SEARCH · Search PubMed

Results for “TYROSINE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,639 records · Page 91Linked to original sources

PubMed1994

Regulatory role of major tyrosine autophosphorylation site of kinase domain of c-Met receptor (scatter factor/hepatocyte growth factor receptor).

Ligand-induced tyrosine kinase activation of the scatter factor/hepatocyte growth factor receptor (c-Met) is thought to be essential for the biological responses of target cells. To assess the regulatory role of the major tyrosine autophosphorylation site (tyrosine 1233) of the mouse c-Met receptor in the tyrosine kinase activation of the receptor, we constructed a mutant receptor in which the tyrosine residue was replaced with phenylalanine. When the cells expressing the mutant receptor were incubated with the ligand, no biological responses were observed, and the level of tyrosine phosphorylation of the receptor was very low compared with that of the wild-type receptor. The in vitro kinase activity of the mutant receptor toward an exogenous substrate and the receptor itself was also low. Furthermore, tyrosine phosphorylation of the cellular proteins by ligand stimulation was not detected in intact cells expressing the mutant receptor. The low level of kinase activity and the lack of biological activity of the mutant receptor indicate that the major autophosphorylation site positively regulates the tyrosine kinase of the c-Met receptor and phosphorylation of cellular substrates in the scatter factor/hepatocyte growth factor signaling pathway.

Animals↗

PubMed1994

Tyrosine phosphorylation of a M(r) 38,000 A/B-type hnRNP protein selectively modulates its RNA binding.

The M(r) 38,000 RNA-binding protein (P38) is the major component of translationally repressed messenger ribonucleoproteins in cryptobiotic gastrulae of the brine shrimp Artemia. Partial elucidation of the amino acid sequence of P38 reveals that it is homologous to A/B-type hnRNP proteins. This was confirmed by immunodetection with antibodies specific for A/B-type hnRNP proteins from Drosophila melanogaster. P38 can be phosphorylated in vitro by a src-related protein tyrosine kinase on multiple tyrosine residues located predominantly in the glycine-rich domain. Tyrosine phosphorylated P38 can be efficiently dephosphorylated by a specific protein tyrosine phosphatase (1B-like) and by protein phosphatase 2A activated by the phosphotyrosyl phosphatase activator. Tyrosine phosphorylation of P38 slightly influences its subsequent phosphorylation by casein kinase II. The latter phosphorylation site is located in the glycine-rich domain of P38. Two-dimensional gel electrophoresis resolves P38 into multiple isoforms which shift to more acidic pI values after phosphorylation by protein tyrosine kinase or casein kinase II. From nitrocellulose filter binding and UV cross-linking analysis, evidence was obtained that tyrosine phosphorylation of P38 impairs its binding to poly(A) but not to poly(U). This demonstrates the involvement of tyrosine residues in polynucleotide-specific RNA binding that can be regulated by phosphorylation/dephosphorylation.

Amino Acid Sequence↗

PubMed1994

Induction of tyrosine phosphorylation of Vav and expression of Pim-1 correlates with Jak2-mediated growth signaling from the erythropoietin receptor.

The receptor for erythropoietin (Epo) belongs to the cytokine receptor family and lacks a tyrosine kinase domain. However, it has been hypothesized that a tyrosine kinase, Jak2, associates with the membrane proximal cytoplasmic region of Epo receptor (EpoR) and mediates the growth signaling from the receptor through tyrosine phosphorylation of cellular substrates. To explore the growth signaling pathways from the EpoR, we analyzed substrates of tyrosine phosphorylation induced by Epo stimulation in cells expressing various mutant EpoRs. The vav proto-oncogene product was found to be tyrosine phosphorylated after Epo stimulation in cells expressing the wild-type EpoR or a truncated receptor, H mutant, that retains the growth signaling function. In these cells, Epo also induced the expression of a serine/threonine kinase, Pim-1. However, Epo stimulation did not have any effect on Vav or Pim-1 in cells expressing a mutant EpoR, PM4 mutant, inactivated by a point mutation, Trp282 to Arg, in the membrane proximal region, which abrogates the interaction with Jak2. On the other hand, both tyrosine phosphorylation of Vav and expression of Pim-1 were observed constitutively in cells expressing a mutant EpoR that is constitutively activated by a point mutation, Arg 129 to Cys, in the extracellular domain. Jak2 was also constitutively tyrosine phosphorylated and activated in cells expressing this mutant, which confirms the crucial role of Jak2 in growth signaling from the EpoR. Taken together, these observations suggest that the tyrosine phosphorylation of Vav and the expression of Pim-1 may play important roles in growth signaling from the EpoR.

