Differential effects of phosphotyrosine phosphatase expression on hormone-dependent and independent pp60 c-src activity

pp60 ^c-src kinase activity can be increased by phosphotyrosine dephosphorylation or growth factor-dependent phosphorylation reactions. Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit growth factor receptor signal transduction (Mooney, RA, Freund, GG, Way, BA and Bordwell, KL (1992) J Biol Chem 267, 23443–23446). Here it is shown that PTPase expression decreased platelet-derived growth factor (PDGF)-dependant activation of pp60 ^c-src but failed to increase hormone independent (basal) pp60 ^c-src activity. PDGF-dependent tyrosine phosphorylation of its receptor was reduced by approximately 60% in cells expressing the PTPase. In contrast, a change in phosphotyrosine content of pp60 ^c-src was not detected in response to PDGF or in PTPase+cells. PDGF increased the intrinsic tyrosine kinase activity of pp60 ^c-src in both control and PTPase+cells but the effect was smaller in PTPase+cells. In anin vitro assay, hormone-stimulate pp60 ^c-src autophosphorylation from PTPase+ cells was decreased 64±22%, and substrate phosphorylation by pp60 ^c-src was reduced 54±16% compared to controls. Hormone-independent pp60 ^c-src kinase activity was unchanged by expression of the PTPase. pp60 ^c-src was, however, anin vitro substrate for CD45, being dephosphorylated at both the regulatory (Tyr⁵²⁷) and kinase domain (Tyr⁴¹⁶) residues. In addition,in vitro dephosphorylation by CD45 increased pp60 ^c-src activity. These findings suggest that the PDGF receptor was anin vivo substrate of CD45 but pp60 ^c-src was not. The lack of activation of pp60 ^c-src in the presence of expressed PTPase may demonstrate the importance of compartmentalization and/or accessory proteins to PTPase-substrate interactions.Abbreviations PTPase phosphotyrosine phosphatase - PDGF platelet-derived growth factor - PMSF phenylmethylsulfonyl fluoride - LCA, CD45 leukocyte common antigen - PBS phosphate buffered saline - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - DTT dithiothreitol - Na₃VO₄ sodium orthovanadate - PV pervanadate - -ME -mercaptoethanol     

Protein tyrosine phosphorylation in cardiovascular system

Treatment of rat parotid acinar cells with sodium orthovanadate (an inhibitor of protein tyrosine phosphatase) caused a dose-dependent inhibition of phosphatase activity as measured by the hydrolysis of para nitrophenylphosphate (pNPP). Inclusion of 50 M sodium orthovanadate inin vitro gland cultures prevented the amylase secretion from both untreated control and isoproterenol-stimulated parotid acinar cells. Four different tyrosine-phosphorylated proteins with M_r 40, 45, 70 and 95 kDa, respectively, were identified in secretory granule preparations from rat parotid glands by immunoblot using a monospecific antibody for phosphotyrosine. An increase in the phosphorylation levels of these phosphoproteins was noted in the presence of 50 M sodium orthovanadate, suggesting that a protein tyrosine phosphatase (PTPase) is involved in parotid gland protein dephosphorylation reactions. Using antibody to Syp (a PTPase belonging to class 1D), a major fraction of subcellular activity was found to be associated with secretory granule membranes. These results suggest the possible involvement of a PTPase (Syp) in parotid gland secretory mechanisms.     

The dephosphorylation of insulin and epidermal growth factor receptors. Role of endosome-associated phosphotyrosine phosphatase(s).

