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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Binding sites for angiotensin II were found, in a line of Swiss 3T3 cells (designated as R3T3 cells), that were insensitive to Dup 753 and dithiothreitol yet were sensitive to PD 123319, making them members of the AT2 class of angiotensin II binding sites. These binding sites appeared not to be coupled to guanine nucleotide-binding proteins, and affinity labeling experiments revealed a specifically labeled protein with an apparent molecular weight of about 100,000. Treatment of cells with angiotensin II revealed no perturbation of common signaling pathways, including stimulation of phosphatidylinositol turnover, effects on levels of cAMP, tyrosine kinase activity, and release of arachidonic acid. Also, angiotensin II or PD 123319 had no effect on cell growth, mitogenesis, or hypertrophy or on mitogenesis or hypertrophy stimulated by several growth factors. These results show that the AT2 binding site is quite distinct from the AT1 site in terms of molecular weight, binding properties, and coupling to second messenger systems. Although the significance of this novel angiotensin II binding site remains obscure, the identification of cell lines selectively expressing it should greatly aid in the understanding of its regulation and function.
Mol Pharmacol 1991 Sep
PMID:Characterization of angiotensin II (AT2) binding sites in R3T3 cells. 189 25

A Saccharomyces cerevisiae lambda gt11 library was screened with antiphosphotyrosine antibodies in an attempt to identify a gene encoding a tyrosine kinase. A subclone derived from one positive phage was sequenced and found to contain an 821-amino-acid open reading frame that encodes a protein with homology to protein kinases. We tested the activity of the putative kinase by constructing a vector encoding a glutathione-S-transferase fusion protein containing most of the predicted polypeptide. The fusion protein phosphorylated endogenous substrates and enolase primarily on serine and threonine. The gene was designated SPK1 for serine-protein kinase. Expression of the Spk1 fusion protein in bacteria stimulated serine, threonine, and tyrosine phosphorylation of bacterial proteins. These results, combined with the antiphosphotyrosine immunoreactivity induced by the kinase, indicate that Spk1 is capable of phosphorylating tyrosine as well as phosphorylating serine and threonine. In in vitro assays, the fusion protein kinase phosphorylated the synthetic substrate poly(Glu/Tyr) on tyrosine, but the activity was weak compared with serine and threonine phosphorylation of other substrates. To determine if other serine/threonine kinases would phosphorylate poly(Glu/Tyr), we tested calcium/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The two kinases had similar tyrosine-phosphorylating activities. These results establish that the functional difference between serine/threonine- and tyrosine-protein kinases is not absolute and suggest that there may be physiological circumstances in which tyrosine phosphorylation is mediated by serine/threonine kinases.
Mol Cell Biol 1991 Feb
PMID:Spk1, a new kinase from Saccharomyces cerevisiae, phosphorylates proteins on serine, threonine, and tyrosine. 189 89

Alveolar macrophages (AM) migrate less well in response to chemotactic ligands than do monocytes and neutrophils. The response of monocytes and neutrophils to chemotactic ligands is mediated at least in part by pertussis toxin-sensitive guanine nucleotide binding proteins (Gi proteins). Whether this is also true in AM is uncertain. We hypothesized that decreased chemotaxis by AM was due in part to diminished Gi protein and/or chemotactic receptor density in AM. G proteins are heterotrimers made up of alpha, beta, and gamma subunits; the predominant pertussis toxin-sensitive Gi proteins are those containing alpha i2 or alpha i3 subunits. Pertussis toxin pretreatment (0.5 microgram/ml) significantly reduced AM, monocyte, and neutrophil chemotaxis to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) and human zymosan-activated serum (P less than 0.05). However, as previously noted, AM chemotaxis was much less than that observed in monocytes and neutrophils. Immunoblots using antibodies that are specific for alpha i2 and alpha i3 showed that AM contained approximately 3-fold less alpha i2 and approximately 10-fold less alpha i3 per microgram of plasma membrane protein than did monocytes or neutrophils. Similar results were obtained in immunoblots made using antibodies to common alpha subunit determinants and to the beta 36 subunit. A comparable approximately 4-fold reduction in density of receptors for [3H]FMLP was found in AM compared to neutrophils. The diminished density of Gi proteins and FMLP receptors was not due to a generally decreased density of plasma membrane proteins in AM, since the density of the membrane-associated tyrosine kinase hck was similar in AM, monocytes, and neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1991 Jul
PMID:Signal transduction in human alveolar macrophages: diminished chemotactic response to FMLP correlates with a diminished density of Gi proteins and FMLP receptors. 190 89

