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At least 415 records · Page 23Linked to original sources
PubMed1997

CD148, a membrane protein tyrosine phosphatase, is able to induce tyrosine phosphorylation on human lymphocytes.

CD148 is a new cluster of differentiation defined in the VI International Workshop on Leucocyte Differentiation Antigens. It has been identified as the hematopoietic form of a formerly described membrane protein tyrosine phosphatase called HPTP eta/ DEP-1. Previous data have demonstrated that this molecule is able to give rise to [Ca2+]i increase. In the present work we show its capability to induce protein tyrosine phosphorylation in human lymphocytes in spite of its intrinsic protein tyrosine phosphatase activity. The induction of kinase activity suggests the involvement of some protein tyrosine kinase based signaling pathway. The activation of this postulated kinase could be carried out through a direct association or via an adapter molecule.

Antigens, CD↗
PubMed2001

Involvement of Hic-5 in platelet activation: integrin alphaIIbbeta3-dependent tyrosine phosphorylation and association with proline-rich tyrosine kinase 2.

Hic-5 and paxillin, members of the LIM protein family, have been shown to be localized in focal adhesion and to have a role in integrin-mediated signalling. In the present study we examined the involvement of Hic-5 in human platelet activation: platelets express Hic-5 but not paxillin, whereas human umbilical-vein vascular endothelial cells and MEG-01 cells express mainly paxillin. When platelets were stimulated with thrombin, collagen or the stable thromboxane A(2) analogue U46619, Hic-5 was markedly tyrosine-phosphorylated, in a manner dependent on integrin alphaIIbbeta3-mediated aggregation. In addition, direct activation of protein kinase C with PMA resulted in tyrosine phosphorylation of Hic-5 only when platelets were fully aggregated with the exogenous addition of fibrinogen. Furthermore, PMA-induced Hic-5 tyrosine phosphorylation was also observed when platelets adhered to immobilized fibrinogen. In studies on immunoprecipitation and immunodepletion, Hic-5 seemed to associate with proline-rich tyrosine kinase 2 (Pyk2) but only marginally with focal adhesion kinase. When platelets were stimulated with thrombin, both Hic-5 and Pyk2 translocated to the cytoskeleton from the cytosol and membrane fractions in a manner dependent on alphaIIbbeta3-mediated aggregation. Finally, on stimulation with PMA, Hic-5, as well as Pyk2, translocated to the cell periphery, where a meshwork of actin filaments assembled after adhesion to immobilized fibrinogen. Our results suggest that Hic-5 might be important in platelet aggregation and adhesion, in a manner dependent on alphaIIbbeta3-mediated outside-in signalling, through association with Pyk2.

Blood Platelets↗
PubMed2001

Role of tyrosine kinases and the tyrosine phosphatase SptP in the interaction of Salmonella with host cells.

Salmonella has evolved an intimate functional interface with its host. Central to this interface is a battery of bacterial proteins delivered into host cells via a specialized organelle termed the type III secretion system. A subset of these bacterial proteins stimulates cellular responses by activating the Rho family GTPases Cdc42 and Rac. Stimulation of these responses leads to actin cytoskeleton reorganization and the activation of cellular transcription factors that result in bacterial uptake and proinflammatory cytokine production. Remarkably, the cellular responses stimulated by Salmonella are quickly reversed by another bacterial protein, SptP, which exerts its function as a GTPase-activating protein (GAP) for Cdc42 and Rac. In addition to its GAP activity located within its amino-terminus, the carboxy-terminal domain of SptP possesses potent tyrosine phosphatase activity. We show here that the tyrosine phosphatase activity of SptP is involved in reversing the MAP kinase activation that results from Salmonella infection. We also demonstrate an important role for tyrosine kinases, including ACK, in the cellular responses induced by Salmonella. We also found that a potential target for the tyrosine phosphatase activity of SptP is the intermediate filament protein vimentin, which is recruited to the membrane ruffles stimulated by Salmonella.

Actins↗
PubMed1997

Protein-tyrosine phosphatase 1B complexes with the insulin receptor in vivo and is tyrosine-phosphorylated in the presence of insulin.

