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PubMed1994

Structurally distinct disintegrins contortrostatin and multisquamatin differentially regulate platelet tyrosine phosphorylation.

Tyrosine phosphorylation of multiple platelet proteins is regulated by the integrin alpha IIb beta 3. In order to further examine integrin-regulated tyrosine phosphorylation, we have used small Arg-Gly-Asp-containing snake venom proteins (termed disintegrins) that inhibit platelet aggregation to competitively block the agonist-induced binding of fibrinogen to alpha IIb beta 3. One structurally unique disintegrin, contortrostatin (which appears to be a disulfide-linked dimer of 13.5 kDa with two Arg-Gly-Asp sites), was found to trigger signaling events typically mediated by fibrinogen cross-linking of alpha IIb beta 3, as demonstrated by tyrosine phosphorylation of the tyrosine kinase pp72syk and a 140-kDa protein. Contortrostatin and another disintegrin, multisquamatin (a monomer of 5.7 kDa with a single Arg-Gly-Asp site), did not affect thrombin-induced platelet shape change, secretion, or integrin-independent tyrosine phosphorylation; however, they inhibited aggregation and aggregation-dependent tyrosine phosphorylation of numerous proteins, including the focal adhesion kinase pp125FAK. Our results suggest that structurally distinct disintegrins have varying effects on tyrosine phosphorylation; while monomeric multisquamatin and dimeric contortrostatin both inhibit aggregation-dependent tyrosine phosphorylation, contortrostatin also possesses a unique functional activity that allows it to activate an intracellular signaling pathway leading to tyrosine phosphorylation. This activity may be involved in the function of this snake venom protein on hemostasis.

Amino Acid Sequence↗
PubMed1995

Zymosan-triggered association of tyrosine phosphoproteins and lyn kinase with cytoskeleton in human monocytes.

Phagocytosis of pathogens and inert particles such as zymosan by macrophages, and related secretory functions require the combination of several intracellular signals and the reorganization of cytoskeleton. We recently reported that zymosan stimulated the tyrosine phosphorylations of several endogenous substrates in human monocytes. In this work, the relationship between zymosan-stimulated tyrosine phosphoproteins and detergent-insoluble material considered as cytoskeleton was investigated. Triton X-100-insoluble fraction contained two proteins of 53 and 56 kDa that were tyrosine phosphorylated after only 5 min of stimulation with zymosan and remained labeled for 30 min. Because 53- and 56-kDa phosphoproteins migrated, as did some components of the src tyrosine kinase family, namely p53-56lyn, we wondered if 53- and 56-kDa phosphoproteins were related to lyn kinase. First, the amount of immunoreactive p53-56lyn increased in Triton X-100-insoluble fraction as did zymosan-stimulated tyrosine phosphoproteins. This property of p53-56lyn was unique, as no other member of the src family was found in this fraction. Second, when the immunoblots were reprobed with anti-phosphotyrosine mAb, the m.w. of p53-56lyn and tyrosine-phosphorylated proteins were identical in apparent size. Third, p53-56lyn was probably activated after cell stimulation with zymosan, because the phosphorylation levels of a synthetic copolymer of glutamine-tyrosine were increased in Triton X-100-insoluble fraction. In addition, we studied the distribution of lyn kinase and tyrosine phosphoproteins in phagocytozing monocytes. By using immunofluorescence, we showed that lyn kinase was located preferentially in the periphagosomal region in a specific manner, as an src tyrosine kinase such as p59hck, which was not associated with cytoskeleton, was not concentrated around the vacuoles. Moreover, periphagosomal phosphoproteins were also detected and found to be colocalized with polymerized actin. Because zymosan interacts with human monocytes via beta 2 integrins, which are known to be cytoskeleton-associated, we suggest that p53-56lyn provides the molecular link between zymosan receptors and cytoskeleton, and directs the cytoskeletal reorganization in the periphagosomal area.

Blotting, Western↗
PubMed1994

Tyrosines 1148 and 1173 of activated human epidermal growth factor receptors are binding sites of Shc in intact cells.