Amino Acid Sequence↗

PubMed1995

Protein tyrosine phosphatase activity associates with the high affinity IgE receptor and dephosphorylates the receptor subunits, but not Lyn or Syk.

Protein tyrosine phosphorylation is an early event in the high affinity IgE receptor (Fc epsilon RI)-mediated signaling cascade leading to secretion in mast cells. Numerous proteins, including the beta- and gamma-subunits of Fc epsilon RI, become tyrosine phosphorylated after receptor aggregation. Dephosphorylation of these proteins may be important to reverse and limit transmembrane signaling. RBL-2H3 mast cell lysates were found to contain protein tyrosine phosphatase activity that dephosphorylated the tyrosine-phosphorylated beta- and gamma-subunits of Fc epsilon RI. The protein tyrosine phosphatase activity associated with Fc epsilon RI and was equally present with receptors from nonactivated and stimulated cells. Moreover, the phosphatase eluted from the immunoprecipitates and, when added back, dephosphorylated both tyrosine-phosphorylated beta- and gamma-subunits, but not tyrosine-phosphorylated Lyn or Syk. These results strongly suggest that the IgE receptor-associated protein tyrosine phosphatase may regulate the steady state level of tyrosylphosphate of the beta- and gamma-subunits and, therefore, may modulate the interaction of these subunits with other downstream molecules, such as Syk.

Animals↗

PubMed1995

Tyrosine phosphatase antagonist-induced activation of the neutrophil NADPH oxidase: a possible role for protein kinase C.

To investigate the role of tyrosine phosphorylation in polymorphonuclear leucocyte (PMN) activation we have examined the effect of the potent tyrosine phosphatase (PTPase) inhibitor, vanadyl hydroperoxide, on PMN function. Western blotting of vanadyl hydroperoxide-treated PMN showed that there was a rapid dose-dependent increase in tyrosine-phosphorylated proteins. Vanadyl hydroperoxide also induced superoxide production in PMN over the range 10-100 microM, similar to the concentrations that also induced tyrosine phosphorylation. The tyrosine kinase inhibitor erbstatin totally inhibited the respiratory burst induced by vandyl hydroperoxide, showing that tyrosine kinase activity was necessary for superoxide production. The protein kinase C (PKC) inhibitors chelerythrine and bisidolylmaleimide inhibited the vanadyl hydroperoxide-induced respiratory burst with an inhibitory concentration of 50% (IC50) close to that for PKC inhibition without affecting tyrosine phosphorylation. These results indicate a possible role for PKC in vanadyl hydroperoxide-mediated superoxide production, and that any PKC involvement is downstream of tyrosine phosphorylation. These results further demonstrate that inhibition of phosphotyrosine phosphatases results in the activation of a functional response, indicating a critical role for phosphotyrosine phosphatases in PMN stimulation.

Animals↗

PubMed1993

Anti-TAPA-1 antibodies induce protein tyrosine phosphorylation that is prevented by increasing intracellular thiol levels.

We studied the signal induced by the anti-TAPA-1 antibody and compared it to the signal induced by anti-IgM antibodies in a human B cell line, OCl-LY8. We found that exposure of these cells to either antibody resulted in a rapid increase in protein tyrosine phosphorylation which was prevented by inhibitors of tyrosine kinases. Tyrosine phosphorylation was an early event in the cascase leading to the antiproliferative effect of the anti-TAPA-1 antibody. However, 2-ME, a reducing agent that is not an inhibitor of tyrosine kinases, prevented both tyrosine phosphorylation and the antiproliferative effect of the antibody. Cells grown in low concentrations of 2-ME did not exhibit an increase in tyrosine phosphorylation in response to the anti-TAPA-1 antibody and were insensitive to the antiproliferative effect of the antibody. In contrast, the same cells maintained in 2-ME were able to induce tyrosine phosphorylation in response to anti-IgM. The use of 2-ME resulted in an increase in intracellular thiols, mostly glutathione. Moreover, compounds that block glutathione synthesis rendered cells susceptible to the antibody, even in the presence of 2-ME. These experiments demonstrate that tyrosine kinases are involved in propagating the antiproliferative signal initiated by the anti-TAPA-1 antibody and suggest that this signal is dependent upon the level of intracellular thiols.