The autophosphorylation, from [gamma-32P]ATP, of insulin and epidermal growth factor receptors in rat liver endosomes peaked at 2-5 min and declined thereafter. When autophosphorylation from either [gamma-32P]ATP or unlabeled ATP was stopped after 5 min by adding excess EDTA +/- ATP, the phosphotyrosine (PY) content of each receptor decreased at 37 degrees C with a t 1/2 of 1.6 min. This was equally so whether the PY content of 32P-labeled receptors was analyzed by autoradiography of KOH-treated gels or by Western blotting with PY antibodies of immunoprecipitated receptors. The dephosphorylation reaction was strictly dependent on the presence of sulfhydryl, was unaffected by the addition of rat liver cytosol, and was temperature-dependent. The phosphotyrosine phosphatase(s) (PTPase(s)) appeared to be tightly anchored to the endosomal membrane, since the dephosphorylation reaction was unaffected by sodium carbonate and 0.6 M KCl treatments. However, treatment with Triton X-100 abolished dephosphorylation, implying an intimate association between the PTPase(s) and its substrate in an intact membrane environment. The powerful insulinomimetic agent pervanadate was the most potent inhibitor (50% inhibition at 1 microM). Increasing the dose of injected ligand augmented the rate of insulin and decreased that of EGF receptor dephosphorylation, respectively. Immunoblotting with specific antibodies failed to identify PTPase 1B or T-cell PTPase in ENs, whereas positive signals were seen in plasma membrane. These studies indicate that the phosphorylation state of receptor tyrosine kinases is dynamically regulated, with dephosphorylation, by closely associated PTPase(s), playing an important role.     

Chemical dissection of the effects of tyrosine phosphorylation of SHP-2

The regulation of the protein tyrosine phosphatase (PTPase) SHP-2 by tyrosine phosphorylation has been difficult to elucidate because of the intrinsic instability of the phosphoprotein. In the past, expressed protein ligation has been used to site-specifically incorporate the phosphotyrosine mimic Pmp (phosphonomethylene phenylalanine) into the two tyrosine phosphorylation sites (542, 580) of SHP-2 one at a time to analyze the effects on catalytic behavior. In this study, we have incorporated two Pmps into the phosphorylation sites simultaneously and examined the effects of double SHP-2 tyrosine phosphorylation. We have found that the Pmp groups show close to additive effects on PTPase stimulation, suggesting dual SH2 domain occupancy. The relative effects of the phosphotyrosine analogue difluoromethylene phosphonophenylalanine (F(2)Pmp) compared to those of Pmp were also examined. It was found that the F(2)Pmp analogue showed slightly enhanced PTPase stimulation compared with the Pmp analogue, consistent with its higher affinity for SH2 domains. Taken together with the bis-Pmp studies, these data suggest that double phosphorylation of the SHP-2 C-terminus could give rise to a 9-fold overall PTPase activation, 30-50% of the value associated with deletion of the SH2 domains. Catalytically inactive forms of phosphorylated SHP-2 proteins were also produced by expressed protein ligation. This allowed for a systematic analysis of intermolecular autodephosphorylation of SHP-2, which revealed how conformational plasticity can modulate phosphotyrosine stability.     

Protein Tyrosine Phosphatase Inhibitors in FcγRI-Induced Myeloid Oxidant Signaling

Fc-receptor stimulation in myeloid cells results in increased oxygen consumption, termed the respiratory burst, which is coupled to a rapid and transient increase in tyrosine phosphorylation of cellular proteins. In a previous paper in this journal we showed that the protein tyrosine phosphatase (PTPase) inhibitors sodium orthovanadate and phenylarsine oxide (PAO) block the FcγRI-induced respiratory burst in interferon-γ-differentiated U937 cells (U937IF) while augmenting the FcγRI-induced tyrosine phosphorylation of cellular proteins. Herein we examine the effects of PTPase inhibitors on specific molecules involved in FcγRI signaling. We show that orthovanadate and PAO augmented the FcγRI-induced tyrosine phosphorylation of the adaptor protein CBL. CBL interactions with other phosphoproteins, among them SHC and CRKL, were also augmented in response to pretreatment with the PTPase inhibitors. SHC was tyrosine phosphorylated in response to FcγRI stimulation of U937IF cells and bound to the SH2 domain of GRB2 in a stimulation-dependent manner. In fusion protein pull-down experiments the interaction of SHC with the SH2 domain of GRB2 was increased in PTPase inhibitor pretreated U937IF cells in response to FcγRI stimulation. Our data support the hypothesis that a tyrosine dephosphorylation event is required for effective transmission of the FcγRI signal to result in activation of the myeloid respiratory burst response.     