Autophosphorylation of receptor tyrosine kinases provides binding sites for signaling proteins containing Src homology 2 (SH2) domains. We determined the binding sites of Shc, SH2-containing adaptor protein, within epidermal growth factor (EGF) receptors, using Chinese hamster ovary cells overexpressing EGF receptor mutants in which autophosphorylation sites, either alone or in combination, were replaced by phenylalanine. Binding of Shc to EGF receptor mutants lacking single tyrosine residues at 1148 or 1173 decreased by approximately 60 or approximately 15%, respectively, whereas other single point mutants bound the wild-type level of Shc. Binding of Shc markedly decreased in mutants lacking both tyrosine residues at 1148 and 1173. In peptide inhibition assay, phosphorylated nonameric peptide representing tyrosine 1148, DNPDpYQQDF, but not pentameric peptide, pYQQDF, inhibited the binding of glutathione S-transferase-Shc SH2 domain fusion protein to in vitro autophosphorylated EGF receptors, suggesting that N-terminal sequences adjacent to phosphotyrosine are necessary for the association of Shc. Based on results of peptide inhibition assays in which phosphorylated peptides representing tyrosines 992, 1148, and 1173 inhibited Shc binding to the receptor, we constructed another EGF receptor mutant in which one of these tyrosine residues was retained. The amount of Shc bound to mutant receptors retaining tyrosines 1148, 1173, or 992 was approximately 80, approximately 40, or approximately 10% of wild-type level, respectively. These results indicate that tyrosine 1148 of activated human EGF receptors is a major binding site of Shc and tyrosine 1173 is a secondary binding site in intact cells.

Amino Acid Sequence↗
PubMed1993

A survey of protein tyrosine kinase mRNAs expressed in normal human melanocytes.

We have used the reverse transcription-polymerase chain reaction to survey the repertoire of protein tyrosine kinases expressed in cultured normal human melanocytes, a differentiated cell type derived from the neural crest. We identified 25 different tyrosine kinase cDNAs among a total of 608 protein tyrosinase kinase-related cDNAs analyzed. Six encode receptor tyrosine kinases for known ligands, several of which have been implicated in controlling melanocyte proliferation in vitro. Two others encode apparent receptor tyrosine kinases for unknown ligands. Four encode known non-receptor tyrosine kinases and five encode previously identified anonymous protein tyrosine kinases. Of the eight other melanocyte-associated protein tyrosine kinases, most or all appear to be novel. These 25 protein tyrosine kinase genes exhibit distinct patterns of expression in cultured human melanocytes, human erythroleukemia cells, and a variety of normal human tissues. We mapped 16 of the corresponding protein tyrosine kinase genes to specific human chromosomes, identifying a total of 19 human genetic loci, some of which may constitute candidate genes for genetic disorders of mammalian development.

Amino Acid Sequence↗
PubMed1994

Erythropoietin-dependent association of phosphatidylinositol 3-kinase with tyrosine-phosphorylated erythropoietin receptor.

Erythropoietin (Epo) regulates the proliferation and differentiation of erythroid precursors. The Epo receptor (EpoR) belongs to the cytokine receptor family and lacks a tyrosine kinase domain. However, Epo induces tyrosine phosphorylation of cellular substrates including the EpoR. To explore the functional significance of receptor tyrosine phosphorylation, we examined the possible interaction of the receptor with the 85-kDa regulatory subunit (p85) of phosphatidylinositol (PI) 3-kinase. After Epo stimulation, p85 was found to associate with the tyrosine-phosphorylated 72-kDa form of EpoR as well as a 92-kDa phosphotyrosyl protein, and PI 3-kinase activity was detectable in anti-EpoR immunoprecipitates. Anti-EpoR blotting of anti-p85 immunoprecipitates revealed that p85 binds specifically to the 72-kDa form of the EpoR and not to unphosphorylated 66- and 64-kDa forms. Association of p85 with the EpoR was Epo dose- and time-dependent and correlated with tyrosine phosphorylation of the receptor. Consistent with a role for tyrosine phosphorylation of the EpoR, PI 3-kinase did not associate with a mitogenically active receptor mutant that lacked tyrosine-phosphorylation sites in the carboxyl-terminal region. A recombinant fusion protein containing the carboxyl-terminal SH-2 domain of p85 was shown to bind to tyrosine-phosphorylated EpoR in vitro. Taken together, these results indicate that, following Epo stimulation, the EpoR recruits PI 3-kinase to the cell membrane by binding between the carboxyl-terminal SH-2 domain of p85 and the tyrosine-phosphorylated carboxyl-terminal region of the receptor. The association with PI 3-kinase is, however, not required for the growth signal transduction from the EpoR.

