Search PubMed⌕ Search

Biomedical subjects

M M Hamawy

Publications and source records attributed to M M Hamawy.

33 records · Page 2Linked to original sources
PubMed1997

Fc epsilon RI aggregation induces tyrosine phosphorylation of a novel 72 kDa protein downstream of Syk.

Tyrosine phosphorylation of proteins is critical for the Fc epsilon RI-induced signal transduction that leads to the release of inflammatory mediators from mast cells. Here we report the isolation of a monoclonal antibody, mAb BD2, to a 72 kDa protein that becomes rapidly tyrosine phosphorylated after Fc epsilon RI aggregation. By immunoprecipitation, immunoblotting and/or protease digestion this 72 kDa protein was different from the previously identified 68-76 kDa tyrosine phosphorylated proteins Btk, paxillin, SLP-76 or Syk. The phosphorylation of this 72 kDa protein was detectable within 15 sec after receptor aggregation and was independent of Ca2+ influx or the activation of protein kinase C. By in vitro kinase reaction, the 72 kDa protein did not autophosphorylate, which suggests that it is not a kinase, but is associated with a 140 kDa protein that was strongly phosphorylated. Studies in Syk deficient and Syk transfected variants of the RBL-2H3 cells demonstrated that the tyrosine phosphorylation of this 72 kDa protein was downstream of Syk. These data indicate that the 72 kDa protein precipitated by mAb BD2 is a novel phosphoprotein involved in Fc epsilon RI signaling.

Animals↗
PubMed1997

Large scale isolation of intact rat basophilic leukemia (RBL-2H3) cells.

The rat basophilic leukemia RBL-2H3 mast cell line is widely used for studies of the structure and function of the high affinity IgE receptor (Fc epsilonRI). Here we report on a simple method to isolate large numbers of intact RBL-2H3 cells from tumors produced by injection of the cells into newborn rats. Collagenase treatment of rat tumors yields approximately 3.5 x 10(8) viable cells/animal. Aggregating Fc epsilonRI on these cells induced tyrosine phosphorylation of proteins including the protein tyrosine kinase Syk. This procedure should prove useful for the isolation and characterization of cellular molecules important for mast cell and basophil function.

Animals↗
PubMed1995

A 77-kDa protein associates with pp125FAK in mast cells and becomes tyrosine-phosphorylated by high affinity IgE receptor aggregation.

The focal adhesion kinase, pp125FAK, is a novel non-receptor protein tyrosine kinase expressed in different cells including mast cells. Here we report that a 77-kDa protein associates with pp125FAK in the mast cell analog, rat basophilic leukemia (RBL-2H3) cells. When pp125FAK immunoprecipitates were subjected to an in vitro kinase assay, there was prominent phosphorylation on tyrosine of pp125FAK and of a 77-kDa protein. By V8 protease digestion mapping and by immunoblotting with two different anti-pp125FAK antibodies, the 77-kDa protein was distinct from pp125FAK. This Fak Associated Protein or FAP was detected in RBL-2H3 cells but not in fibroblasts. The aggregation of the high affinity IgE receptor, Fc epsilon RI, induced the in vivo tyrosine phosphorylation of FAP. However, there was a marked decrease in the in vitro phosphorylation of FAP in the immunoprecipitates from Fc epsilon RI aggregated cells. Both of these Fc epsilon RI-mediated effects were enhanced by cell adhesion. There was strong association of FAP with non-tyrosine-phosphorylated pp125FAK. Thus this interaction does not appear to be mediated by the Src homology 2 domain. Together the data indicate that FAP associates with pp125FAK and suggest that FAP may play a role in Fc epsilon RI signaling.

3T3 Cells↗
PubMed1995

Protein tyrosine phosphorylation as a mechanism of signalling in mast cells and basophils.

Tyrosine phosphorylation of proteins is a mechanism of signalling for different receptors and is important for cell growth and differentiation. Mast cells and basophils are secretory cells that play a role in inflammatory and immediate allergic reactions. The activation/aggregation of different surface receptors on these cells induces tyrosine phosphorylation of proteins. Because these signals are essential for the function of basophils and mast cells, characterizing these pathways could provide methods to specifically regulate the function of these cells. Here we discuss the signals generated by three receptors: the high affinity IgE receptor (Fc epsilon RI) the growth factor receptor, Kit, and integrins.

Animals↗
PubMed1994

The aggregation of the high affinity IgE receptor induces tyrosine phosphorylation of paxillin, a focal adhesion protein.

