Fc-receptor mediated protein phosphorylation in murine peritoneal macrophages

The effect of Fc receptor engagement on protein phosphorylation in murine peritoneal macrophages has been investigated. Treatment of macrophage cultures with insoluble immune complexes resulted in enhanced phosphorylation of six proteins at 73, 66, 53, 37, 31 and 25 kD. Comparison of the protein phosphorylation patterns induced by immune complexes with those induced by agents which mimic the actions of well known intracellular second messengers (i.e., A23187, dibutyryl cAMP, or phorbol myristate acetate) revealed substantial similarity between Fc receptor induced events and those induced in response to phorbol diesters. There were, however, two phosphorylated proteins which were only seen following stimulation with immune complexes. Thus, more than one kind of protein kinase activity appears to be involved in Fc receptor mediated stimulation of macrophage function.     

Platelet-derived growth factor induces multisite phosphorylation of pp60c-src and increases its protein-tyrosine kinase activity.

We have shown previously that pp60c-src is a substrate for protein kinase C in vivo and that the target of protein kinase C phosphorylation in mammalian pp60c-src is serine 12. We now demonstrate that in addition to tumor promoters, all activators of phosphatidylinositol turnover that we have tested in fibroblasts (platelet-derived growth factor, fibroblast growth factor, serum, vasopressin, sodium orthovanadate, and prostaglandin F2 alpha) lead to the phosphorylation of pp60c-src at serine 12. In addition to stimulating serine 12 phosphorylation in pp60c-src, platelet-derived growth factor treatment of quiescent fibroblasts induces phosphorylation of one or two additional serine residues and one tyrosine residue within the N-terminal 16 kilodaltons of the enzyme and activates its immune complex protein-tyrosine kinase activity.     

Spred-2 steady-state levels are regulated by phosphorylation and Cbl-mediated ubiquitination

Spred proteins modulate growth factor receptor signaling by inhibiting the Ras-MAPK cascade. Here, we show that Spred-1, Spred-2, and Spred-3 are ubiquitinated in HEK293T cells stimulated with epidermal growth factor (EGF) or pervanadate. Spred-2 tyrosines Y228 and/or Y231 in the Kit binding domain were identified as putative phosphorylation site(s) critical for Spred-2 ubiquitination. Depletion of Cbl and Cbl-b E3 ubiquitin ligases by RNA interference, or overexpression of a Cbl dominant inhibitory mutant (Cbl-N), inhibited Spred-2 ubiquitination, while conversely, wild type Cbl enhanced Spred-2 ubiquitination. Interaction of Spred-2 with Cbl-N was detectable by co-immunoprecipitation and required the Cbl SH2 domain and Spred-2 Y228 and Y231 residues. Studies on endogenous Spred-2 in ME4405 melanoma cells showed that pervanadate induced Spred-2 ubiquitination and a marked reduction in Spred-2 steady-state levels that was partially blocked by the proteasomal inhibitor, MG-132. These results suggest a role for Spred-2 tyrosine phosphorylation and ubiquitination in controlling Spred-2 expression levels.     

Angiotensin II stimulates protein-tyrosine phosphorylation in a calcium-dependent manner.

Cellular responses to epidermal growth factor (EGF) are dependent on the tyrosine-specific protein kinase activity of the cell-surface EGF receptor. Previous studies using WB rat liver epithelial cells have detected at least 10 proteins whose phosphotyrosine (P-Tyr) content is increased by EGF. In this study, we have examined alternate modes of activating tyrosine phosphorylation. Treatment of WB cells with hormones linked to Ca2+ mobilization and protein kinase C (PKC) activation, including angiotensin II, [Arg8]vasopressin, or epinephrine, stimulated rapid (less than or equal to 15-s) and transient increases in the P-Tyr content of several proteins (p120/125, p75/78, and p66). These proteins, detected by anti-P-Tyr immunoblotting, were similar in molecular weight to a subset of EGF-sensitive P-Tyr-containing proteins (P-Tyr-proteins). The increased P-Tyr content was confirmed by [32P]phosphoamino acid analysis of proteins recovered by anti-P-Tyr immunoprecipitation. Elevating intracellular [Ca2+] with the ionophore A23187 or ionomycin or with the tumor promoter thapsigargin mimicked the effects of hormones on tyrosine phosphorylation, whereas treatment with a PKC-activating phorbol ester did not. In addition, responses to angiotensin II were not diminished in PKC-depleted cells. Ca2+ mobilization, measured by fura-2 fluorescence, was coincident with the increase in tyrosine phosphorylation in response to angiotensin II or thapsigargin. Loading cells with the intracellular Ca2+ chelator bis-(o-aminophenoxy)ethane-N ,N ,N' , N'-tetraacetic acid (BAPTA) inhibited the appearance of all P-Tyr-proteins in response to angiotensin II, thapsigargin, or ionophores, as well as two EGF-stimulated P-Tyr-proteins. The majority of EGF-stimulated P-Tyr-proteins were not affected by BAPTA. These studies indicate that angiotensin II can alter protein-tyrosine phosphorylation in a manner that is secondary to, and apparently dependent on, Ca2+ mobilization. Thus, ligands such as EGF and angiotensin II, which act through distinct types of receptors, may activate secondary pathways involving tyrosine phosphorylation. These results also raise the possibility that certain growth-promoting effects of Ca2+ -mobilizing agents such as angiotensin II may be mediated via tyrosine phosphorylation.     