Binding Sites↗
PubMed1993

Tyrosine phosphorylation and cytoskeletal reorganization in platelets are triggered by interaction of integrin receptors with their immobilized ligands.

Agonist-induced platelet activation causes fibrinogen binding to integrin alpha IIb beta 3 (glycoprotein (GP) IIb-IIIa) and platelet aggregation. This is associated with the phosphorylation of specific platelet proteins on tyrosine residues. Since fibrinogen immobilized on a solid matrix can bind platelet GP IIb-IIIa without the need for exogenous agonists, we examined whether platelet adhesion to a fibrinogen matrix induces tyrosine phosphorylation. Platelets adhered to fibrinogen in a GP IIb-IIIa-dependent manner and assumed a spread morphology. This change in cell shape was associated with tyrosine phosphorylation of multiple proteins, most prominently two unidentified proteins (101 and 105 kDa) and pp125FAK, a focal adhesion protein-tyrosine kinase. Pretreatment of platelets with prostaglandin I2 to increase cAMP, with cytochalasin D to inhibit actin polymerization, or with ADP scavengers to remove ADP did not affect initial adhesion, but inhibited both platelet spreading and tyrosine phosphorylation of pp125FAK and the 101- and 105-kDa proteins. This suggested that adhesion to fibrinogen caused cytoskeletal reorganization and the local release of ADP from platelet-dense granules, which potentiated the biochemical and morphological responses of the platelets to fibrinogen. Platelet adhesion to a collagen matrix also led to the induction of tyrosine phosphorylation of the 101- and 105-kDa proteins and pp125FAK. In this case, tyrosine phosphorylation was dependent on the interaction of collagen with integrin alpha 2 beta 1 (GP Ia-IIa), and it was independent of both GP IIb-IIIa and ADP. These results indicate that platelet adhesion to fibrinogen or collagen induces signal transduction that is initiated through integrins GP IIb-IIIa and alpha 2 beta 1, respectively. In both cases, tyrosine phosphorylation is accompanied by cytoskeletal reorganization and changes in cell shape. However, different regulatory components may be interposed between each of these integrins and the enzymes that control the level of protein tyrosine phosphorylation.

Blood Platelets↗
PubMed1993

Identification, cloning, and characterization of a novel human T-cell-specific tyrosine kinase located at the hematopoietin complex on chromosome 5q.

Signal transduction through the T-cell receptor and cytokine receptors on the surface of T lymphocytes occurs largely via tyrosine phosphorylation of intracellular substrates. Because neither the T-cell receptor nor cytokine receptors contain intrinsic kinase domains, signal transduction is thought to occur via association of these receptors with intracellular protein tyrosine kinases. Although several members of the SRC and SYK families of tyrosine kinases have been implicated in signal transduction in lymphocytes, it seems likely that additional tyrosine kinases involved in signal transduction remain to be identified. To identify unique T-cell tyrosine kinases, we used polymerase chain reaction-based cloning with degenerate oligonucleotides directed at highly conserved motifs of tyrosine kinase domains. We have cloned the complete cDNA for a unique human tyrosine kinase that is expressed mainly in T lymphocytes (EMT) and natural killer (NK) cells. The cDNA of EMT predicts an open reading frame of 1866 bp encoding a protein with a predicted size of 72 Kd, which is in keeping with its size on Western blotting. A single 6.2-kb EMT mRNA and 72-Kd protein were detected in T lymphocytes and NK-like cell lines, but were not detected in other cell lineages. EMT contains both SH2 and SH3 domains, as do many other intracellular kinases. EMT does not contain the N-terminal myristylation site or the negative regulatory tyrosine phosphorylation site in its carboxyterminus that are found in the SRC family of tyrosine kinases. EMT is related to the B-cell progenitor kinase (BPK), which has recently been implicated in X-linked hypogammaglobulinemia, to the TECI mammalian kinase, which has been implicated in liver neoplasia, to the more widely expressed TECII mammalian kinase, and to the Drosophila melanogaster Dsrc28 kinase. Sequence comparison suggests that EMT is likely the human homologue of a recently identified murine interleukin-2 (IL-2)-inducible T cell kinase (ITK). However, unlike ITK, EMT message and protein levels do not vary markedly on stimulation of human IL-2-responsive T cells with IL-2. Taken together, it seems that EMT is a member of a new family of intracellular kinases that includes BPK, TECI, and TECII. EMT was localized to chromosome 5q31-32, a region that contains the genes for several growth factors and receptors as well as early activation genes, particularly those involved in the hematopoietic system. Furthermore, the 5q31-32 region is implicated in the genesis of the 5q- syndrome associated with myelodysplasia and development of leukemia.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation of the focal adhesion-associated protein paxillin in Swiss 3T3 cells.