Lipopolysaccharide-induced priming of the human neutrophil is not associated with a change in phosphotyrosine phosphatase activity.

The activation of the neutrophil respiratory burst is a two-step process involving an initial 'priming' phase followed by a 'triggering' event. The biochemical mechanisms which underlie these events are yet to be fully elucidated, but the evidence suggests a crucial role for stimulus-induced tyrosine phosphorylation. The enhanced tyrosine phosphorylation observed upon triggering primed cells may reflect an increase in tyrosine kinase activity or a reduction in the levels of the opposing phosphotyrosine phosphatases (PTPases). We have investigated the latter by examining the possibility that lipopolysaccharide (LPS)-induced priming of the neutrophil respiratory burst involves the suppression of cellular PTPase activity. Purified human neutrophils were incubated for 60 min with and without LPS. Priming of the respiratory burst was confirmed by fMet-Leu-Phe-induced cytochrome c reduction. The level of PTPase activity was assessed by dephosphorylation of [32P]RR-src peptide as substrate. Pretreatment of human neutrophils with 200 ng/ml LPS induced a 2.9 +/- 0.3 (mean +/- SEM, n = 3, P = 0.022) fold increase in the fMet-Leu-Phe-triggered respiratory burst. In the same cells, LPS did not induce a significant change in the total cellular PTPase activity (1.02 +/- 0.02-fold, mean +/- SEM, n = 3, P = 0.63). Similarly, stimulation of neutrophils with fMet-Leu-Phe or phorbol myristate acetate did not significantly affect the cellular PTPase activity (P = 0.94 and 0.68, respectively). Our results suggest that suppression of PTPase activity is not the mechanism underlying the priming and/or triggering of the neutrophil respiratory burst.     

Cloning and expression of a yeast protein tyrosine phosphatase.

To study the regulation of tyrosine phosphorylation/dephosphorylation in Saccharomyces cerevisiae, a protein tyrosine phosphatase (PTPase) was cloned by the polymerase chain reaction (PCR). Conserved amino acid sequences within the mammalian PTPases were used to design primers which generated a yeast PCR fragment. The sequence of the PCR fragment encoded a protein with homology to the mammalian PTPases. The PCR fragment was used to identify the yeast PTP1 gene which has an open reading frame encoding a 335-amino acid residue protein. This yeast PTPase shows 26% sequence identity to the rat PTPase, although highly conserved residues within the mammalian enzymes are invariant in the yeast protein. The yeast PTP1 is physicallt linked to the 5'-end of a heat shock gene SSB1. This yeast PTP1 gene was expressed in Escherichia coli and obtained in a highly purified form by a single affinity chromatography step. The recombinant yeast PTPase hydrolyzed phosphotyrosine containing substrates approximately 1000 times faster than a phosphoserine containing substrate. Gene disruption of yeast PTP1 has no visible effect on vegetative growth.     

Characterization ofEntamoeba histolytica-induced dephosphorylation in Jurkat cells

Entamoeba histolytica killing of host cells is contact dependent and mediated by a Gal/GalNAc lectin. Upon contact with amoeba a rapid and extensive dephosphorylation of tyrosine phosphorylated host cell proteins is observed. This effect is mediated by the Gal/GalNAc lectin. However, it requires intact cells, as purified lectin failed to induce dephosphorylation in Jurkat cells. The nonpathogenic, but morphologically identical amoeba,Entamoeba moshkovskii also did not induce dephosphorylation in target cells. Treatment of Jurkat cells with phosphotyrosine phosphatase inhibitors has shown that a host phosphatase is responsible for dephosphorylation. However, it was found that the CD45 phosphotase was not necessary for dephosphorylation of host cell proteins.     