Antigens, CD↗

PubMed1994

Regulation of the protein tyrosine kinase pp72syk by platelet agonists and the integrin alpha IIb beta 3.

Agonist stimulation of platelets induces multiple waves of tyrosine phosphorylation, several of which are dependent on the integrin alpha IIb beta 3. At least two classes of protein tyrosine kinases are activated during various stages of platelet activation, 1) Src family tyrosine kinases are activated during an early phase of platelet activation by an integrin-independent mechanism and 2) pp125FAK is activated during a late stage of platelet activation, and it is dependent on platelet aggregation mediated by fibrinogen binding to alpha IIb beta 3. In this report, we examined the mechanism of agonist-induced phosphorylation and activation of the tyrosine kinase pp72syk, which is known to couple with immune response receptors in B cells and mast cells. pp72syk was found to be regulated by both agonist and integrin receptors in a pattern distinct from that of pp60src and pp125FAK. Specifically, thrombin induced the tyrosine phosphorylation and activation of pp72syk independent of platelet aggregation. However, full activation of pp72syk required integrin engagement since treatment with antibodies that block fibrinogen binding to alpha IIb beta 3 reduced pp72syk phosphorylation by 40%. Furthermore, fibrinogen binding to alpha IIb beta 3 stimulated directly with an anti-beta 3 antibody activated pp72syk 3-fold and stimulated its tyrosine phosphorylation. Thus, pp72syk is the only platelet tyrosine kinase identified to date that can be directly activated through integrin ligation. In addition, we found that the activation of pp72syk is dependent upon the state of actin polymerization and that pp72syk redistributes to actin-rich cytoskeletal complexes in an aggregation-dependent manner. These results suggest a role for pp72syk in both early, integrin-independent tyrosine phosphorylation events as well as those dependent upon subsequent integrin engagement.

Blood Platelets↗

PubMed1994

Correlations among tyrosine phosphorylation of Shc, p72syk, PLC-gamma 1, and [Ca2+]i flux in Fc gamma RIIA signaling.

Tyrosine phosphorylation plays a critical role in Fc gamma RIIA signaling. In a mouse macrophage cell line transfected with human Fc gamma RIIA, cross-linking Fc gamma RIIA led to the transient generation of inositol 1, 4, 5-trisphosphate (IP3), [Ca2+]i flux, and rapid tyrosine phosphorylation of cellular substrates, including Shc, PLC-gamma 1, and a tyrosine kinase p72syk. In addition, tyrosine phosphorylated Fc gamma RIIA was co-precipitated with activated PLC-gamma 1. In contrast, no tyrosine phosphorylation of Shc or PLC-gamma 1 was detected in cells transfected with mutant receptors that failed to trigger [Ca2+]i flux. PMA inhibits both tyrosine phosphorylation of Shc and IP3 production leading to [Ca2+]i flux. However, PMA does not affect tyrosine phosphorylation of PLC-gamma 1 and p72syk. These results suggest that tyrosine phosphorylation of Shc and PLC-gamma 1 is important for the initiation of [Ca2+]i flux, and that activation of protein kinase C may modulate the activity of PLC-gamma 1 through serine/threonine phosphorylation.

Amino Acid Sequence↗

PubMed1994

Autophosphorylation modulates the kinase activity and oncogenic potential of the Met receptor tyrosine kinase.