Treatment of Swiss 3T3 cells with bombesin caused a striking increase (21-fold) in the tyrosine phosphorylation of the cytoskeleton-associated protein paxillin, as judged by anti-phosphotyrosine Western blots of anti-paxillin immunoprecipitates. Vasopressin and endothelin also stimulated paxillin tyrosine phosphorylation. Bombesin-stimulated tyrosine phosphorylation of paxillin was detectable within 1 min and was concentration-dependent (half-maximum effect at 0.09 nM). Bombesin stimulation of paxillin tyrosine phosphorylation could be dissociated from both protein kinase C (PKC) activation and the mobilization of Ca2+ from intracellular stores. Activation of PKC in quiescent Swiss 3T3 cells using the tumor promoter phorbol 12,13-dibutyrate (PDB) increased the tyrosine phosphorylation of paxillin in a time-dependent manner but was less effective than bombesin and stimulated detectable phosphorylation only within 5 min, considerably slower than bombesin-induced tyrosine phosphorylation of paxillin. Furthermore, the selective PKC inhibitor, GF109203X, or down-regulation of PKC using prolonged treatment with PDB markedly inhibited the stimulation of paxillin tyrosine phosphorylation by PDB but had little effect on the response to bombesin. In contrast, cytochalasin D, an agent that selectively disrupts the network of actin microfilaments, completely inhibited bombesin- and PDB-induced paxillin tyrosine phosphorylation. This is the first report to identify paxillin as a substrate for neuropeptide-stimulated tyrosine phosphorylation.

3T3 Cells↗
PubMed1995

Platelet-activating factor induces cell growth through tyrosine phosphorylation pathway in cultured rat mesangial cells.

Accumulating evidence suggests that platelet-activating factor (PAF) may play a role in renal pathophysiology. Therefore, in order to investigate this notion further, the effects of PAF on cell growth and tyrosine phosphorylation were analyzed in cultured rat mesangial cells. PAF was found to enhance a time and concentration-dependent increase in phosphotyrosine in several proteins and stimulate 3H-thymidine incorporation. Tyrosine phosphorylation was also enhanced by PAF in protein kinase C (PKC) depleted cells, whereas a tyrosine kinase inhibitor, genistein, inhibited tyrosine phosphorylation of these proteins at the concentration of 1 microgram/ml. PAF stimulated 3H-thymidine incorporation at concentrations below 10(-6) M, but exerted progressive inhibition at concentrations above 10(-6) M. Pre-treatment with phorbol 12-myristate 13-acetate (PMA) did not affect PAF-enhanced incorporation at lower concentrations of PAF, and reversed the inhibitory effects of PAF at higher concentrations. Finally, genistein pre-treatment completely inhibited PAF-induced cell growth at the concentration of 1 microgram/ml. Both tyrosine phosphorylation and 3H-thymidine incorporation induced by PAF were completely inhibited by pre-treatment with the PAF-receptor antagonist, CV-6209, at the concentration of 10(-5)M. These results suggest that PAF enhancement of tyrosine phosphorylation occurred in a PKC-independent manner and that a tyrosine kinase was associated with PAF-induced tyrosine phosphorylation. Moreover, they indicate that the phosphoinositide hydrolysis-PKC pathway is not essential for PAF-induced cell proliferation, and that PKC activation may play an inhibitory rather than a stimulatory role in mitogenesis in response to PAF. Our results indicate that the tyrosine phosphorylation pathway induced by PAF may participate critically in downstream mitogenic signaling through the PAF receptor.

