphorylated receptor. Zn2+ was also observed to inhibit phosphotyrosyl-protein phosphatase activity present in preparations of partially purified insulin receptor. These inhibitory effects of Zn2+ should be considered in the design of protocols for the isolation and handling of insulin receptor and possibly other tyrosine kinases. Additionally, the possible physiological significance of the inhibition of insulin receptor kinase by Zn2+ is discussed in light of the fact that Zn2+ is accumulated in and secreted from pancreatic islet cells together with insulin.
Epidermal growth factor stimulates a cyclic AMP-independent protein kinase associated with membrane vesicles derived from human epidermoid carcinoma cells (Carpenter, G., King, L., Jr., and Cohen, S. (1979) J. Biol. Chem. 254,. 4884-4891). The kinase specifically phosphorylates tyrosyl residues in a Mr = 150,000 membrane protein (Ushiro, H., and Cohen, S. (1980) J. Biol. Chem. 255, 8363-8365). We show that the reverse reaction, catalyzed by a phosphotyrosyl-protein phosphatase associated with the membrane, is inhibited by Zn2+. Dephosphorylation of phosphotyrosyl residues in the Mr = 150,000 protein is completely inhibited by Zn2+ at concentrations as low as 10 microM, whereas other divalent cations have no substantial effect. Inhibition of the phosphatase was reversed by EDTA and the activity in membrane preparations was increased by EDTA or fluoride, agents commonly thought to be phosphatase inhibitors. Acid hydrolysis of the membrane proteins followed by analysis of phosphoamino acids by two-dimensional electrophoresis revealed that the phosphatase hydrolyzed phosphotyrosyl in preference to phosphoseryl residues. The specific inhibition of this phosphatase activity by low concentrations of Zn2+ may be indicative of the physiological importance of Zn2+ in the regulation of cellular phosphotyrosyl-protein levels.
Several sarcoma-inducing viruses encode protein kinases that phosphorylate tyrosine residues. Such enzymatic activities can be detected within the detergent-insoluble matrix of transformed fibroblasts. We have analysed the protein kinase activities in two murine lymphoma cell lines ( MBL2 and LSTRA) induced by Moloney murine leukemia virus (Mo-MuLV). After incubation of the detergent-insoluble matrix of these cells with [gamma-32P]ATP, several alkali-resistant phosphoproteins, including a very heavily labelled 55 000 mol. wt. protein ( p55 ), have been detected in LSTRA, reflecting the activity of a protein kinase specific to this cell line. This protein kinase activity shares some of the distinctive properties of the protein kinases of transforming viruses, i.e., specificity for tyrosine residues, association with membranous and/or cytoskeletal structures, and inhibition by a synthetic peptide derived from the phosphorylation site of pp60src. In view of the absence of a transforming gene in MoMuLV , it is likely that the high level of protein kinase detected in the LSTRA cell line arises from the expression of a cellular gene.
The particulate fraction from a lymphoma cell line, LSTRA, was found to contain an apparent high level of tyrosine protein kinase activity. When this fraction was incubated with [gamma-32P]ATP in the presence of 10 mM MnCl2, hydrolyzed, and assayed, 70--80% of the radioactivity recovered in phosphoamino acids was in phosphotyrosine. Gel electrophoresis of the proteins showed that a large portion of the 32P was in a single protein with a molecular weight of approximately 58,000. The phosphorylated residue in this protein was identified as phosphotyrosine. Detergent extracts of the particulate fraction from LSTRA cells contained both the Mr 58,000 protein and the enzyme responsible for its phosphorylation. These extracts were found to catalyze the phosphorylation of the tyrosine residue in the synthetic peptide, Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gln-Gly, corresponding to the sequence around the tyrosine that is phosphorylated in pp60src; the Km for the peptide in this reaction was 5 mM. High-performance liquid chromatography was used to assay for this phosphorylation. A second peptide was synthesized that contained two additional arginine residues whose presence permitted the phosphorylation of the peptide to be measured by a simple assay using phosphocellulose paper. The Km for this peptide was 3--4 mM, indicating that the presence of the additional arginine residues did not alter the apparent affinity of the kinase for the peptide.