Tyrosine phosphorylation of proteins is an essential component of high affinity IgE receptor (Fc epsilon RI) signaling and secretion. This signaling and secretion is also dependent on the organization of the cytoskeleton. Here we report that the aggregation of Fc epsilon RI on rat basophilic leukemia cells results in tyrosine phosphorylation of the cytoskeletal protein, paxillin. Tyrosine phosphorylation of paxillin is a relatively late event after Fc epsilon RI aggregation. Both the direct increase in intracellular Ca2+ with calcium ionophore and the activation of protein kinase C (PKC) with PMA induced tyrosine phosphorylation of paxillin. The optimal tyrosine phosphorylation of paxillin by Fc epsilon RI aggregation required PKC and extracellular Ca2+. However, there was also Fc epsilon RI-mediated tyrosine phosphorylation of paxillin independent of Ca2+ influx or PKC activation. By fluorescent microscopy, cell stimulation induced a redistribution of paxillin toward the periphery of the cells. Although Fc epsilon RI aggregation induced tyrosine phosphorylation of paxillin in nonadherent cells, adherence markedly enhanced this phosphorylation. Together, the data suggest a role for paxillin in Fc epsilon RI signaling.

Animals↗
PubMed1994

The anti-ganglioside monoclonal antibody AA4 induces protein tyrosine phosphorylations, but not degranulation, in rat basophilic leukemia cells.

The monoclonal antibody AA4 (mAb AA4) recognizes novel alpha-galactosyl derivatives of GD1b on rat basophilic leukemia (RBL-2H3) cells. The binding of mAb AA4 induced protein tyrosine phosphorylations without histamine release. Several of the same proteins including Fc epsilon RI beta, Fc epsilon RI gamma, p72syk, and phospholipase C-gamma 1 were tyrosine-phosphorylated by mAb AA4 binding and by the activation of the high affinity IgE receptor, Fc epsilon RI. There was also activation of the p53/56lyn and p72syk protein-tyrosine kinases, but compared to direct Fc epsilon RI activation, mAb AA4 did not result in increased tyrosine phosphorylation of pp105-115 or pp125FAK, and the receptor subunits (Fc epsilon RI beta and Fc epsilon RI gamma) were more heavily phosphorylated. Furthermore, the time course of the phosphorylations with mAb AA4 was slower than that induced by Fc epsilon RI aggregation. By immunofluorescence, the tyrosine-phosphorylated proteins after mAb AA4 stimulation were localized in patches at the cell membrane and in areas of cell-cell contact, whereas after Fc epsilon RI activation, there was a reticular cytoplasmic pattern. There were no protein tyrosine phosphorylations either when Fc epsilon RI was saturated with IgE or when F(ab')2 fragments of mAb AA4 were used, although the F(ab')2 fragments still induced morphological changes. There was also coprecipitation of the beta and gamma subunits of Fc epsilon RI with the anti-ganglioside antibody. These data strongly suggest the involvement of Fc epsilon RI in the antiganglioside-induced protein tyrosine phosphorylations. Moreover, phosphorylations of these proteins including the beta and gamma chains of Fc epsilon RI and activation of p53/56lyn and p72syk did not result in histamine release.

Animals↗
PubMed1994

Src family tyrosine kinase Lyn binds several proteins including paxillin in rat basophilic leukemia cells.

Aggregation of the high affinity IgE receptors on rat basophilic leukemia (RBL-2H3) cells results in protein tyrosine phosphorylation although the receptor has no intrinsic enzymatic activity. The Src related protein tyrosine kinase p53/56lyn present in RBL-2H3 cells could play a role in this reaction. Here we have isolated the cDNA for rat Lyn and found it to be very homologous at the amino acid level to both the human and mouse proteins. A bacterially expressed maltose binding protein-Lyn (MBP-Lyn) fusion protein was already tyrosine phosphorylated and had tyrosine kinase activity. In a filter-binding assay, MBP-Lyn fusion protein (at 0.1 microM) specifically bound to several proteins of RBL-2H3 cells. In lysates of IgE receptor-activated cells, there was increased binding of MBP-Lyn to 65, 72, 78 and 110 kDa tyrosine phosphorylated proteins. The 72, 78 and 110 kDa tyrosine phosphorylated proteins were precipitated by a fusion protein containing the Lyn Src Homology 2 (SH2) domain. The 72 kDa Lyn binding protein was different from p72syk. Furthermore, paxillin, a cytoskeletal protein, was identified as one of the Lyn binding proteins. Thus Fc epsilon RI mediated signal transduction in RBL-2H3 cells may result from the interaction of p53/56lyn with paxillin, pp72, pp110 and other proteins.

Amino Acid Sequence↗
PubMed1994

Adhesion molecules as regulators of mast-cell and basophil function.