Animals↗
PubMedDate not supplied

Design and synthesis of novel tyrosine kinase inhibitors using a pharmacophore model of the ATP-binding site of the EGF-R.

One of the most promising targets for the rational design of anti-cancer drugs is the family of the EGF-receptor protein tyrosine kinases. Despite the high sequence homology within the ATP-binding region of protein tyrosine and/or serine threonine kinases, ATP-competitive compounds have the potential to be selective inhibitors of protein kinases. Dianilino-phthalimides CGP 52 411 and CGP 53,353 have been identified as potent and ATP-competitive inhibitors of the EGF-R tyrosine kinase with no or only minor activity against a panel of tyrosine and serine/threonine kinases. Using a calculated 3-D computer model of the catalytic domain of the EGF-R-tyrosine kinase together with CGP 52 411 as example of an ATP-competitive inhibitor, a pharmacophore model for ATP-competitive inhibitors in the active site of the EGF-R PTK was developed. With the help of this model, 4-phenylamino-7H-pyrrolo[2,3-d]pyrimidines were then identified as new potent EGF-R PTK inhibitors. In an interactive process, the class of the 4-phenylamino-pyrrolo-pyrimidines was optimized and structure-activity-relationship of a series of derivatives thereof are discussed. In vitro, the most active compounds (CGP 59 326, CGP 60 261, CGP 62 706) inhibited the EGF-R tyrosine kinase with IC50 value between 6-30 nM. High selectivity towards a panel of non-receptor tyrosine kinases (c-SRC, v-Abl) and serine/threonine kinases (PKC alpha, PKA) was observed. Kinetic analysis revealed competitive type kinetics relative to ATP. In cells, EGF-stimulated cellular tyrosine phosphorylation was inhibited by these compounds at IC50 values between 0.1-0.3 microM, whereas the ligand-induced receptor autophosphorylation of the PDGR-R was not effected by concentrations up to 100 microM. Furthermore, CGP 59 326, CGP 60 261, CGP 62 706 were able to selectively inhibit c-fos mRNA expression in EGF-dependent cell lines with (IC50) approx. 0.1-1 microM) but not in EGF-independent cell systems (IC50 > 100 microM). Proliferation of the EGF-dependent MK cell line was inhibited with similar IC50 values. In addition, CGP 59 326 and CGP 62 706 showed good in vivo efficacy at low doses after oral or subcutaneous administration in nude mice tumor models using xenografts of the EGF-dependent A431 cell lines. The ED50 values were between 1.5-2 mg/kg. Phenylamino-pyrrolo-pyrimidines therefore represent a new series of tyrosine kinase inhibitors which preferentially inhibit the EGF-mediated signal transduction pathway and have the characteristics for further evaluation as anticancer agents.

Adenosine Triphosphate↗
PubMed1998

Impaired Ca2+-induced tyrosine phosphorylation and defective lipid scrambling in erythrocytes from a patient with Scott syndrome: a study using an inhibitor for scramblase that mimics the defect in Scott syndrome.

Scott syndrome is an hereditary bleeding disorder characterized by a deficiency in platelet procoagulant activity. Unlike normal blood cells, Scott platelets, as well as erythrocytes and lymphocytes, are strongly impaired in their ability to scramble their membrane phospholipids when challenged with Ca2+. In normal cells this collapse of membrane asymmetry leads to surface exposure of phosphatidylserine. Here we report that Scott erythrocytes show an apparent defect in tyrosine phosphorylation on treatment with Ca2+-ionophore. Diminished tyrosine phosphorylation was also apparent in activated Scott platelets, but much less pronounced than observed in red blood cells. On the other hand, tyrosine phosphorylation profiles observed in Scott red blood cell ghosts after sealing in the presence of adenosine triphosphate (ATP) were indistinguishable from those obtained from normal ghosts. Several observations argue in favor of a mechanism in which tyrosine phosphorylation in red blood cells is facilitated by, rather than required for scrambling of membrane lipids. Staurosporin blocks tyrosine phosphorylation in normal red blood cells, but does not inhibit the lipid scrambling process. White ghosts from normal erythrocytes, resealed in the absence of ATP, exhibit Ca2+-induced lipid scrambling without tyrosine phosphorylation. A selective inhibitor of Ca2+-induced lipid scrambling also showed an apparent inhibition of tyrosine phosphorylation in ionophore-treated normal red blood cells, similar to that observed in Scott erythrocytes. While this inhibitor also suppressed Ca2+-induced lipid scrambling in ghosts that were sealed in the presence of ATP, it did not inhibit tyrosine kinase activity. We conclude that the apparent deficiency in tyrosine phosphorylation in Scott cells is an epiphenomenon, possibly associated with a defect in phospholipid scrambling, but not causal to this defect.