In response to insulin, protein-tyrosine phosphatase 1B (PTPase 1B) dephosphorylates 95- and 160-180-kDa tyrosine phosphorylated (PY) proteins (Kenner, K. A., Anyanwu, E., Olefsky, J. M., and Kusari, J. (1996) J. Biol. Chem. 271, 19810-19816). To characterize these proteins, lysates from control and insulin-treated cells expressing catalytically inactive PTPase 1B (CS) were immunoadsorbed and subsequently immunoblotted using various combinations of phosphotyrosine, PTPase 1B, and insulin receptor (IR) antibodies. Anti-PTPase 1B antibodies coprecipitated a 95-kDa PY protein from insulin-stimulated cells, subsequently identified as the IR beta-subunit. Similarly, anti-IR antibodies coprecipitated the 50-kDa PY-PTPase 1B protein from insulin-treated cells. To identify PTPase 1B tyrosine (Tyr) residues that are phosphorylated in response to insulin, three candidate sites (Tyr66, Tyr152, and Tyr153) were replaced with phenylalanine. Replacing Tyr66 or Tyr152 and Tyr153 significantly reduced insulin-stimulated PTPase 1B phosphotyrosine content, as well as its association with the IR. Studies using mutant IRs demonstrated that IR autophosphorylation is necessary for the PTPase 1B-IR interaction. These results suggest that PTPase 1B complexes with the autophosphorylated insulin receptor in intact cells, either directly or within a complex involving additional proteins. The interaction requires multiple tyrosine phosphorylation sites within both the receptor and PTPase 1B.

Insulin↗
PubMed2003

Interaction of SAP-1, a transmembrane-type protein-tyrosine phosphatase, with the tyrosine kinase Lck. Roles in regulation of T cell function.

SAP-1 is a transmembrane-type protein-tyrosine phosphatase that is expressed in most tissues but whose physiological functions remain unknown. The cytoplasmic region of SAP-1 has now been shown to bind directly the tyrosine kinase Lck. Overexpression of wild-type SAP-1, but not that of a catalytically inactive mutant of SAP-1, inhibited both the basal and the T cell antigen receptor (TCR)-stimulated activity of Lck in human Jurkat T cell lines. Lck served as a direct substrate for dephosphorylation by SAP-1 in vitro. Overexpression of wild-type SAP-1 in Jurkat cells also: (i) inhibited both the activation of mitogen-activated protein kinase and the increase in cell surface expression of CD69 induced by TCR stimulation; (ii) reduced the extent of the TCR-induced increase in the tyrosine phosphorylation of ZAP-70 or that of LAT; (iii) reduced both the basal level of tyrosine phosphorylation of p62dok, as well as the increase in the phosphorylation of this protein induced by CD2 stimulation; and (iv) inhibited cell migration. These results thus suggest that the direct interaction of SAP-1 with Lck results in inhibition of the kinase activity of the latter and a consequent negative regulation of T cell function.

Animals↗
PubMed2000

v-Src induces tyrosine phosphorylation of focal adhesion kinase independently of tyrosine 397 and formation of a complex with Src.

The non-receptor tyrosine kinase FAK plays a key role at sites of cellular adhesion. It is subject to regulatory tyrosine phosphorylation in response to a variety of stimuli, including integrin engagement after attachment to extracellular matrix, oncogene activation, and growth factor stimulation. Here we use an antibody that specifically recognizes the phosphorylated form of the putative FAK autophosphorylation site, Tyr(397). We demonstrate that FAK phosphorylation induced by integrins during focal adhesion assembly differs from that induced by activation of a temperature-sensitive v-Src, which is associated with focal adhesion turnover and transformation. Specifically, although v-Src induces tyrosine phosphorylation of FAK, there is no detectable phosphorylation of Tyr(397). Moreover, activation of v-Src results in a net decrease in fibronectin-stimulated phosphorylation of Tyr(397), suggesting possible antagonism between v-Src and integrin-induced phosphorylation. Our mutational analysis further indicates that the binding of v-Src to Tyr(397) of FAK in its phosphorylated form, which is normally mediated, at least in part, by the SH2 domain of Src, is not essential for v-Src-induced cell transformation. We conclude that different stimuli can induce phosphorylation of FAK on distinct tyrosine residues, linking specific phosphorylation events to ensuing biological responses.