In Prasad’s initial description of the extraordinarily complex syndrome produced by profound zinc deficiency in humans, evidence of increased susceptibility to infection was one manifestation of this nutritional deficiency (Prasad et al. 1961). Holstein—Friesian cattle bearing the highly lethal A46 mutation also have severe zinc deficiency that is attributable to failure to absorb zinc normally from the gastrointestinal tract. These cattle have a profound immune deficiency disease and frequently die from infections. Their immunodeficiency disease is characterized by failure of development of normal T cell-mediated immunities, defective delayed hypersensitivity reactions, feeble allograft immunity, failure of normal development of the thymus, deficiencies of T lymphocytes in blood and thymus-dependent regions of the lymphoid tissues and defective defences against viruses, fungi and bacteria. In addition, these genetically defective cattle exhibit striking malfunctions of the gastrointestinal tract, and a pleurioroficial dermatitis characterized by parakeratoses. All of these manifestations, including all of the immunodeficiencies which occur in these zinc-deficient cattle, are corrected by administration of zinc orally or parenterally (Brummerstedt et al. 1971; Andresen et al. 1974).
This chapter describes viral protein-tyrosine kinases and discusses their roles in viral transformation. For this, the properties of the individual viral protein-tyrosine kinases are described in the chapter and are contrasted to the cognate cellular enzymes that are encoded by the cellular genes homologous to the viral oncogenes. The chapter discusses a number of transformed cell types where altered tyrosine phosphorylation is evident and describes the general properties of protein-tyrosine kinases and their common structural features. In addition, the chapter includes a progress report on the identification of substrates for the viral protein-tyrosine kinases. The chapter discusses the way in which viral protein-tyrosine kinases differ from their cellular counterparts—enzymes that clearly coexist peaceably with normal cells. In the chapter, the properties of the viral enzymes are compared and contrasted to those of the growth factor receptor protein-tyrosine kinases, particularly because of the abnormal growth state of transformed cells.
Zinc is an essential element in The development and function of the immune system. This article reviews some of the newest data on its role in immunity.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
p56lck, a lymphocyte-specific member of the src family of cytoplasmic protein-tyrosine kinases, is associated noncovalently with the cell surface glycoproteins CD4 and CD8, which are expressed on functionally distinct subpopulations of T cells. Using transient coexpression of p56lck with CD4 or CD8 alpha in COS-7 cells, we show that the unique N-terminal region of p56lck binds to the membrane-proximal 10 and 28 cytoplasmic residues of CD8 alpha and CD4, respectively. Two cysteine residues in each of the critical sequences in CD4, CD8 alpha, and p56lck are required for association. Our results suggest a novel role for cysteine-mediated interactions between unrelated proteins and provide a model for the association of other src-like cytoplasmic kinases with transmembrane proteins.
The CD4 (T4) antigen is a cell-surface glycoprotein that is expressed predominantly on the surface of helper T cells and has been implicated in the regulation of T-cell activation and in the associative recognition of class II antigens of the major histocompatibility complex. In addition, the CD4 antigen appears to serve as a receptor for the human immunodeficiency virus (HIV). An important question has been whether the CD4 receptor is linked to an intracellular mediator that could regulate the activation of the CD4+ subset. In this paper, we provide preliminary evidence that the CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (PTK) of 55-60 kDa, which is expressed specifically in T cells. The PTK is the human analogue of the murine pp56LSTRA (pp56lck) and has significant homology with c-src, c-yes, and other members of the src family. The identification of the PTK associated with CD4 receptor was made by use of an antiserum to a synthetic peptide that was deduced from the DNA sequence of PTK. Two-dimensional nonequilibrium pH gradient gel electrophoresis/NaDodSO4/PAGE revealed the kinase to focus as a heterogeneous collection of spots in the pH range of 4.0-5.0. Furthermore, in vitro phosphorylation revealed the phosphorylation of two additional polypeptides at 40 and 80 kDa, in addition to the autophosphorylation of the PTK at 55-60 kDa. The potential importance of the association between the CD4 receptor and the PTK of T cells is discussed in relation to T-cell activation and HIV infectivity.
In human platelet membranes enhanced tyrosine phosphorylation of certain proteins was observed when Zn2+ instead of Mg2+ or Mn2+ was used as a divalent cation for the kinase reaction. An enhanced level of phosphate incorporation into tyrosine residues occurred into a 68 kDa polypeptide besides the 45 kDa and 105 kDa proteins. Preincubation of platelet membranes with TBR-IgG showed a concentration-dependent inhibition of the phosphorylation of the 45, 68 and 105 kDa proteins. Moreover, pp60c-src, representing the major protein tyrosine kinase activity in platelets, was found to be stimulated by Zn2+. The data, thus, support the assumption that pp60c-src kinase is responsible for Zn2+ stimulated tyrosine phosphorylation.