Analysis of changes in tyrosine and serine phosphorylation of red cell membrane proteins induced by P. falciparum growth

Phosphorylation of erythrocyte membrane proteins has been previously documented following infection and intracellular growth of the malarial parasite, Plasmodium falciparum in red cells. Much of this data dealt with phosphorylation of serine residues. In this study, we report detailed characterization of phosphorylation of serine and tyrosine residues of red cell membrane proteins following infection by P falciparum. Western blot analysis using anti‐phosphotyrosine and anti‐phosphoserine antibodies following 2‐DE in conjunction with double channel laser‐induced infrared fluorescence enabled accurate assessment of phosphorylation changes. Tyrosine phosphorylation of band 3 represented the earliest modification observed during parasite development. Band 3 tyrosine phosphorylation observed at the ring stage appears to be under the control of Syk kinase. Serine and tyrosine phosphorylation of additional cytoskeletal, trans‐membrane and membrane associated proteins was documented as intracellular development of parasite progressed. Importantly, during late schizont stage of parasite maturation, we observed widespread protein dephosphorylation. In vitro treatments that caused distinct activation of red cell tyrosine and serine kinases elicited phosphorylative patterns similar to what observed in parasitized red blood cell, suggesting primary involvement of erythrocyte kinases. Identification of tyrosine phosphorylations of band 3, band 4.2, catalase and actin which have not been previously described in P. falciparum infected red cells suggests new potential regulatory mechanisms that could modify the functions of the host cell membrane.     

Site-specific incorporation of a phosphotyrosine mimetic reveals a role for tyrosine phosphorylation of SHP-2 in cell signaling.

The regulation of protein tyrosine phosphatase (PTPase) SHP-2 is proposed to involve tyrosine phosphorylation on two tail tyrosine residues. Using "expressed protein ligation", nonhydrolyzable phosphotyrosine analogs were introduced at known phosphorylation sites in SHP-2. Biochemical analysis suggests that a phosphonate at Tyr542 interacts intramolecularly with the N-terminal SH2 domain to relieve basal inhibition of the PTPase, whereas a phosphonate at Tyr-580 stimulates the PTPase activity by interaction with the C-terminal SH2 domain. Microinjection experiments indicate that a single phosphorylation of Tyr-542 of SHP-2 is sufficient to activate the MAP kinase pathway in living cells. These studies support a novel mechanism explaining how tyrosine phosphorylation of a PTPase is important in signal transduction.     

Temperature-sensitive mutants of Abelson murine leukemia virus deficient in protein tyrosine kinase activity.

The effect of two missense mutations in abl on transformation by Abelson murine leukemia virus was evaluated. These mutations led to the substitution of a histidine for Tyr-590 and a glycine for Lys-536. Both changes gave rise to strains that were temperature dependent for transformation of both NIH 3T3 cells and lymphoid cells when expressed in the context of a truncated Abelson protein. In the context of the prototype P120 v-abl protein, the Gly-536 substitution generated a host range mutant that induced conditional transformation in lymphoid cells but had only a subtle effect on NIH 3T3 cells. The combination of both substitutions gave rise to a P120 strain that was temperature sensitive for both NIH 3T3 and lymphoid cell transformation. The Abelson proteins encoded by the temperature-sensitive strain displayed in vitro kinase activities that were reduced when compared with those of wild-type proteins. In vivo, levels of phosphotyrosine were reduced only at the restrictive temperature. Analysis of cells expressing either the wild-type P160 v-abl protein or the P210 bcr/abl protein and an Abelson protein encoded by a temperature-sensitive strain failed to correct this defect, suggesting either that tyrosine phosphorylation in vivo is an intramolecular reaction or that the protein encoded by the temperature-sensitive strain is a poor substrate for tyrosine phosphorylation in vivo. These results raise the possibility that tyrosine phosphorylation of Abelson protein plays a role in transformation.     