Amino Acid Sequence↗
PubMed1993

The CD3 chains of the T cell antigen receptor associate with the ZAP-70 tyrosine kinase and are tyrosine phosphorylated after receptor stimulation.

Recent work indicates that signaling events resulting from stimulation of the T cell antigen receptor (TCR) can be initiated by the CD3 complex (gamma, delta, epsilon) as well as the zeta chains of the receptor. To help characterize the signaling function of CD3 we examined its associated tyrosine kinase activity since induction of tyrosine phosphorylation is one of the earliest signaling events. Our results indicate that at least two kinases, lck and ZAP-70, contribute to the CD3-associated kinase activity. A likely target of this activity is the CD3 complex itself since we observed that TCR stimulation resulted in rapid tyrosine phosphorylation of the CD3 epsilon and delta chains. To examine the function of the CD3 epsilon chain in particular, we constructed a chimera that fused the extracellular and transmembrane domains of CD8 to the cytoplasmic domain of CD3 epsilon. This chimera demonstrated that CD3 epsilon was independently capable of associating with proteins having tyrosine kinase activity, including ZAP-70. Our results show that the kinase activity that associates with the CD3 complex has characteristics that are quite similar to the previously characterized zeta-associated kinase activity. This finding suggests that both these components of the TCR initiate signaling events using a common mechanism. However, differences in their signaling function could result from recognition of distinct substrates.

Antibodies, Monoclonal↗
PubMed2006

Suppression of the phosphorylation of receptor tyrosine phosphatase-alpha on the Src-independent site tyrosine 789 by reactive oxygen species.

Oxidation of receptor protein tyrosine phosphatase-alpha (RPTPalpha) is emerging as an important yet poorly characterized regulatory mechanism for RPTPalpha signaling in cells. RPTPalpha has been shown to be reversibly oxidized and inhibited by reactive oxygen species. However, it is not known whether oxidative stress could regulate the phosphorylation of Tyr789, a critical tyrosine residue for RPTPalpha signaling that modulates the function of Grb2 and the activation of Src family kinases. In the present study, we have taken advantage of a phosphospecific antibody against Tyr789-phosphorylated RPTPalpha and characterized the phosphorylation of RPTPalpha Tyr789 in various cultured cells, including SYF cells lacking all three ubiquitously expressed members (Src, Yes, and Fyn) of Src family kinases. We have obtained substantial evidence indicating that the phosphorylation of RPTPalpha Tyr789 is regulated predominantly by an Src kinase inhibitor, protein phosphatase 1 (PP1)-sensitive but Src/Yes/Fyn-independent tyrosine kinase, in cells. We further reported a novel finding that, besides the inhibition of RPTPalpha's activity, H(2)O(2) at low to moderate concentrations (50-250 microM) markedly suppressed the phosphorylation of RPTPalpha Tyr789 and the association of RPTPalpha with Grb2 in cultured cells, which may result from inhibition of such a PP1-sensitive but Src/Yes/Fyn-independent tyrosine kinase. Because Tyr789 plays an important role in RPTPalpha signaling, our findings may provide new insights into the functional regulation of RPTPalpha by oxidative stress in cells.

Cells, Cultured↗
PubMed2001

Effects of protein tyrosine kinase and protein tyrosine phosphatase on apical K(+) channels in the TAL.