A major protein tyrosine phosphatase (PTPase 1B) has been isolated in essentially homogeneous form from the soluble and particulate fractions of human placenta. Unexpectedly, partial amino acid sequences displayed no homology with the primary structures of the protein Ser/Thr phosphatases deduced from cDNA clones. However, the sequence is strikingly similar to the tandem C-terminal homologous domains of the leukocyte common antigen (CD45). A 157-residue segment of PTPase 1B displayed 40% and 33% sequence identity with corresponding regions from cytoplasmic domains I and II of human CD45. Similar degrees of identity have been observed among the catalytic domains of families of regulatory proteins such as protein kinases and cyclic nucleotide phosphodiesterases. On this basis, it is proposed that the CD45 family has protein tyrosine phosphatase activity and may represent a set of cell-surface receptors involved in signal transduction. This suggests that the repertoire of signal transduction mechanisms may include the direct control of an intracellular protein tyrosine phosphatase, offering the possibility of a regulatory balance with those protein tyrosine kinases that act at the internal surface of the membrane.
A large number of serum growth factors stimulate cellular mitogenesis by interaction with a family of cell-surface receptors that possess an intrinsic, ligand-sensitive, protein tyrosine kinase activity. These receptor tyrosine kinases (RTKs) comprise an extracellular ligand-binding domain that is linked directly to a cytoplasmically oriented, catalytic domain, which not only transduces a hormonal signal, but also generates a biochemical message. While it is widely appreciated that these proteins play a key role in cellular growth regulation, little is known about the biochemical cascades that RTKs trigger. Even the first steps, which must include tyrosine phosphorylation of cytoplasmic substrate polypeptides, are only beginning to be elucidated. An understanding of the connection between protein phosphorylation and transcriptional activation of specific genes seems distant, at best. Two recent advances have already brought us substantially closer to an elucidation of RTK function. The primary structures of a number of RTKs have recently become available from cloned cDNA sequences, and comparative analysis has offered clues to receptor domain function. This family of cell-surface glycoproteins can now be classified into three distinct structural subclasses that may reflect unique molecular pathways of cellular activation. In combination with classical biochemical approaches, the availability of cloned cDNAs for RTKs and their respective ligands opens the way for detailed dissection of the mechanism(s) of growth signal generation. A second conceptual breakthrough has come from the realization that a number of oncogene products represent altered cellular RTKs. Such oncogenic homologs provide invaluable clues to the mechanisms of normal growth factor receptors, in addition to enhancing our understanding of cellular transformation and carcinogenesis. In the future, a combination of these biochemical and genetic approaches will undoubtedly bring a fuller understanding of the mitogenic response in both normal and transformed cells.
Tyrosine protein kinases are important both in the normal regulation of cellular proliferation and in the oncogenic transformation of cells by several tumour viruses. The LSTRA Moloney murine leukaemia virus (M-MuLV)-induced thymoma cell line contains approximately 20-fold more phosphotyrosine in protein than do typical haematopoietic cell lines; this seems to result from the expression of an abnormally high level of a cellular tyrosine protein kinase termed p56tck (refs 3, 4). This kinase is normally expressed at low levels in most, but not all, murine T cells. The elevated levels of p56tck could contribute to the malignant properties of LSTRA cells. Therefore, we have isolated cloned complementary DNAs encoding the whole of p56tck. Sequence analysis shows it to be a novel cellular tyrosine protein kinase which is distinct from all others described to date. p56tck is encoded in LSTRA cells by a hybrid messenger RNA; approximately 200 nucleotides at the 5' end of the mRNA are identical to the 5' end of the genome of M-MuLV. The three- to ninefold transcriptional activation of the gene therefore results from retroviral promoter insertion.
The CD4 and CD8 T cell antigens are thought to transduce an independent signal during the process of T cell activation. We report our evaluation of the possible involvement of the lymphocyte-specific tyrosine kinase p56lck in these transduction pathways. Our data demonstrate that p56lck is specifically modulated with either CD4 or CD8 following antibody-mediated cross-linking of these molecules and that a large fraction of the total cellular lck protein can be coimmunoprecipitated with these surface glycoproteins. These results suggest that p56lck is functionally and physically associated with CD4/CD8 in normal murine T lymphocytes and support the concept that an independent signal is transduced by the interaction of these surface molecules with major histocompatibility complex determinants.