The MET proto-oncogene encodes a transmembrane tyrosine kinase of 190 kDa (p190MET), which has recently been identified as the receptor for hepatocyte growth factor/scatter factor. p190MET is a heterodimer composed of two disulfide-linked chains of 50 kDa (p50 alpha) and 145 kDa (p145 beta). We have produced four different monoclonal antibodies that are specific for the extracellular domain of the Met receptor. These antibodies immunoprecipitate with p190MET two additional Met proteins of 140 and 130 kDa. The first protein (p140MET) is membrane bound and is composed of an alpha chain (p50 alpha) and an 85-kDa C-terminal truncated beta chain (p85 beta). The second protein (p130MET) is released in the culture supernatant and consists of an alpha chain (p50 alpha) and a 75-kDa C-terminal truncated beta chain (p75 beta). Both truncated forms lack the tyrosine kinase domain. p140MET and p130MET are consistently detected in vivo, together with p190MET, in different cell lines or their culture supernatants. p140MET is preferentially localized at the cell surface, where it is present in roughly half the amount of p190MET. The two C-terminal truncated forms of the Met receptor are also found in stable transfectants expressing the full-length MET cDNA, thus showing that they originate from posttranslational proteolysis. This process is regulated by protein kinase C activation. Together, these data suggest that the production of the C-terminal truncated Met forms may have a physiological role in modulating the Met receptor function.
Mol Cell Biol 1991 Dec
PMID:C-terminal truncated forms of Met, the hepatocyte growth factor receptor. 194 72

The mutant c-fgr protein (p58c-fgr/F523) containing Phe-523 instead of Tyr-523 exhibited transforming activity in NIH 3T3 cells like other protein-tyrosine kinases of the src family, but normal p58c-fgr (p58c-fgr/wt) did not. The mutant protein showed tyrosine kinase activity threefold higher than that of the normal protein in vitro. Surprisingly, transfection of the normal c-fgr gene into NIH 3T3 cells resulted in induction of sodium fluoride (NaF)-sensitive alpha-naphthyl butyrate esterase (alpha-NBE), a marker enzyme of cells of monocytic origin, which was not induced in v-src-, v-fgr-, or lyn-transfected NIH 3T3 cells. The NaF-sensitive alpha-NBE induced in c-fgr transfectants was shown by isoelectric focusing to have a pI of 5.2 to 5.4, a range which was the same as those for thioglycolate-induced murine peritoneal macrophages and 1 alpha,25-dihydroxyvitamin D3-treated WEHI-3B cells. Immunoblotting studies with antiphosphotyrosine antibodies revealed that 58-, 62-, 75-, 120-, 200-, and 230-kDa proteins were commonly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in cells transfected with the mutated c-fgr. These findings suggest that tyrosine phosphorylation of specific cellular substrate proteins is important in induction of NaF-sensitive alpha-NBE and cell transformation by p58c-fgr.
Mol Cell Biol 1991 Dec
PMID:Human c-fgr induces a monocyte-specific enzyme in NIH 3T3 cells. 194 88

We have isolated a hamster fibroblast cDNA clone that encodes a serotoninergic receptor whose deduced amino acid sequence displays 94% identity with the rat brain serotonin (5-HT) type 2 receptor. When expressed in Xenopus oocytes, the hamster receptor efficiently couples to the phosphoinositide second messenger system and leads to intracellular Ca2+ mobilization in response to 5-HT. To determine the pharmacological properties of this receptor, and to evaluate the role of phospholipase C (PLC) activation in growth modulation by 5-HT, we have expressed it in hamster fibroblasts. Transfected cells that express 5-HT receptors were selected using a novel method based on coexpression of the Na+/H+ antiporter gene as a selectable marker. After co-transfection of the 5-HT receptor and Na+/H+ antiporter cDNAs in fibroblasts lacking antiporter activity (variants of the CCL39 line), 50% of the clones resistant to an acute acid load express functional receptors. The pharmacological profile of the transfected receptor is consistent with it being of the 5-HT2 subtype, and the extent of 5-HT-stimulated PLC activation in independent clones correlates with their relative level of cRNA expression. In cells in where addition of 5-HT leads to strong activation of PLC, and inhibition of adenylate cyclase via endogenous 5-HT1b receptors, 5-HT alone has little effect on DNA synthesis stimulation. Thus we conclude that activation of the PLC signalling pathway in these cells is not sufficient to trigger G0/G1 to S phase transition. Strong activation of PLC via 5-HT2 receptors does however contribute to the synergy observed between 5-HT (Gi-coupled pathway) and fibroblast growth factor (tyrosine kinase-activated pathway) on DNA synthesis reinitiation in transfected cells.
Mol Endocrinol 1991 Jul
PMID:Cloning, functional expression and role in cell growth regulation of a hamster 5-HT2 receptor subtype. 194 95