The met oncogene is activated by a genomic rearrangement that generates a hybrid protein containing tpr sequences at its amino terminus fused directly to the met (hepatocyte growth factor/scatter factor) receptor tyrosine kinase domain. The resultant p65Tpr-Met hybrid protein possesses tyrosine kinase activity and is constitutively phosphorylated on tyrosine in vivo. A substitution of the conserved lysine in the catalytic domain abolished kinase activity and the transforming potential of Tpr-Met demonstrating the requirement for kinase activity for transformation. To study the role of autophosphorylation at particular tyrosine residues on the transforming activity of Tpr-Met, the major autophosphorylation sites were identified by two dimensional phosphopeptide mapping. Two tyrosine residues in the catalytic domain, tyrosine 365 and tyrosine 366, were found to be the major autophosphorylation sites both in vitro and in vivo. Mutation of these sites singly or together modulates the biochemical and biological properties of Tpr-Met. Compared with the wild-type Tpr-Met, mutants have a reduced in vitro kinase activity when measured by levels of autophosphorylation and by their ability to phosphorylate an exogenous substrate and have little or no transforming activity. These results indicate that autophosphorylation at critical tyrosine residues is required for activation of the kinase activity of Tpr-Met and for cell transformation by this oncogene.

3T3 Cells↗

PubMed1996

Sensitization of HER-2/neu-overexpressing non-small cell lung cancer cells to chemotherapeutic drugs by tyrosine kinase inhibitor emodin.

Overexpression of the HER-2/neu proto-oncogene which encodes tyrosine kinase receptor p185neu, has been observed frequently in many human cancers, including non-small cell lung cancer (NSCLC), and is correlated with poor patient survival in these cancers. In addition, HER-2/neu overexpression in NSCLC is known to induce chemoresistance. Recently, we demonstrated that emodin, a tyrosine kinase inhibitor, suppresses HER-2/neu tyrosine kinase activity in HER-2/neu-overexpressing breast cancer cells and preferentially represses proliferation of these cells. The work described here was carried out to examine (1) whether the tyrosine kinase activity of p185neu is required for resistance to chemotherapeutic drugs of HER-2/neu-overexpressing NSCLC cells and (2) whether the tyrosine kinase inhibitor emodin can sensitize these cells to chemotherapeutic drugs. We found that emodin decreased tyrosine phosphorylation of HER-2/neu and preferentially suppressed proliferation of HER-2/neu-overexpressing NSCLC cells. Furthermore, the combination of emodin with cisplatin, doxorubicin or etoposide (VP16) synergistically inhibited the proliferation of HER-2/neu-overexpressing lung cancer cells, whereas low doses of emodin, cisplatin, doxorubicin, or VP16 alone had only minimal antiproliferative effects on these cells. These results indicate that tyrosine kinase activity is required for the chemoresistant phenotype of HER-2/neu-overexpressing NSCLC cells and that tyrosine kinase inhibitors such as emodin can sensitize these cells to chemotherapeutic drugs. The results may have important implications in chemotherapy for HER-2/neu-overexpressing cancers.

Antineoplastic Agents↗

PubMed1996

Increase of protein tyrosine phosphorylation in rat retina after ischemia-reperfusion injury.

PURPOSE: This study was conducted to examine the effect of retinal ischemia-reperfusion injury on protein tyrosine phosphorylation, the production of angiogenic growth factors, and the activation of signal proteins in tyrosine kinase pathways. METHODS: Ischemia-reperfusion injury was induced in rats by compression of the optic nerve for 2 hours. The rats were killed, and the retinas were collected at 0, 1, 6, 24, 48, 96, or 168 hours of reperfusion. Tyrosine phosphorylation of proteins in the retina was examined by Western blot analysis and immunohistochemistry. Angiogenic growth factors and their receptors, such as basic fibroblast growth factor (bFGF) and Flg, vascular endothelial growth factor (VEGF) and Flk-1, platelet-derived growth factor (PDGF)-B chain and PDGF-beta receptor, and five intracellular signal proteins (phosphatidylinositol 3-kinase [PI3K], phospholipase C gamma [PLC gamma], C-Src, SHC, and mitogen-activated protein kinase [MAPK]) were examined by Western blot analysis. RESULTS: Protein tyrosine phosphorylation increased after ischemia-reperfusion injury, reaching a peak at 48 hours of reperfusion. Increased staining of tyrosine-phosphorylated proteins in the inner retina were evident on immunohistochemical examination. The amount of bFGF decreased after injury, but the amounts of VEGF and PDGF-B chain increased. Tyrosine phosphorylation of PLC gamma, SHC, and MAPK was increased at 48 hours of reperfusion, and tyrosine phosphorylation of PDGF-beta receptor and PI3K was increased at 168 hours of reperfusion. CONCLUSIONS: Ischemia-reperfusion injury in the rat retina leads to activation of the tyrosine kinase pathway, increasing the amounts of angiogenic growth factors. The resultant activation of signal proteins PLC gamma, SHC, MAPK, PI3K, and PDGF-beta receptor may play an important role in ischemia-induced retinal changes such as cell proliferation.