Adenosine Triphosphate↗
PubMed1994

CD28 signal transduction: tyrosine phosphorylation and receptor association of phosphoinositide-3 kinase correlate with Ca(2+)-independent costimulatory activity.

The interaction of CD28 with its counter-receptor, B7, induces a cosignal in T cells required to prevent clonal anergy and to promote antigen-dependent interleukin-2 production. The molecular basis of the CD28 cosignal is not well understood but involves the activation of protein tyrosine kinase(s) (PTK). In this report we demonstrate that CD28 cross-linking on Jurkat T leukemic cells causes the activation of at least two PTK pathways. A CD28-induced, p56lck kinase-independent pathway causes tyrosine-phosphorylation of a 110-kDa substrate while recruitment of p56lck kinase activity is apparently required for CD28-induced tyrosine-phosphorylation of 97- and 68-kDa substrates as well as CD28-induced increases in intracellular calcium. The tyrosine phosphorylation of p110, but not p97 or p68, correlated with CD28 calcium-independent costimulatory activity. The pp110 molecule was tentatively identified as the catalytic subunit of phosphoinositide (PI)-3 kinase based upon its coimmunoprecipitation with the p85 regulatory subunit of PI-3 kinase. PI-3 kinase protein and catalytic activity were found complexed with the CD28 receptor if the receptor was "activated" by cross-linking on the surface of intact cells prior to detergent solubilization. The kinetics of association of PI-3 kinase with the "activated" CD28 receptor was rapid, occurring within 30 s of receptor cross-linking and was stable for at least 30 min. Analysis of the CD28 cytoplasmic peptide sequence revealed a putative PI-3 kinase src homology 2 binding motif and CD28 tyrosine phosphorylation site, DYMNM. Tyrosine phosphorylation of CD28 was detected in pervanadate-treated Jurkat B2.7 cells, but not untreated cells. Pervanadate-induced tyrosine phosphorylation of CD28 correlated with receptor association of PI-3 kinase in the absence of CD28 cross-linking, suggesting that CD28 association with PI-3 kinase uses a tyrosine phosphorylation-dependent mechanism. These data provide a model for CD28 signal transduction and support a role for PI-3 kinase in mediating the CD28 calcium-independent, cyclosporin A-insensitive costimulatory signal.

Amino Acid Sequence↗
PubMed1991

Constitutively activated neu oncoprotein tyrosine kinase interferes with growth factor-induced signals for gene activation.

The neu receptor oncoprotein tyrosine kinase, capable of transforming cultured fibroblasts and causing mammary carcinomas in transgenic mice, carries a point mutation in its transmembrane domain and shows a constitutive tyrosine kinase activity. We analyzed the neu tyrosine kinase and its substrates in transfected NIH 3T3 fibroblasts by phosphotyrosine immunoblotting. Tyrosine phosphorylated proteins were similar but not identical in epidermal growth factor (EGF)-stimulated cells expressing the human EGF receptor (EGFR) or a chimeric EGFR/neu receptor but differed from phosphotyrosyl proteins constitutively expressed in neu oncogene-transformed cells. The neu oncoprotein in the latter cells was phosphorylated in tyrosine in a ligand-independent manner and had a shortened half-life in comparison with the normal neu protein. Tumor promoter pretreatment inhibited ligand-induced receptor tyrosine phosphorylation and decreased tyrosine phosphorylated neu oncoprotein. Prolonged pretreatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also prevented the induction of immediate early growth factor-regulated genes in response to neu activation. Expression of the neu oncogene but not the protooncogene in NIH 3T3 cells was associated with enhanced levels of the jun and fos oncoproteins and loss of serum growth factor induction of immediate early mRNA responses. The constitutively activated neu oncoprotein tyrosine kinase thus deregulates cellular genomic responses to growth factors.