Basophils and mast cells play a role both in immediate allergic reactions and in inflammation. Both types of cells have surface adhesion receptors that can mediate binding to other cells and to extracellular matrix glycoproteins. Here Majed Hamawy and colleagues discuss the importance of these adhesion molecules in regulating basophil and mast-cell functions.

Animals↗
PubMed1993

Tyrosine phosphorylation of pp125FAK by the aggregation of high affinity immunoglobulin E receptors requires cell adherence.

The aggregation of the high affinity IgE receptor (Fc epsilon RI) in adherent rat basophilic leukemia (RBL-2H3) cells induces the tyrosine phosphorylation of several proteins. We examined whether focal adhesion-associated tyrosine kinase, pp125FAK, is one of these proteins. Anti-pp125FAK monoclonal antibody immunoblotted and precipitated a 115-kDa tyrosine-phosphorylated protein. In the absence of Fc epsilon RI aggregation, pp125FAK was tyrosine-phosphorylated only in adherent cells. Aggregating Fc epsilon RI in adherent cells markedly enhanced tyrosine phosphorylation of pp125FAK. This increase was detectable within 1 min of Fc epsilon RI aggregation and was maximal by 15 min. In contrast, in nonadherent cells Fc epsilon RI aggregation did not induce tyrosine phosphorylation of pp125FAK. The enhanced influx of calcium by calcium ionophore or the activation of protein kinase C by phorbol myristate acetate induced tyrosine phosphorylation of pp125FAK only in adherent cells. Thus, Fc epsilon RI-induced tyrosine phosphorylation of pp125FAK could be mediated by the activation of protein kinase C and/or the induction of calcium influx. The data indicate that cell adherence is essential for Fc epsilon RI-induced tyrosine phosphorylation of pp125FAK.

Animals↗
PubMed1993

Cell adherence to fibronectin and the aggregation of the high affinity immunoglobulin E receptor synergistically regulate tyrosine phosphorylation of 105-115-kDa proteins.

Adherence of cells to extracellular matrix components modulates cellular responses. Here we compared the array of tyrosine phosphorylated proteins induced by the aggregation of the high affinity receptor for IgE (Fc epsilon RI) in fibronectin-adherent and in nonadherent rat basophilic leukemia (RBL-2H3) cells. Adherence to fibronectin in the absence of Fc epsilon RI aggregation induced tyrosine phosphorylation of 105-115-kDa proteins. This phosphorylation was reversed by EDTA and by a synthetic peptide containing the sequence Arg-Gly-Asp, demonstrating a requirement for fibronectin-integrin interaction. Aggregation of Fc epsilon RI in fibronectin-adherent cells markedly enhanced the tyrosine phosphorylation of the same 105-115-kDa proteins. There were minimal differences in tyrosine phosphorylation of other proteins induced by the aggregation of Fc epsilon RI in nonadherent and in fibronectin-adherent cells. Direct activation of protein kinase C and/or increase in calcium influx induced the phosphorylation of the 105-115-kDa proteins only in fibronectin-adherent cells. The magnitude of the phosphorylation of the 105-115-kDa proteins induced by the aggregation of Fc epsilon RI in fibronectin-adherent cells was substantially greater than the sum of that due to adherence to fibronectin and the aggregation of Fc epsilon RI in nonadherent cells. Therefore, cell adherence and the aggregation of Fc epsilon RI synergistically regulate tyrosine phosphorylation of the 105-115 kDa proteins.

Amino Acid Sequence↗
PubMed1993

Mast cells and their microenvironment: the influence of fibronectin and fibroblasts on the functional repertoire of rat basophilic leukemia cells.

To determine how the microenvironment in which mast cells are located may influence their function, we explored the effects of fibronectin and fibroblasts on histamine secretion in vitro from a mast cell model, the rat basophilic leukemia (RBL-2H3) cell line. RBL-2H3 cells bound specifically to fibronectin-coated surfaces. Binding was maximal by 1 hour, was not detectable at 0 degrees C or in the absence of Ca++, and was inhibited by preincubating the cells with a synthetic peptide containing the RGD sequence. Adherence to fibronectin stimulated RBL-2H3 cell spreading with a concomitant reorganization of the cytoskeleton and a repositioning of the cytoplasmic granules to the cell periphery. Although adherence to fibronectin did not by itself induce histamine release, when stimulated by either immunologic or non-immunologic means, fibronectin-adherent cells released dramatically more histamine than cells plated in wells coated with BSA only. Thus, RBL-2H3 cells bind specifically to fibronectin, and in so doing are stimulated to undergo changes in morphology and enhanced responsiveness to secretory stimuli. RBL-2H3 cells grown in coculture with 3T3 fibroblasts, but not RBL-2H3 cells grown alone, became responsive to the polymeric synthetic secretagogue Compound 48/80 and the neuropeptide Substance P. Maximum sensitivity to Compound 48/80 was attained by the second week in coculture. Histamine release was dose-dependent, noncytotoxic and occurred even in the absence of extracellular Ca++. Contact between the 2 cell types appeared to be a critical factor. RBL-2H3 cells, separated from 3T3 cells by a 0.45 micron filter, failed to secrete histamine in response to Compound 48/80.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Adherence of rat basophilic leukemia (RBL-2H3) cells to fibronectin-coated surfaces enhances secretion.