Animals↗

PubMed1996

Leishmania-induced tyrosine phosphorylation in the host macrophage and its implication to infection.

Tyrosine phosphorylation is an important mechanism of cell regulation and has been recently implicated in defense strategies against a variety of pathogens. We have investigated the involvement of protein tyrosine kinase activity in the Leishmania attachment, invasion and survival within macrophages, as well as promastigote ability to trigger tyrosine phosphorylation, which could contribute to leishmanicidal activity. Treatment of murine macrophage monolayers with genistein, herbimycin A, tyrphostin 25 or staurosporine prior to infection decreased parasite invasion in a dose-dependent manner. Contrary, addition of sodium orthovanadate, a protein tyrosine phosphatase inhibitor, phosphotyrosine and p-nitrophenyl phosphate to the interaction medium, significantly increased parasite binding and internalization, whereas phosphoserine and phosphothreonine had no effect. The phosphatase activity of intact promastigotes was greater than that of macrophages. Western blot analysis revealed tyrosine-phosphorylated bands from 198 to 28 kDa following macrophage challenge with promastigotes. Uninfected macrophages displayed no detectable tyrosine phosphorylated proteins, possibly indicating an inducible process, while in parasites it was constitutive, as seen by the presence of 42, 40 and 35 kDa phosphoproteins on the Leishmania lysates. Immunofluorescence and immunogold detection of phosphotyrosine residues in some promastigote-macrophage attachment areas, but not in the vicinity of ingested parasites, suggest that Leishmania-induced tyrosine phosphorylation is an early, local and short-lived event. Genistein treatment of Leishmania-infected cells significantly enhanced the parasite burden. This antagonist also diminished nitric oxide production in resting and interferon gamma/lipopolysaccharide-activated infected macrophages, which may account for the increased parasite survival. We propose that protein tyrosine kinase-linked pathways regulate the Leishmania promastigote invasion and the macrophage microbicidal activity.

Animals↗

PubMedDate not supplied

Modulation of cell adhesion by tyrosine kinases and phosphatases inhibitors.

Integrin-mediated activation of monocytes is an important aspect involved in the increase of proinflammatory cytokine messages. Tyrosine phosphorylation of proteins is one of the earliest events involved in these processes: Therefore, we selected two inhibitors, one for tyrosine kinases (quercitin) and another for tyrosine phosphatases (sodium orthovanadate) and we studied their capacity to modulate monocyte adhesion to fibronectin. Our results showed that quercitin strongly inhibits both tyrosine phosphorylation and cell adhesion. Sodium orthovanadate induces a modest increase of tyrosine phosphorylation and a weak enhancement of cell adhesion. When a combination of the two inhibitors was used, the tyrosine phosphorylation level displayed a strong enhancement. In contrast, cell adhesion was inhibited, but to the same degree. These observations indicate that tyrosine kinases may be more important than tyrosine phosphatases in the modulation of cell adhesion by flavonoid compounds.

Cell Adhesion↗

PubMed2001

The protein tyrosine kinase syk activity is reduced by clustering the mast cell function-associated antigen.

The mast cell function-associated antigen (MAFA) is a glycoprotein first identified on the membrane of rat mucosal-type mast cells (RBL-2H3 line). MAFA clustering causes a dose-dependent inhibition of these cells' secretory response to the type I Fcepsilon receptor (FcepsilonRI) stimulus. The inhibition has earlier been shown to take place upstream to the production step of inositol phosphates in the FcepsilonRI coupling cascade. To resolve further the mechanism of action of MAFA, we have investigated the events prior to the activation of phospholipase C. Activities of the non-receptor protein tyrosine kinases Lyn and Syk in untreated cells were compared with those where the FcepsilonRI, MAFA or both were clustered. Syk tyrosine phosphorylation and activation, as well as LAT (linker for activation of T cells) tyrosine phosphorylation, both induced by FcepsilonRI clustering, were found to be reduced upon MAFA clustering. In contrast, the activity of the Src homology domain 2 (SH2)-containing protein tyrosine phosphatase (SHP-2) increased. MAFA clustering also enhanced the co-isolation of SHP-2 and Syk with tyrosine-phosphorylated MAFA in both untreated and FcepsilonRI-stimulated cells. SHP-2 caused a decline in the FcepsilonRI-induced tyrosine phosphorylation of Syk, at least under in vitro conditions. Taken together, these results suggest that one possible mechanism by which MAFA affects the FcepsilonRI stimulation cascade is suppression of Syk activity, i.e. MAFA clustering leads SHP-2 to act on Syk, thereby reducing its tyrosine phosphorylation and its activity.