Animals↗
PubMed1995

Regulation of apoptosis of interleukin 2-dependent mouse T-cell line by protein tyrosine phosphorylation and polyamines.

We examined the effect of inhibitors of tyrosine kinase and tyrosine phosphatase on DNA fragmentation, protein tyrosine phosphorylation, and polyamine metabolism in the murine T-cell line CTLL-2. When cells were exposed to herbimycin A, a specific inhibitor of tyrosine kinase (Uehara et al., 1989, Biochem. Biophys. Res. Commun., 163:803-809), in the presence of interleukin 2 (IL-2), DNA was degraded into oligonucleosomal fragments in a dose-dependent fashion. Genistein, another inhibitor of tyrosine kinase (Akiyama et al., 1987, J. Biol. Chem., 262:5592-5596), had similar effects. Exposure of CTLL-2 cells to vanadate, a tyrosine phosphatase inhibitor, blocked with the DNA fragmentation induced by herbimycin A. Tyrosine phosphorylation of 55 Kd protein was inhibited by herbimycin A, and the inhibition was reduced by vanadate. Ornithine decarboxylase (ODC) activity decreased rapidly after herbimycin A was added to CTLL-2 cell cultures, while vanadate increased ODC activity. The exogenous addition of putrescine or spermine, but not that of spermidine, attenuated herbimycin A-induced DNA fragmentation. These findings suggest that phosphorylation of tyrosine residues of 55 Kd protein prevents DNA fragmentation and that polyamines are involved in regulation of apoptosis.

Animals↗
PubMed2002

Phosphorylation of the N-terminal and C-terminal CD3-epsilon-ITAM tyrosines is differentially regulated in T cells.

Tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) within CD3 chains is crucial for the recruitment of protein tyrosine kinases and effector molecules into the T cell receptor. Thus, phenylalanine substitution at the N-terminal tyrosine residue of the CD3-epsilon-ITAM abolished signal transduction functions of this ITAM, including phosphorylation at the C-terminal ITAM tyrosine, and its association with ZAP-70. In contrast, mutation at the C-terminal tyrosine of CD3-epsilon-ITAM did not prevent phosphorylation at the N-terminal tyrosine, nor its association with Lck, or p85 PI 3-K regulatory subunit. In contrast to the ZAP-70/diphosphorylated CD8-epsilon-ITAM interaction, the Lck/monophosphorylated CD8-epsilon-ITAM interaction was sensitive to octylglucoside, an agent that disrupts Lck interaction with membrane rafts. Therefore, association of Lck with membrane rafts seems to be essential for stabilization of Lck/CD3-epsilon protein-protein interactions. Overall, the data suggest that the sequential and coordinated phosphorylation of CD3-epsilon-ITAM tyrosines provides to CD3-epsilon the potential to interact with multiple downstream effectors and signaling pathways.

Amino Acid Motifs↗
PubMed2002

Mitogen-induced, FAK-dependent tyrosine phosphorylation of the SSeCKS scaffolding protein.

The ability of mitogens to rapidly induce tyrosine phosphorylation of cellular proteins has been taken as evidence of participation in subsequent signaling pathways. SSeCKS, a major protein kinase C (PKC) substrate with protein scaffolding and tumor suppressive properties, becomes tyrosine phosphorylated in NIH3T3 and rodent embryo fibroblasts after short-term treatment with epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or fetal calf serum in the presence of pervanadate, but not by treatment with insulin or insulin-like growth factor-1. The relative phosphotyrosine level on SSeCKS was higher in actively dividing cells than in confluent cultures. Tyrosine phosphorylation of SSeCKS was apparent in cells deficient in Src, Fyn, Yes, or Abl tyrosine kinases or in NIH3T3 cells expressing a temperature-sensitive v-Src allele, but not in FAK-deficient embryo fibroblasts. Purified FAK or Src enzyme failed to directly phosphorylate SSeCKS in vitro. EGF failed to induce SSeCKS tyrosine phosphorylation in FAK-/- fibroblasts, indicating that the EGF receptor is probably not the direct kinase of SSeCKS. Phosphorylation under these conditions was rescued by the transient reexpression of wt-FAK but not FAK mutated at Y397, a major autophosphorylation and SH2-based docking site. Adhesion of FAK+/+ cells to fibronectin failed to significantly induce SSeCKS tyrosine phosphorylation although FAK was activated, suggesting that SSeCKS phosphorylation is mediated through a growth factor receptor-FAK rather than an integrin-FAK pathway. Moreover, PDGF could induce SSeCKS tyrosine phosphorylation in the absence of FAK activation, suggesting a role for FAK SH2-based docking rather than kinase activity. Immunofluorescence analysis showed that in FAK-/- cells, SSeCKS costains along F-actin stress fibers, in contrast to FAK+/+ cells, where most SSeCKS stains at the cell edge and along a cortical cytoskeletal matrix. This correlated with increased coprecipitation of SSeCKS with biotin-phalloidin-bound F-actin from FAK-/- compared to FAK+/+ cell lysates. Similarly, bacterially expressed, unphosphorylated SSeCKS cosedimented with F-actin in ultracentrifugation assays. These data suggest that mitogen-induced, FAK-dependent tyrosine phosphorylation of SSeCKS modulates its binding to the actin-based cytoskeleton, suggesting a role for SSeCKS in mitogen-induced cytoskeletal reorganization.