DMSO increases tyrosine residue phosphorylation in membranes from murine erythroleukemia cells

Phosphorylation of membranes from murine erythroleukemia cells was performed in the presence and absence of the polar solvent dimethyl sulfoxide. Quantitation of the phosphoamino acid content revealed that DMSO stimulated phosphotyrosine accumulation by three-fold; serine and threonine phosphorylation decreased significantly. We had previously shown that DMSO stimulated tyrosine residue phosphorylation of the hepatic epidermal growth factor receptor. EGF had little effect in MEL membranes; therefore, DMSO results in accumulation of phosphotyrosine in cell membranes that do not exhibit significant EGF-dependent phosphorylation.     

Nitric oxide regulates human sperm capacitation and protein-tyrosine phosphorylation in vitro.

The aim of the present study was to investigate whether the generation of nitric oxide by human spermatozoa is associated with human sperm capacitation and with the tyrosine phosphorylation of sperm proteins. Human spermatozoa were capacitated in the presence or absence of nitric oxide-releasing compounds or nitric oxide synthase inhibitors, and then the percentage of acrosome loss induced by human follicular fluid or by calcium ionophore was determined. The presence of the nitric oxide-releasing compounds primed spermatozoa to respond earlier to human follicular fluid whereas nitric oxide synthase inhibitors decreased the percentage of acrosome reaction. Moreover, nitric oxide modulated tyrosine phosphorylation of sperm proteins. A tight correlation between capacitation and tyrosine phosphorylation regulated by nitric oxide was observed. Results indicate that nitric oxide is involved in human sperm capacitation and emphasize the importance of oxidoreduction reactions in the fine control of sperm physiology.     

Developmentally regulated protein-tyrosine kinase genes in Dictyostelium discoideum.

Dictyostelium discoideum, an organism that undergoes development and that is amenable to biochemical and molecular genetic approaches, is an attractive model organism with which to study the role of tyrosine phosphorylation in cell-cell communication. We report the presence of protein-tyrosine kinase genes in D. discoideum. Screening of a Dictyostelium cDNA expression library with an anti-phosphotyrosine antibody identifies fusion proteins that exhibit protein-tyrosine kinase activity. Two distinct cDNAs were identified and isolated. Though highly homologous to protein kinases in general, these kinases do not exhibit many of the hallmarks of protein-tyrosine kinases of higher eucaryotes. In addition, these genes are developmentally regulated, which suggests a role for tyrosine phosphorylation in controlling Dictyostelium development.     

Recombinant granulocyte-macrophage colony-stimulating factor increases adenylate cyclase activity in murine peritoneal macrophages

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotrophic cytokine which stimulates the function and proliferation of macrophage populations. Although the effects of GM-CSF are diverse and GM-CSF has entered into clinical trials, relatively little is known about signal transduction pathways utilized by GM-CSF. In view of previous studies which have suggested that some of the effects of GM-CSF on monocyte-macrophages can be mimicked by agents which increase intracellular cAMP, we investigated the effect of rGM-CSF on adenylate cyclase (AC) activity in murine peritoneal macrophages. Adenylate cyclase activity was quantitated in macrophage membrane preparations and in intact cells. In seven separate experiments, GM-CSF (50 U/ml) increased AC activity by 61(6)% relative to macrophages treated with carrier medium alone. A dose-dependent increase in AC activity was observed (10 to 100 U/ml) which peaked within 1 to 5 min after the addition of GM-CSF and returned to basal levels by 10 to 20 min. Lineweaver-Burk analysis revealed that the Vmax of macrophage AC was increased from 0.40 to 0.52 pmoles cAMP/min by GM-CSF but the Km was unchanged. Intracellular cAMP was increased by GM-CSF to 129(27)% of control values by 1 min of treatment (n = 6). Under similar experimental conditions, GM-CSF did not increase the activity of PK C (n = 14) or phospholipase A2 (n = 3) in peritoneal macrophages. These data show that macrophage adenylate cyclase activity is rapidly stimulated by GM-CSF. Moreover, these findings support further study of the role of cAMP in transmitting the intracellular signals initiated by GM-CSF in tissue macrophages.     