We have previously demonstrated that the protein level of c-Src, a nonreceptor type of protein tyrosine kinase (PTK), was higher in the renal medulla from rats on a K-deficient (KD) diet than that in rats on a high-K (HK) diet (Wang WH, Lerea KM, Chan M, and Giebisch G. Am J Physiol Renal Physiol 278: F165-F171, 2000). We have now used the patch-clamp technique to investigate the role of PTK in regulating the apical K channels in the medullary thick ascending limb (mTAL) of the rat kidney. Inhibition of PTK with herbimycin A increased NP(o), a product of channel number (N) and open probability (P(o)), of the 70-pS K channel from 0.12 to 0.42 in the mTAL only from rats on a KD diet but had no significant effect in tubules from animals on a HK diet. In contrast, herbimycin A did not affect the activity of the 30-pS K channel in the mTAL from rats on a KD diet. Moreover, addition of N-methylsulfonyl-12,12-dibromododec-11-enamide, an agent that inhibits the cytochrome P-450-dependent production of 20-hydroxyeicosatetraenoic acid, further increased NP(o) of the 70-pS K channel in the presence of herbimycin A. Furthermore, Western blot detected the presence of PTP-1D, a membrane-associated protein tyrosine phosphatase (PTP), in the renal outer medulla. Inhibition of PTP with phenylarsine oxide (PAO) decreased NP(o) of the 70-pS K channel in the mTAL from rats on a HK diet. However, PAO did not inhibit the activity of the 30-pS K channel in the mTAL. The effect of PAO on the 70-pS K channel was due to indirectly stimulating PTK because pretreatment of the mTAL with herbimycin A abolished the inhibitory effect of PAO. Finally, addition of exogenous c-Src reversibly blocked the activity of the 70-pS K channel in inside-out patches. We conclude that PTK and PTP have no effect on the low-conductance K channels in the mTAL and that PTK-induced tyrosine phosphorylation inhibits, whereas PTP-induced tyrosine dephosphorylation stimulates, the apical 70-pS K channel in the mTAL.

Amides↗
PubMed2003

Tyrosine phosphorylation of the beta3-subunit of the alphaVbeta3 integrin is required for embrane association of the tyrosine phosphatase SHP-2 and its further recruitment to the insulin-like growth factor I receptor.

Ligand occupancy of the alphaVbeta3 integrin is required for IGF-I receptor (IGF-IR) phosphorylation of an appropriate duration and for stimulation of IGF-I actions. In vascular smooth muscle cells (SMCs), the tyrosine phosphatase SHP-2 regulates the duration of IGF-IR phosphorylation and biological actions. We determined the role of ligand occupancy of the alphaVbeta3 integrin on beta3 phosphorylation and studied the role of beta3 phosphorylation in regulating both SHP-2 recruitment to the cell membrane and IGF-I-dependent biological responses. Vitronectin binding to alphaVbeta3 induced tyrosine phosphorylation of the beta3-subunit in subconfluent SMCs and was accompanied by increased association of SHP-2 with beta3. In confluent SMCs, the beta3-subunit was constitutively phosphorylated leading to basal binding of SHP-2. The Src kinase inhibitor PP2 caused a concentration-dependent decrease in beta3 phosphorylation and resulted in decreased SHP-2 association with beta3 and with the cell membrane. In contrast to control cells, SMCs expressing a mutant beta3 that had two tyrosines changed to phenylalanines showed a 89.9 +/- 1.2% decrease in beta3 phosphorylation. This decrease was associated with reduced SHP-2 binding to nonphosphorylated beta3 and a corresponding decrease in the membrane association of SHP-2. When IGF-I was added to cells expressing mutant beta3, SHP-2 was not recruited to the Src homology 2 domain-containing tyrosine phosphatase substrate-1 or to IGF-IR. This was associated with prolonged IGF-IR phosphorylation and an impaired cellular DNA synthesis response to IGF-I. These results define a mechanism by which ligand occupancy of alphaVbeta3 regulates the SMC response to IGF-I.

Cell Membrane↗
PubMed2005

T cell activation-induced CrkII binding to the Zap70 protein tyrosine kinase is mediated by Lck-dependent phosphorylation of Zap70 tyrosine 315.