Inhibition of phosphotyrosine phosphatases reveals candidate substrates of the PDGF receptor kinase

In normal fibroblasts stimulated by platelet derived growth factor (PDGF), PDGF receptors are transiently phosphorylated on tyrosine and represent the major phosphotyrosine containing protein. The phosphate of the phosphotyrosine groups turns over rapidly, and extensive evidence indicates a dynamic balance between phosphorylation and dephosphorylation reactions. Thus, the effect of an inhibitor of phosphatases, orthovanadate, on the pattern of the tyrosine phosphorylations induced by PDGF in Swiss 3T3 fibroblasts was investigated. Western blot analysis with antibodies against phosphotyrosine indicated that whereas in unstimulated cells no phosphotyrosine containing proteins were detected, treatment of cells with orthovanadate alone elicited the slow phosphorylation of several proteins including a 170 kDa component that was recognized to be the phosphorylated PDGF receptor. Addition of PDGF to cells shortly pretreated with vanadate highly increased the intensity of the 170 kDa band corresponding to the phosphorylated receptor and caused its stabilization during time. In addition, the phosphorylation on tyrosine of other proteins (molecular mass 116, 80, 73, 60, 50 and 39 kDa) was also induced. Both the receptor and the other tyrosine phosphorylated proteins appeared to be associated with the detergent insoluble matrix.     

Regulation of the human sperm tyrosine kinase c-yes. Activation by cyclic adenosine 3',5'-monophosphate and inhibition by Ca(2+)

During the process of capacitation, spermatozoa go through a whole set of signaling cascade events in order to become fully competent at fertilizing the egg. An increase in sperm protein tyrosine phosphorylation has been described during this final maturational event in different animal species as well as in humans. Although the phosphotyrosine content of sperm protein is modulated by cAMP, Ca(2+), BSA, oxygen derivatives, and cholesterol, no protein tyrosine kinase (PTK) nor the phosphotyrosine protein phosphatase (PTPase) directly involved in the control of the phosphotyrosine content of sperm protein has been identified. Therefore, the goal of the present study was to identify the tyrosine kinases putatively responsible for the increases in sperm protein phosphotyrosine content. In the present study, we show that the src-related tyrosine kinase c-yes is present in the head of human spermatozoa in both membranes and Triton X-100-insoluble extracts. Our hypothesis was that c-yes is a tyrosine kinase responsible for at least some of the capacitation-induced increase in protein tyrosine phosphorylation. When spermatozoa were previously incubated in the presence of 3-isobutyl-1-methylxanthine or 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, treatments known to increase the phosphotyrosine content of human sperm proteins, an increase in the kinase activity of immunoprecipitated yes was measured using enolase as a substrate. These results suggest that cAMP activates while Ca(2+) inhibits human sperm c-yes kinase activity.     

Heterologous expression of the cytochrome P450cam hydroxylase operon and the repressor gene of Pseudomonas putida in Escherichia coli

Abstract Cytoplasmic proteins from the antarctic psychrotrophic bacterium Pseudomonas syringae showed two phosphorylated proteins of molecular mass 66 kDa and 62 kDa. The phosphorylation of 66 kDa protein was enhanced in the presence of Triton X-100 solubilised membrane proteins at a higher temperature (30°C) only. Western blot analysis and phosphoamino acid analysis indicated that the 66 kDa protein is phosphorylated at a tyrosine residue. Surprisingly, sodium orthovanadate, which is a known phosphotyrosine phosphatase (PTPase) inhibitor, inhibited the phosphorylation of the protein. The possible importance of this tyrosine phosphorylated protein to growth at low temperature is suggested.     