Lipopolysaccharide increases glucocorticoid receptor expression in murine macrophages. A possible mechanism for glucocorticoid-mediated suppression of endotoxicity.

In a previous study we demonstrated that IFN-gamma induced an increase in the number of glucocorticoid receptors (GR) in murine macrophages. To examine further the environmental signals involved in regulation of macrophage GR availability, we asked whether another classical macrophage-activating factor, LPS, would induce an increase in GR number in the macrophage cell line, RAW 264.7, and in primary macrophages from C3H mice. We report that treatment of RAW 264.7 cells and peritoneal exudate macrophages from C3H/OuJ mice with protein-free, phenol water-extracted LPS (PW-LPS) induced an increase in the number of GR. A significant increase in GR number was observed as early as 4 h after PW-LPS treatment, was maximal at 12 h, and remained heightened through 48 h. Optimal induction of the GR by PW-LPS was observed when murine macrophages were treated with 10 ng/ml of PW-LPS. The LPS-induced increase in macrophage GR number could be inhibited by polymyxin B. Macrophages obtained from the LPS hyporesponsive C3H/HeJ strain did not respond to PW-LPS, but did respond to protein-rich, butanol-extracted LPS with a modest increase in GR number after treatment with 2 micrograms/ml. Moreover, taxol, an antineoplastic agent with LPS mimetic activity, also increased GR number in murine macrophages. These results suggest that LPS is not only an important macrophage-activating signal, but may also be important in sensitizing the cell for negative regulatory events such as feedback inhibition by glucocorticoids.     

Chelation of cytoplasmic Ca2+ increases plasma membrane permeability in murine macrophages

Cytoplasmic free Ca2+ (Ca2+i) was chelated to 10-20 nM in the macrophage cell line J774 either by incubation with quin2 acetoxymethyl ester in the absence of external Ca2+ (Di Virgilio, F., Lew, P.D., and Pozzan, T. (1984) Nature 310, 691-693) or by loading [ethyl-enebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) into the cytoplasm via reversible permeabilization of the plasma membrane with extracellular ATP (Steinberg, T.H., Newman, A.S., Swanson, J.A., and Silverstein, SS.C. (1987) J. Biol. Chem. 262, 8884-8888; Di Virgilio, F., Meyer, B.C., Greenberg, S., and Silverstein, S.C. (1988) J. Cell Biol. 106, 657-666). After removal of ATP from the incubation medium, ATP-permeabilized Ca2+i-depleted macrophages recovered a near-normal plasma membrane potential which slowly depolarized over a 2-4 h incubation at low [Ca2+]i. In both ATP-treated and quin2-loaded cells, depolarization of plasma membrane potential was paralleled by an increase in plasma membrane permeability to low molecular weight aqueous solutes such as eosin yellowish (Mr 692), ethidium bromide (Mr 394), and lucifer yellow (Mr 463). This increased plasma membrane permeability was not accompanied by release of the cytoplasmic marker lactic dehydrogenase for incubations up to 4 h and was likely a specific effect of Ca2+i depletion since it was not caused by: (i) the mere incubation of macrophages with extracellular EGTA, i.e. at near-normal [Ca2+]i; and (ii) loading into the cytoplasm of diethylenetriaminepentaacetic acid, a specific chelator of heavy metals with low affinity for Ca2+. Treatment of Ca2+i-depleted cells with direct (phorbol 12-myristate 13-acetate) or indirect (platelet-activating factor) activators of protein kinase C prevented the increase in plasma membrane permeability. Down-regulation of protein kinase C rendered Ca2+i-depleted macrophages refractory to the protective effect of phorbol 12-myristate 13-acetate. This report suggests a role for Ca2+i and possibly protein kinase C in the regulation of plasma membrane permeability to low molecular weight aqueous solutes.     

Dual mechanism of protein-tyrosine phosphorylation in concanavalin A-stimulated platelets

Treatment of human platelets with the lectin Concanavalin A (Con A) resulted in the tyrosine phosphorylation of several proteins with molecular masses 65, 80, 85, 95, 120, 135, and 150 kDa. These proteins were divided in two groups: the first group included the 65-, 85-, 95-, and 120-kDa bands, which were tyrosine phosphorylated also in thrombin-stimulated platelets; the second group (80-, 135-, and 150-kDa bands) included proteins whose tyrosine phosphorylation was exclusively promoted by Con A, but not by thrombin. Members of the second group were rapidly dephosphorylated when the lectin was displaced from the cell surface by methyl α-D -mannopyranoside. Pretreatment of intact platelets with the prostacyclin analog iloprost, inhibited Con A-induced tyrosine phosphorylation of the first group of proteins, but had no effect on the tyrosine phosphorylation of the proteins of the second group. Succinyl-Con A, a dimeric derivative of the lectin, which binds to the platelet surface but does not promote clustering of the receptor, did not induce tyrosine phosphorylation of the second group of proteins, although phosphorylation of some members of the first group was observed. Our results demonstrate the presence of two different mechanisms leading to protein-tyrosine phosphorylation in Con A-stimulated platelets, and identify a new signal transduction pathway, promoted by the clustering of membrane glycoproteins, that produces tyrosine phosphorylation of specific substrates. This new pathway may be activated by platelet interaction with multivalent ligands, such as adhesive proteins, during adhesion, spreading, and aggregation.     