The Zap70 protein tyrosine kinase controls TCR-linked signal transduction pathways and is critical for T cell development and responsiveness. Following engagement of TCR, the Zap70 undergoes phosphorylation on multiple tyrosine residues that are implicated in the regulation of its catalytic activity and interaction with signaling effector molecules downstream of the TCR. We have shown previously that the CT10 regulator of kinase II (CrkII) adapter protein interacts with tyrosine-phosphorylated Zap70 in TCR-engaged T cells, and now extend these studies to show that Tyr315 in the Zap70 interdomain B region is the site of interaction with CrkII. A point mutation of Tyr315 (Y315F) eliminated the CrkII-Zap70 interaction capacity. Phosphorylation of Tyr315 and Zap70 association with CrkII were both dependent upon the Lck protein tyrosine kinase. Previous studies demonstrated the Tyr315 is the Vav-Src homology 2 (SH2) binding site, and that replacement of Tyr315 by Phe impaired the function of Zap70 in TCR signaling. However, fluorescence polarization-based binding studies revealed that the CrkII-SH2 and the Vav-SH2 bind a phosphorylated Tyr315-Zap70-derived peptide with affinities of a similar order of magnitude (Kd of 2.5 and 1.02 microM, respectively). The results suggest therefore that the biological functions attributed to the association of Zap70 with Vav following T cell activation may equally reflect the association of Zap70 with CrkII, and further support a regulatory role for CrkII in the TCR-linked signal transduction pathway.

Binding Sites↗
PubMed1994

Identification by electrospray ionization mass spectrometry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP-70.

We have developed a rapid and sensitive two capillary-column chromatography and mass spectrometry-based method for the determination of protein phosphorylation sites following recovery of individual phosphopeptides from two-dimensional phosphopeptide maps. With a standard phosphopeptide, we demonstrate detection sensitivity of at least 250 fmol for this system. We applied this technique to the analysis of in vitro sites of tyrosine phosphorylation induced on the T cell-specific protein tyrosine kinase ZAP-70 in the absence and presence of p56lck. We show that ZAP-70 has a primary autophosphorylation site at Tyr-292, with a secondary site at Tyr-126. We also show additional phosphorylation at Tyr-69, Tyr-178, Tyr-492, and Tyr-493 upon the addition of the protein tyrosine kinase, p56lck. By comparative two-dimensional phosphopeptide mapping, we show that ZAP-70 isolated from Jurkat T cells also autophosphorylates at Tyr-292 and Tyr-126. Similar analysis of 32P-labeled Jurkat cells stimulated with anti-T cell receptor antibodies reveals Tyr-492 and Tyr-493 as the principal sites of T cell antigen receptor-induced tyrosine phosphorylation, with additional phosphorylation at the Tyr-292, but not the Tyr-126 autophosphorylation site. The high degree of sensitivity achieved with this technology should greatly facilitate the direct biochemical determination of inducible protein phosphorylation events, an experimental strategy that until now has been both time consuming and difficult.

Amino Acid Sequence↗
PubMed1993

In vitro activation of the transcription factor ISGF3 by interferon alpha involves a membrane-associated tyrosine phosphatase and tyrosine kinase.

Interaction of interferon alpha (IFN alpha) with its cell surface receptor rapidly activates the formation of the transcription complex ISGF3, which subsequently translocates to the nucleus and stimulates the expression of a variety of early response genes. We have recently developed a cell-free system where IFN alpha can activate the formation of ISGF3 in vitro. This system has enabled us to demonstrate that the component of the ISGF3 transcription complex which is modified by IFN alpha treatment (ISGF3 alpha) is membrane-associated and that its activation involves a protein kinase. Using a combination of specific tyrosine kinase and phosphatase inhibitors and monoclonal anti-phosphotyrosine antibodies we now are able to demonstrate that IFN alpha-activated transcription involves at least a two-step process where a membrane-associated tyrosine phosphatase and a tyrosine kinase lead to modification of ISGF3 alpha and subsequent formation of the complete complex. Furthermore, formation of the ISGF3 complex is specifically disrupted by protein tyrosine phosphatase and can be reversibly dissociated by the phosphotyrosine analogue phenylphosphate. The latter observation suggested that SH2 and/or SH3 domains may be required for the stable formation of this transcription complex.

Base Sequence↗
PubMedDate not supplied

Enzymatic polymerization of tyrosine derivatives. Peroxidase- and protease-catalyzed synthesis of poly(tyrosine)s with different structures.