Tyrosine phosphorylation translocates beta-catenin from cell-->cell interface to the cytoplasm, but does not significantly enhance the LEF-1-dependent transactivating function

beta-catenin plays an essential role in cells, not only as a cadherin-associated complex, but also as a signaling molecule in the nucleus. Tyrosine phosphorylation of beta-catenin has been shown to correlate with tumorigenesis, cell migration, and developmental processes. However, its exact effects on downstream targets in the nucleus are not yet clear. In this study, we used HCT-15 colon carcinoma and NIH 3T3 fibroblasts as models to investigate the effects of a phosphotyrosine phosphatase (PTPase) inhibitor on the localization of beta-catenin, the binding affinity to LEF-1 (Lymphoid Enhancer Factor), and on LEF-1-dependent transactivation function. Treatment with a PTPase inhibitor, pervanadate, increased the tyrosine phosphorylation of beta-catenin in a time-dependent manner and led to its relocation from cell-cell interfaces to the cytoplasm. This phosphorylation/dephosphorylation of beta-catenin does not require its presence at cell-cell interfaces. However, tyrosine phosphorylation of beta-catenin does not change its binding affinity to LEF-1 nor enhance cyclin D1 transactivation, a nuclear target of beta-catenin/LEF-1. This result suggests that tyrosine phosphorylation of beta-catenin has effects on the binding to cadherins in the cytoplasm but not on its LEF-1-dependent transactivating function in the nucleus.     

Alteration of phosphotyrosine-containing proteins during differentiation of chicken erythroleukemia cells (HD3)

After treatment of HD3 cells with erythroid-inducing agents (hemin and butyric acid) at 42°C, the profile of phosphotyrosine-containing proteins was altered. Upon induction the overall level of phosphotyrosine-containing proteins increased. To examine the role of protein phosphorylation in HD3 cells differentiation, the cells were treated with specific inhibitors. In the presence of okadaic acid, cell proliferation was arrested and accompanied by a marked increase in haemoglobin synthesis, a differentiation marker of erythroid cells. Okadaic acid caused decrease of the phosphotyrosine-containing proteins, presumably to maintain a balance between phosphorylation/dephosphorylation processes in the cells. Addition of 3-isobutyl-l-methyl-xanthine, an activator of phosphatases, caused a decrease or disappearance of almost all phosphotyrosine-containing proteins and, at the same time, prevented the erythroid differentiation of HD3 cells. Sodium orthovanadate, a specific inhibitor of phosphotyrosine phosphatase, increased the level of phosphotyrosine proteins and induced differentiation of HD3 cells. These results indicate that phosphorylation of cellular proteins is coupled with a reaction(s) which is responsible for triggering the differentiation of HD3 cells. The phosphorylation/dephosphorylation processes are associated with an early event(s) during the differentiation of HD3 cells and may not be connected to tyrosine residues. Copyright © 1998 John Wiley & Sons, Ltd.     

Insulin receptor protein-tyrosine phosphatases. Leukocyte common antigen-related phosphatase rapidly deactivates the insulin receptor kinase by preferential dephosphorylation of the receptor regulatory domain.

A number of protein-tyrosine phosphatase(s) (PTPases) have been shown to dephosphorylate the insulin receptor in vitro; however, it is not known whether any individual PTPase has specificity for certain phosphotyrosine residues of the receptor that regulate its intrinsic tyrosine kinase activity. We evaluated the deactivation of the insulin receptor kinase by three candidate enzymes that are expressed in insulin-sensitive rat tissues, including the receptor-like PTPases LAR and LRP, and the intracellular enzyme, PTPase1B. Purified insulin receptors were activated by insulin and receptor dephosphorylation, and kinase activity was quantitated after incubation with recombinant PTPases from an Escherichia coli expression system. When related to the level of overall receptor dephosphorylation, LAR deactivated the receptor kinase 3.1 and 2.1 times more rapidly than either PTPase1B or LRP, respectively (p less than 0.03). To assess whether these effects were associated with preferential dephosphorylation of the regulatory (Tyr-1150) domain of the receptor beta-subunit, we performed tryptic mapping of the insulin receptor beta-subunit after dephosphorylation by PTPases. Relative to the rate of initial loss of 32P from receptor C-terminal sites, LAR dephosphorylated the Tris-phosphorylated Tyr-1150 domain 3.5 and 3.7 times more rapidly than either PTPase1B or LRP, respectively (p less than 0.01). The accelerated deactivation of the insulin receptor kinase by LAR and its relative preference for regulatory phosphotyrosine residues further support a potential role for this transmembrane PTPase in the physiological regulation of insulin receptors in intact cells.     