Following their separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, labeled proteins obtained from cultured canine prostatic epithelial cells incubated with [35S]-methionine and [32P]phosphate were subjected to alkali treatment, a method that is currently used to detect phosphotyrosine-containing proteins. Significant amounts of 35S-labeled material were lost during the alkali treatment. The crosslinking of proteins within the gels by glutaraldehyde treatment eliminated protein losses and did not alter the efficiency of phosphoester bond hydrolysis by alkali treatment. Consequently, the time required to detect alkali-resistant phosphoproteins by autoradiography was greatly reduced. Prostatic phosphoproteins were also shown to contain phosphotyrosine, indicating the presence of tyrosine protein kinase activity in these proliferating epithelial cells.
Rat liver cytosol casein kinases 1 and 2 were stimulated by free Mg2+, but the optimal concentration of cation varied with both the casein kinase and the protein substrate used. Mn2+, but neither Ca2+ nor Zn2+, could efficiently substitute for Mg2+ in forming the bivalent-cation-ATP complex used as substrate, but free Mn2+ was inhibitory. The magnitude of these effects depended on the type of casein kinase and the protein substrate used. These results support the idea that, besides the effects of Mg2+ as a component of the Mg-ATP complex, or through interaction with the protein substrate, free Mg2+ is an allosteric effector of both casein kinases.
The regulation of protein phosphorylation by Zn2+ ions and by other divalent cations was studied in membrane vesicles from a normal mouse epithelial cell line, MMC-E (Mus musculus castaneous). Four major phosphoacceptor polypeptides were found in these membranes. Micromolar concentrations of Zn2+ ions inhibited the phosphorylation of the epidermal growth factor (EGF) receptor and of threonine residues in a 47,000-dalton polypeptide. In contrast, two polypeptides with molecular weights of 54,000 and 57,000 showed increased phosphorylation, mainly of serine residues, in the p.esence of Zn2+ ions. These results were not obtained using similar concentrations of other divalent cations and were apparently not due to an effect of Zn2+ ions on phosphoprotein phosphatases. Thus, the effects of Zn2+ ions on protein phosphorylation in membrane vesicles are complex and are not restricted to an inhibition of a single protein phosphatase or kinase.
Although alteration of dietary protein and lipid content has been demonstrated to modify the progression of murine autoimmune disease, relatively little is known regarding the influence of trace metal nutriture on such immunologic phenomena. To investigate such interactions, groups of NZB mice at 6 wk and 6 mo of age were fed diets containing 100 ppm zinc (control), 9 ppm zinc (designated marginal deficiency), 5 ppm zinc (designated moderate deficiency), and 2.5 ppm zinc (designated severe deficiency). Further, to control for the inanition of mice fed the zinc deficient diets, a group of mice were pair-fed the control diet in amounts equal to the intake of mice fed 5 ppm zinc. NZB mice who began the study at 6 wk of age and who were fed 9 ppm zinc experienced a progression of autoimmune disease similar to that observed in the ad libitum-fed control mice. In contrast, young mice fed either 5 or 2.5 ppm zinc showed a delay in the onset of autoimmune hemolytic anemia, as reflected by significantly higher packed cell volume and hemoglobin values, lower titers of anti-erythrocyte autoantibodies and serum immunoglobulins, and prolonged lifespan. The delay in the progression of these features was partially accounted for by depression of food intake and consequent caloric deprivation. However, mice fed 5 ppm zinc had higher packed cell volume and hemoglobin values, and lower anti-erythrocyte autoantibody titers and serum immunoglobulin levels than did their pair-fed controls, indicating that deprivation of zinc accounted for a significant share of the alteration in autoimmunity by nutritional manipulation. In contrast, NZB mice that began to consume the diets at 6 mo of age showed a moderate slowing but not a reversal of the autoimmune hemolytic anemia. The retardation of autoimmunity in older NZB mice was almost entirely due to the inanition that accompanied zinc deprivation, as reflected by a similar slowing of the onset of autoimmune hemolytic anemia in pair-fed controls. These results reaffirm the possibility of utilizing nutritional factors to alter murine autoimmune disease, and further establish the potential importance of trace metals in the development and maintenance of immunocompetence.
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Krebs The Enzymes 17
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