Mutual regulation of protein-tyrosine phosphatase 20 and protein-tyrosine kinase Tec activities by tyrosine phosphorylation and dephosphorylation

PTP20, also known as HSCF/protein-tyrosine phosphatase K1/fetal liver phosphatase 1/brain-derived phosphatase 1, is a cytosolic protein-tyrosine phosphatase with currently unknown biological relevance. We have identified that the nonreceptor protein-tyrosine kinase Tec-phosphorylated PTP20 on tyrosines and co-immunoprecipitated with the phosphatase in a phosphotyrosine-dependent manner. The interaction between the two proteins involved the Tec SH2 domain and the C-terminal tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20, which were also necessary for tyrosine phosphorylation/dephosphorylation. Association between endogenous PTP20 and Tec was also tyrosine phosphorylation-dependent in the immature B cell line Ramos. Finally, the Tyr-281 residue of PTP20 was shown to be critical for deactivating Tec in Ramos cells upon B cell receptor ligation as well as dephosphorylation and deactivation of Tec and PTP20 itself in transfected COS7 cells. Taken together, PTP20 appears to play a negative role in Tec-mediated signaling, and Tec-PTP20 interaction might represent a negative feedback mechanism.     

Relationship between exopolysaccharide production and protein-tyrosine phosphorylation in gram-negative bacteria

The phosphorylation of proteins at tyrosine residues is known to play a key role in the control of numerous fundamental processes in animal systems. In contrast, the biological significance of protein-tyrosine phosphorylation in bacteria, which has only been recognised recently, is still unclear. Here, we have analysed the role in Escherichia coli cells of an autophosphorylating protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb, by performing knock-out experiments on the corresponding genes, wzc and wzb, and looking at the metabolic consequences induced. The results demonstrate that the phosphorylation of Wzc, as regulated by Wzb, is directly connected with the production of a particular capsular polysaccharide, colanic acid. Thus, when Wzc is phosphorylated on tyrosine, no colanic acid is synthesised by bacteria, but when dephosphorylated by Wzb, colanic acid is produced. This process is rather specific to the pair of proteins Wzc/Wzb. Indeed, a much lesser effect, if any, on colanic acid synthesis is observed when knock-out experiments are performed on another pair of genes, etk and etp, which also encode respectively a protein-tyrosine kinase, Etk, and a phosphotyrosine-protein phosphatase, Etp, in E. coli. In addition, the analysis of the phosphorylation reaction at the molecular level reveals differences between Gram-negative and Gram-positive bacteria, namely in the number of protein components required for this reaction to occur.     

Citral and eugenol modulate DNA damage and pro-inflammatory mediator genes in murine peritoneal macrophages

Citral and eugenol have been broadly studied because of their anti-inflammatory, antioxidant and antiparasitic potentials. In this study, the effects of citral (25, 50 and 100 µg/mL) and eugenol (0.31, 0.62, 1.24 and 2.48 µg/mL) on the expression (RT-PCR) of the pro-inflammatory mediator genes NF-κB1, COX-2 and TNF-α were evaluated in mouse peritoneal macrophages with or without activation by a bacterial lipopolysaccharide (LPS). Additionally, the genotoxic potentials of two compounds and their capacities to modulate the DNA damage induced by doxorubicin (DXR) were investigated using the comet assay. The data revealed that neither citral nor eugenol changed COX-2, NF-κB1 or TNF-α expression in resting macrophages. However, in LPS-activated cells, citral induced the hypoexpression of COX-2 (100 µg/mL) and TNF-α (50 and 100 µg/mL). Hypoexpression of TNF-α was also detected after cellular exposure to eugenol at the highest concentration (2.48 µg/mL). Both compounds exhibited genotoxic potential (citral at 50 and 100 µg/mL and eugenol at all concentrations) but also showed chemopreventive effects, in various treatment protocols. Both citral and eugenol might modulate inflammatory processes and DXR-induced DNA damage, but the use of these compounds must be viewed with caution because they are also able to induce primary DNA lesions.