Animals↗

PubMed2000

Shp-2 specifically regulates several tyrosine-phosphorylated proteins in brain-derived neurotrophic factor signaling in cultured cerebral cortical neurons.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophins, promotes differentiation and survival and regulates plasticity of various types of neurons. BDNF binds to TrkB, a receptor tyrosine kinase, which results in the activation of a variety of signaling molecules to exert the various functions of BDNF. Shp-2, a Src homology 2 domain-containing cytoplasmic tyrosine phosphatase, is involved in neurotrophin signaling in PC12 cells and cultured cerebral cortical neurons. To examine the roles of Shp-2 in BDNF signaling in cultured rat cerebral cortical neurons, the wild-type and phosphatase-inactive mutant (C/S mutant) forms of Shp-2 were ectopically expressed in cultured neurons using recombinant adenovirus vectors. We found that several proteins tyrosine-phosphorylated in response to BDNF showed enhanced levels of tyrosine phosphorylation in cultured neurons infected with C/S mutant adenovirus in comparison with those infected with the wild-type Shp-2 adenovirus. In addition, in immunoprecipitates with anti-Shp-2 antibody, we also observed at least four proteins that displayed enhanced phosphorylation in response to BDNF in cultured neurons infected with the C/S mutant adenovirus. We found that the Shp-2-binding protein, brain immunoglobulin-like molecule with tyrosine-based activation motifs (BIT), was strongly tyrosine-phosphorylated in response to BDNF in cultured neurons expressing the C/S mutant of Shp-2. In contrast, the level of BDNF-induced phosphorylation of mitogen-activated protein kinase and coprecipitated proteins with anti-Trk and Grb2 antibodies did not show any difference between neurons infected with these two types of Shp-2. Furthermore, the survival effect of BDNF was enhanced by the wild type of Shp-2, although it was not influenced by the C/S mutant of Shp-2. These results indicated that in cultured cerebral cortical neurons Shp-2 is specifically involved in the regulation of several tyrosine-phosphorylated proteins, including BIT, in the BDNF signaling pathway. In addition, the phosphatase Shp-2 may not influence the level of BDNF-induced activation of mitogen-activated protein kinase in cultured cortical neurons. Further, Shp-2 may have potential to positively regulate BDNF-promoting neuronal survival.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Characterization and cloning of a 58/53-kDa substrate of the insulin receptor tyrosine kinase.

A monoclonal antibody has been produced which immunoprecipitates 58- and 53-kDa proteins which are rapidly tyrosine phosphorylated in insulin-treated cells. These proteins can also be tyrosine phosphorylated in vitro by the isolated human insulin receptor. Increased tyrosine phosphorylation of these proteins is also observed in cells expressing a transforming chicken c-Src (mutant Phe-527) and in cells with the activated tyrosine kinase domains of the Drosophila insulin receptor, human insulin-like growth factor I receptor, and human insulin receptor-related receptor. P58/53 did not appear to associate with either the GTPase activating protein of Ras (called GAP) or the phosphatidylinositol 3-kinase by either co-immunoprecipitation experiments or in Far Westerns with the SH2 domains of these two proteins. Since p58/53 did not appear, by immunoblotting, to be related to any previously described tyrosine kinase substrate such as the SH2 containing proteins SHC and the tyrosine phosphatase Syp, the protein was purified in sufficient amounts to obtain peptide sequence. This sequence was utilized to isolate a cDNA clone that encodes a previously uncharacterized 53-kDa protein which, when expressed in mammalian cells, is tyrosine phosphorylated by the insulin receptor.

Amino Acid Sequence↗

PubMed1997

Regulation of Src homology 2-containing tyrosine phosphatase 1 during activation of human neutrophils. Role of protein kinase C.