Polymerization of tyrosine derivatives has been carried out by using two enzymes, peroxidase and protease, as catalyst to give poly(tyrosine)s with different structures. Tyrosine ester hydrochlorides were oxidatively polymerized by a peroxidase in a buffer. Using a high buffer concentration produced the polymer in good yields. The resulting polymer was soluble in N,N-dimethylformamide, dimethyl sulfoxide, and methanol but was insoluble in acetone, tetrahydrofuran, and water. The ester moiety of the polymer was subjected to the alkaline hydrolysis, yielding a water-soluble polymer having the amino acid group in the side chain. The peroxidase also catalyzed the oxidative polymerization of N-acetyltyrosine to give the polymer soluble in water. The polymerization of tyrosine ester hydrochlorides proceeded in the presence of papain catalyst to give a polymer of alpha-peptide structure. The polymerization in the buffer of high phosphate concentration efficiently produced the polymer. On the other hand, the polymer formation was not observed in the low buffer concentration. The molecular weight was several thousands and almost constant during the reaction. The morphology of the precipitated polymer was examined. The product of the initial reaction stage was amorphous. After 24 h, the precipitates exhibiting clear birefringence were formed. Scanning electron microscopy observation of the polymer after 72 h showed the formation of a globular crystal in a diameter larger than 50 microm, which was not found by recrystallization of poly(tyrosine).

Biocompatible Materials↗
PubMed1994

Skin peptide tyrosine-tyrosine, a member of the pancreatic polypeptide family: isolation, structure, synthesis, and endocrine activity.

Pancreatic polypeptide, peptide tyrosine-tyrosine (PYY), and neuropeptide tyrosine (NPY), three members of a family of structurally related peptides, are mainly expressed in the endocrine pancreas, in endocrine cells of the gut, and in the brain, respectively. In the present study, we have isolated a peptide of the pancreatic polypeptide family from the skin of the South American arboreal frog Phyllomedusa bicolor. The primary structure of the peptide was established as Tyr-Pro-Pro-Lys-Pro-Glu-Ser-Pro-Gly-Glu10-Asp-Ala-Ser-Pro-Glu-Glu- Met-Asn- Lys-Tyr20-Leu-Thr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Val-Thr- Arg-Gln-Arg-Tyr-NH2 . This unusual peptide, named skin peptide tyrosine-tyrosine (SPYY), exhibits 94% similarity with PYY from the frog Rana ridibunda. A synthetic replicate of SPYY inhibits melanotropin release from perifused frog neurointermediate lobes in very much the same way as NPY. These results demonstrate the occurrence of a PYY-like peptide in frog skin. Our data also suggest the existence of a pituitary-skin regulatory loop in amphibians.

Amino Acid Sequence↗
PubMed1998

The 38-amino-acid form of pituitary adenylate cyclase-activating polypeptide induces neurite outgrowth in PC12 cells that is dependent on protein kinase C and extracellular signal-regulated kinase but not on protein kinase A, nerve growth factor receptor tyrosine kinase, p21(ras) G protein, and pp60(c-src) cytoplasmic tyrosine kinase.

The 38-amino-acid isoform of pituitary adenylate cyclase-activating polypeptide (PACAP38) elicits a robust outgrowth of neurites in cultured PC12 cells. Initiation of neurite outgrowth occurs within 4-8 hr after the addition of PACAP38. Treatment with PACAP38 does not elicit collateral activation of p140(trk) nerve growth factor receptor tyrosine kinase activity, nor is it associated with tyrosine phosphorylation of suc1-associated neurotrophic factor target, a selective target of neurotrophin tyrosine kinase receptors. Coadministration of epidermal growth factor with PACAP38 elicits an enhanced response. Induction of neurites is also observed on the addition of PACAP38 to dominant negative Src and Ras PC12 cell variants. PACAP38 stimulates extracellular signal-regulated kinase (Erk) activity >10-fold within 5 min, and the effect is augmented by cotreatment with epidermal growth factor. Pretreatment with the cAMP-dependent protein kinase-selective inhibitor, H-89, is ineffective as an antagonist of PACAP38-induced neurite outgrowth, whereas down-regulation of protein kinase C (PKC) by phorbol ester or incubation with PKC-selective inhibitors GF109203X and calphostin C effectively blocks PACAP38-stimulated neurite formation. Stimulation of Erk activity is inhibited by incubation with PD90859, a pharmacological antagonist of the threonine/tyrosine dual-specificity Erk. Inhibition of ligand-stimulated Erk activation prevents PACAP38-induced neurite outgrowth. Collectively, these findings indicate that PACAP38-stimulated neuritogenesis requires PKC and Erk activation but is independent of cAMP-dependent protein kinase, nerve growth factor receptor tyrosine kinase, p21(ras) G protein, and pp60(c-src) cytoplasmic tyrosine kinase.