3T3 Cells↗
PubMed1996

Tyrosine phosphorylation of measles virus P-phosphoprotein in persistently infected neuroblastoma cells.

Replication and encapsidation of measles virus (MV) requires the interaction between the nuclear protein (N) and the phosphoprotein (P). It is known that both proteins are phosphorylated on serine and threonine residues. Recently we have shown that N is phosphorylated on tyrosine in persistently-infected mouse neuroblastoma cells (NS20Y/MS). Here, we show that P in NS20Y/MS is also phosphorylated on tyrosine. To investigate whether cellular tyrosine kinases can bind and phosphorylate P, a solid phase kinase assay was employed. We show that bacterially-expressed MV P fragments, were phosphorylated on tyrosine by purified mouse c-Src protein-tyrosine kinase and when mixed with uninfected neuroblastoma cell (NS20Y) extracts, these P fragments were phosphorylated on tyrosine in addition to serine and threonine. These results imply that MV P is a substrate for tyrosine phosphorylation by cellular tyrosine kinase(s).

Animals↗
PubMed2002

Synthesis and biological activity of a novel class of small molecular weight peptidomimetic competitive inhibitors of protein tyrosine phosphatase 1B.

Protein tyrosine phosphatase 1B (PTP1B) negatively regulates insulin signaling in part by dephosphorylating key tyrosine residues within the regulatory domain of the beta-subunit of the insulin receptor (IR), thereby attenuating receptor tyrosine kinase activity. Inhibition of PTP1B is therefore anticipated to improve insulin resistance and has recently become the focus of discovery efforts aimed at identifying new drugs to treat type II diabetes. We previously reported that the tripeptide Ac-Asp-Tyr(SO(3)H)-Nle-NH(2) is a surprisingly effective inhibitor of PTP1B (K(i) = 5 microM). With the goal of improving the stability and potency of this lead, as well as attenuating its peptidic character, an analogue program was undertaken. Specific elements of the initial phase of this program included replacement of the N- and C-termini with non-amino acid components, modification of the tyrosine subunit, and replacement of the tyrosine sulfate with other potential phosphate mimics. The most potent analogue arising from this effort was triacid 71, which inhibits PTP1B competitively with a K(i) = 0.22 microM without inhibiting SHP-2 or LAR at concentrations up to 100 microM. Overall, the inhibitors generated in this work showed little or no enhancement of insulin signaling in cellular assays. However, potential prodrug triester 70 did induce enhancements in 2-deoxyglucose uptake into two different cell lines with concomitant augmentation of the tyrosine phosphorylation levels of insulin-signaling molecules. Key elements of the overall SAR reported herein include confirmation of the effectiveness and remarkable PTP1B-specificity of the novel tyrosine phosphate bioisostere, O-carboxymethyl salicylic acid; demonstration that the tyrosine skeleton is optimal relative to closely related structures; replacement of the p-1 aspartic acid with phenylalanine with little effect on activity; and demonstration that inhibitory activity can be maintained in the absence of an N-terminal carboxylic acid. An X-ray cocrystal structure of an analogue bearing a neutral N-terminus (69) bound to PTP1B is reported that confirms a mode of binding similar to that of peptidic substrates.

Cell Line↗