Mechanical Strain Increases Protein Tyrosine Phosphorylation in Airway Smooth Muscle Cells

Mechanical stress contributes to normal structure and function of the lung as well as pathology in such diseases as bronchopulmonary dysplasia and adult respiratory distress syndrome. Stress-related increases in airway smooth muscle (ASM) quantity are reflectedin vitrowhere cultured ASM cells respond to cyclic deformational strain with increased proliferation, cell reorientation, protein production, stress fibers, and focal adhesions. To understand the mechanisms of mechanical signaling in ASM cells, we investigated whether strain increased tyrosine phosphorylation of focal adhesion-related proteins. ASM cells were grown to confluence on collagen type I and subjected to 30 min of cyclic deformation strain (2 s of 25% deformation of the substratum, 2 s relaxation) and compared at various time points with identical cells not subjected to strain for phosphotyrosine content of three focal adhesion-concentrated proteins (pp125^FAK, paxillin, and talin) by Western blotting. Strain caused a rapid increase in tyrosine phosphorylation of pp125^FAKand paxillin. Tyrosine phosphorylation decreased by 4 h in pp125^FAKafter discontinuing strain but remained elevated in paxillin at 24 h. Increases in tyrosine phosphorylation of talin were not found. In separate studies, when cells were strained in the presence of tyrosine kinase inhibitors (genistein and herbimycin A), strain-induced reorientation and elongation were inhibited. Mechanochemical signal transduction appears to mediate cell morphologic changes through quantitative and possibly qualitative changes in tyrosine phosphorylation of adhesion-related proteins.     

Binding of IgG-opsonized particles to Fc gamma R is an active stage of phagocytosis that involves receptor clustering and phosphorylation

Fc gammaR mediate the phagocytosis of IgG-coated particles and the clearance of IgG immune complexes. By dissecting binding from internalization of the particles, we found that the binding stage, rather than particle internalization, triggered tyrosine phosphorylation of Fc gammaR and accompanying proteins. High amounts of Lyn kinase were found to associate with particles isolated at the binding stage from J774 cells. PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), an Src kinase inhibitor, but not piceatannol, an inhibitor of Syk kinase, reduced the amount of Lyn associated with the bound particles and simultaneously diminished the binding of IgG-coated particles. Studies of baby hamster kidney cells transfected with wild-type and mutant Fc gammaRIIA revealed that the ability of the receptor to bind particles was significantly reduced when phosphorylation of the receptor was abrogated by Y298F substitution in the receptor signaling motif. Under these conditions, binding of immune complexes of aggregated IgG was depressed to a lesser extent. A similar effect was exerted on the binding ability of wild-type Fc gammaRIIA by PP2. Moreover, expression of mutant kinase-inactive Lyn K275R inhibited both Fc gammaRIIA phosphorylation and IgG-opsonized particle binding. To gain insight into the mechanism by which protein tyrosine phosphorylation can control Fc gammaR-mediated binding, we investigated the efficiency of clustering of wild-type and Y298F-substituted Fc gammaRIIA upon binding of immune complexes. We found that a lack of Fc gammaRIIA phosphorylation led to an impairment of receptor clustering. The results indicate that phosphorylation of Fc gammaR and accompanying proteins, dependent on Src kinase activity, facilitates the clustering of activated receptors that is required for efficient particle binding.