Animals↗
PubMed1998

Role for tyrosine phosphorylation and Lyn tyrosine kinase in fas receptor-mediated apoptosis in eosinophils.

Fas ligand/Fas receptor molecular interactions have been implicated as having an important function for the regulation of eosinophil apoptosis. The purpose of the present study was to investigate biochemical events triggered by the engagement of the Fas receptor in freshly isolated human and mouse eosinophils. Activation of the Fas receptor on eosinophils with the agonistic anti-Fas monoclonal antibody (MoAb) resulted in increased tyrosine phosphorylation of several intracellular proteins. The tyrosine kinase inhibitors lavendustin A and genistein inhibited Fas receptor-induced cell death in both human and mouse eosinophils in vitro and prevented, at least partially, Fas receptor-mediated resolution of eosinophilic inflammation in a mouse in vivo model of lung eosinophilia. In addition, in freshly purified human eosinophils, lavendustin A prevented anti-Fas MoAb-induced proteolytic cleavage of lamin B, suggesting that tyrosine kinases may amplify the proteolytic signaling cascade within interleukin-1beta converting enzyme (ICE) family proteases. Moreover, the tyrosine kinase Lyn was identified as being involved in Fas receptor-mediated cell death. Collectively, these results demonstrate that tyrosine phosphorylation is an important step in the generation of the Fas receptor-linked transmembrane death signal in eosinophils and that Lyn participates in this pathway.

Animals↗
PubMed1994

The CD45 tyrosine phosphatase regulates specific pools of antigen receptor-associated p59fyn and CD4-associated p56lck tyrosine in human T-cells.

A newly isolated T-cell line (CB1) derived from a T-acute lymphoblastic leukaemia (T-ALL) patient contained cells (40% of total) which did not express the CD45 phosphotyrosine phosphatase. The cells were sorted into CD45- and CD45+ populations and shown to be clonal in origin. T-cell receptor (TCR) cross-linking or coligation of the TCR with its CD4/CD8 co-receptors induced tyrosine phosphorylation and calcium signals in CD45+ but not in CD45- cells. Unexpectedly, whole cell p56lck and p59fyn tyrosine kinase activities were not reduced in CD45- compared to CD45+ cells. A novel technique was therefore developed to isolated specific pools of aggregated receptors expressed at the cell surface, together with their associated tyrosine kinases. Using this technique it was shown that cell surface CD4-p56lck kinase activity was 78% lower in CD45- than in CD45+ cells. Phosphorylation of TCR zeta- and gamma-chains occurred in TCR immunocomplexes from CD45+ but not CD45- cells, despite comparable levels of p59fyn and TCR proteins. Furthermore, TCR-associated tyrosine kinase activity towards an exogenous substrate was 84% lower in CD45- than in CD45+ cells. Addition of recombinant p59fyn to TCR immunocomplexes isolated from CD45-cells restored the phosphorylation of the TCR zeta- and gamma-chains. Our results demonstrate that CD45 selectively regulates the pools of p59fyn and p56lck kinases which are associated with the TCR and CD4 at the cell surface. Activation by CD45 of these receptor-associated kinase pools correlates with the ability of the TCR and its coreceptors to couple to intracellular signalling pathways.

CD4 Antigens↗