Rat basophilic leukemia (RBL-2H3) cells are a useful in vitro model for studies of mast cells and basophils. We examined the adherence of RBL-2H3 cells to different extracellular matrix proteins and the effect of such attachment on secretion. The cells bound to fibronectin-coated surfaces with maximum binding by 1 h at 37 degrees C. There was less attachment to laminin, collagen type I, and collagen type IV. There was no adherence to uncoated surfaces or in the absence of Ca2+. Binding to fibronectin was blocked by a synthetic peptide containing the sequence Arg-Gly-Asp. Therefore, the binding of RBL-2H3 cells to fibronectin may be mediated by surface molecules that belong to the integrin family. Adherence to fibronectin-coated surfaces resulted in cell spreading, a reorganization of the cytoskeletal elements, and a redistribution of the secretory granules. Attachment to fibronectin also dramatically enhanced high affinity IgE receptor-mediated histamine release. This enhancement was maximum by 1 h of adherence and lasted for at least 6 h. There was also enhanced secretion by the Ca2+ ionophore A23187. Thus, adherence to fibronectin can enhance both receptor and non-receptor-mediated release. Addition of soluble fibronectin to RBL-2H3 cells in suspension had no effect on secretion. Therefore, enhanced histamine release required cell attachment to immobilized fibronectin. These results suggest that secretion from mast cells/basophils may be modulated by their interaction with the extracellular matrix.

Animals↗
PubMed1992

Inhibition of IgE binding to RBL-2H3 cells by a monoclonal antibody (BD6) to a surface protein other than the high affinity IgE receptor.

A mAb was isolated (mAb BD6) that recognized a surface glycoprotein on rat basophilic leukemia cells (RBL-2H3). The antibody bound to 2 x 10(6) molecules/cell and specifically blocked IgE binding (50% inhibition with 3.48 +/- 0.51 micrograms/ml; mean +/- SEM), although neither IgE nor anti-high affinity IgE receptor (anti-Fc epsilon RI) mAb blocked mAb BD6 binding to the cells. mAb BD6 did not affect the rate of dissociation of cell-bound IgE, nor did it induce or inhibit the internalization of IgE. mAb BD6 did not release histamine. However, it did cause rapid spreading of the cells. By 1 h the cells had retracted to a spherical shape with their surface covered with membranous spikes, and they could easily be detached from the tissue culture plate. These changes differed from those observed after Fc epsilon RI activation. mAb BD6 immunoprecipitated a complex of two proteins, 38 to 50 kDa and 135 kDa from 125I-surface labeled rat basophilic leukemia cells that are not subunits of Fc epsilon RI. Chemical cross-linking studies showed that these molecules are associated on the cell surface. By immunoblotting, mAb BD6 reacted with a 40-kDa protein. Therefore, mAb BD6 binds to a surface protein that is close to the Fc epsilon RI and sterically inhibits 125I-IgE binding.

Animals↗
PubMed1992

Antigen enhances neuronally induced contraction of intrapulmonary bronchi from IgE-producing rabbits.

The effect of in vitro antigen exposure on contraction induced by electrical field stimulation (EFS) was examined in bronchial rings isolated from rabbits producing specific IgE antibodies. After exposure to antigen, tissues showed an enhanced isometric contractile response to EFS especially at low frequencies, leading to a significant change in the mean slope factor (p less than 0.05) derived from modeling the log frequency response curve using a 4-parameter logistic function. Also, the mean log EF20 +/- SEM decreased from 1.03 +/- 0.05 to 0.88 +/- 0.07 Hz (p less than 0.02). This antigen-induced effect was blocked by pretreatment with 3 microM chlorpheniramine and not observed in unsensitized tissues. Antigen challenge of tissues passively sensitized with IgE (but not IgG) antibodies led to a similar EFS-enhancing effect, significantly reducing the mean slope factor (p less than 0.025). Substituting EFS with exogenous acetylcholine resulted in no antigen-induced enhancement of contraction. The data suggest that antigen-IgE interaction leads to local histamine release sufficient to enhance the function of excitatory airway neurons.

Animals↗