The tyrosine phosphorylation of several proteins induced in neutrophils by soluble and particulate stimuli is thought to be crucial for initiating antimicrobial responses. Although activation of tyrosine kinases is thought to mediate this event, the role of tyrosine phosphatases in the initiation and modulation of neutrophil responses remains largely undefined. We investigated the role of Src homology 2-containing tyrosine phosphatase 1 (SHP-1; also known as protein tyrosine phosphatase 1C (PTP1C), hematopoetic cell phosphatase, PTP-N6, and SHPTP-1), a phosphatase expressed primarily in hemopoietic cells, in the activation of human neutrophils. SHP-1 mRNA and protein were detected in these cells, and the enzyme was found to be predominantly localized to the cytosol in unstimulated cells. Following stimulation with neutrophil agonists such as phorbol ester, chemotactic peptide, or opsonized zymosan, a fraction of the phosphatase redistributed to the cytoskeleton. Agonist treatment also induced significant decreases (30-60%) in SHP-1 activity, which correlated temporally with increases in the cellular phosphotyrosine content. Phosphorylation of SHP-1 on serine residues was associated with the inhibition of its enzymatic activity, suggesting a causal relationship. Accordingly, both the agonist-evoked phosphorylation of SHP-1 and the inhibition of its catalytic activity were blocked by treatment with bisindolylmaleimide I, a potent and specific inhibitor of protein kinase C (PKC) activity. Immunoprecipitated SHP-1 was found to be phosphorylated efficiently by purified PKC in vitro. Such phosphorylation also caused a decrease in the phosphatase activity of SHP-1. Together, these data suggest that inhibition of SHP-1 by PKC-mediated serine phosphorylation plays a role in facilitating the accumulation of tyrosine-phosphorylated proteins following neutrophil stimulation. These findings provide a new link between the PKC and tyrosine phosphorylation branches of the signaling cascade that triggers antimicrobial responses in human neutrophils.

Enzyme Activation↗

PubMed1997

Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of platelet/endothelial cell adhesion molecule-1 (PECAM-1) that are required for the cellular association and activation of the protein-tyrosine phosphatase, SHP-2.

Recent studies have shown that the Src homology-2 (SH2) domain-containing protein-tyrosine phosphatase, SHP-2, associates with the cytoplasmic domain of PECAM-1 as it becomes tyrosine-phosphorylated during platelet aggregation: a process that can be mimicked in part by small synthetic phosphopeptides corresponding to the cytoplasmic domain of PECAM-1 encompassing tyrosine residues Tyr-663 or Tyr-686. To further examine the molecular requirements for PECAM-1/SHP-2 interactions, we generated human embryonic kidney (HEK)-293 cell lines that stably expressed mutant forms of PECAM-1 harboring tyrosine to phenylalanine (Tyr --> Phe) mutations in the cytoplasmic domain. Y663F and Y686F forms of PECAM-1 were tyrosine-phosphorylated to a somewhat lesser extent than wild-type PECAM-1, and a doubly substituted Y663,686F form of PECAM-1 failed to become tyrosine-phosphorylated, suggesting that the PECAM-1 cytoplasmic domain tyrosine residues 596, 636 and 701 do not serve as substrates for cellular kinases. Interestingly, SHP-2 binding was lost when either Tyr-663 or Tyr-686 were changed to phenylalanine, indicating that both residues are required for SHP-2/PECAM-1 association. Although PECAM-1 phosphopeptides NSDVQpY663TEVQV and DTETVpY686SEVRK stimulated the catalytic activity of the phosphatase to a similar extent, surface plasmon resonance studies revealed that the Tyr-663-containing peptide had approximately 10-fold higher affinity for SHP-2 than did the Tyr-686 peptide. Finally, peptido-precipitation analysis showed that the NH2-terminal SH2 domain of SHP-2 reacted preferentially with the Tyr-663 PECAM-1 phosphopeptide, while the Tyr-686 phosphopeptide associated only with the COOH-terminal SH2 domain of the phosphatase. Together, these data provide a molecular model for PECAM-1/SHP-2 interactions that may shed light on the downstream events that follow PECAM-1-mediated interactions of vascular cells.

Binding Sites↗