The ortho, meta, and para forms of hydroxyphenyl acetate were found to be inhibitory in the order of ortho greater than para greater than meta in three distinct biological assays: (a) insulin-dependent assimilation of glucose into lipids in intact adipocytes, (b) growth and proliferation of Nb2 rat lymphoma cells, and (c) tyrosine phosphorylation of copolymer (Glu4Tyr) under cell-free conditions. Although relatively high concentrations of o-hydroxyphenyl acetate (OHPA) were required to inhibit these processes, the inhibitor exhibited a low index of cytotoxicity and high specificity toward inhibiting tyrosine- (but not serine-) specific kinases. Cell cycle analysis of the DNA histograms in Nb2 cells revealed that exposure to OHPA did not change the initiation of the G0/G1----S transition but drastically reduced its rate and a subsequent cell proliferation. Kinetic experiments in which the inhibitor was added or withdrawn through different phases of cell cycle confirmed this conclusion. OHPA inhibition of cell growth appears to be limited to eukaryotic cells as the growth of either gram-positive or gram-negative bacteria was unaffected by the presence of the inhibitor. The study supports the following conclusions: (a) Events that are dependent on tyrosine phosphorylation are indeed essential for mammalian cell growth and proliferation. (b) Neither the initial nor intermediate events of the proliferative cascade that occur in the Nb2 cells prior to DNA synthesis are dependent on the activity of protein tyrosine kinase(s) that are inhibited by OHPA. (c) Cell growth of prokaryotic cells and yeast may lack protein tyrosine kinase activity or be less dependent on events requiring tyrosine phosphorylation. (d) Inhibition of the insulin-dependent lipogenesis is subsequent to the inhibition of insulin receptor tyrosine kinase activity.
Mol Cell Endocrinol 1991 Sep
PMID:Hydroxyphenyl acetate derivatives inhibit protein tyrosine kinase activity and proliferation in Nb2 rat lymphoma cells and insulin-induced lipogenesis in rat adipocytes. 195 77

Based on the sequence of cDNA encoding the intracellular domain of the insulin receptor beta-subunit, we recently defined a heterozygous point mutation causing a Ser for Trp substitution at position 1200 in the tyrosine kinase domain of a patient (BI-2) with the type A syndrome of insulin resistance. We have now sequenced the remainder of BI-2's insulin receptor cDNA-coding region and find no additional alterations in the encoded proreceptor protein. The nucleotide sequence of cDNA encoding the portion of the beta-subunit which includes Trp1200 was normal in BI-2's unaffected mother. Hybridization of a mutant allele-specific oligonucleotide to polymerase chain reaction-amplified cDNA confirmed the presence of the mutant allele in the proband and excluded it in her unaffected sister and mother, 18 normal control subjects, and six other subjects with insulin resistance. To determine whether this mutation had functional consequences for receptor signalling, we reconstructed it into a full-length insulin receptor cDNA expression vector. Chinese hamster ovary cells were transfected with mutant cDNA, and the expressed insulin receptors were compared to receptors expressed by cells transfected with wild-type receptor cDNA. Both mutant and wild-type receptors were properly processed into receptor alpha- and beta-subunits, were expressed on the cell surface, and displayed similar insulin-binding affinity. In contrast, insulin-stimulated autophosphorylation of the mutant receptors was severely impaired, whether assessed in intact cells or with a partially purified receptor preparation.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1990 Aug
PMID:Functional properties of a naturally occurring Trp1200----Ser1200 mutation of the insulin receptor. 196 73

Mitogen-activated protein (MAP) kinase is a serine/threonine-specific protein kinase which is activated in response to various mitogenic agonists (e.g., epidermal growth factor, insulin, and the tumor promoter tetradecanoyl phorbol acetate [TPA]) and requires both threonine and tyrosine phosphorylation for activity. This enzyme has recently been shown to be identical or closely related to pp42, a protein which becomes tyrosine phosphorylated in response to mitogenic stimulation. Neither the kinases which regulate MAP kinase/pp42 nor the in vivo substrates for this enzyme are known. Because MAP MAP kinase is activated and phosphorylated in response both to agents which stimulate tyrosine kinase receptors and to agents which stimulate protein kinase C, a serine/threonine kinase, we have examined the regulation and phosphorylation of this enzyme in 3T3-TNR9 cells, a variant cell line partially defective in protein kinase C-mediated signalling. In this communication, we show that in the 3T3-TNR9 variant cell line, TPA does not cause the characteristically rapid phosphorylation of pp42 or the activation and phosphorylation of MAP kinase. This defective response is not due to the absence of the MAP kinase/pp42 protein itself because both tyrosine phosphorylation of MAP kinase/pp42 and its enzymatic activation could be induced by platelet-derived growth factor in the 3T3-TNR9 cells. Thus, the defect in these variant cells apparently resides in some aspect of the regulation of MAP kinase phosphorylation. Since the 3T3-TNR9 cells are also defective with respect to the TPA-induced increase in ribosomal protein S6 kinase, these in vivo results reinforce the earlier in vitro finding that MAP kinase can regulate S6 kinase activity. These findings suggest a key role for MAP kinase in a kinase cascade cascade involved in the control of cell proliferation.
Mol Cell Biol 1991 Feb
PMID:Defective regulation of mitogen-activated protein kinase activity in a 3T3 cell variant mitogenically nonresponsive to tetradecanoyl phorbol acetate. 199 Feb 61

Raf-1 serine- and threonine-specific protein kinase is transiently activated in cells expressing the epidermal growth factor (EGF) receptor upon treatment with EGF. The stimulated EGF receptor coimmunoprecipitates with Raf-1 kinase and mediates protein kinase C-independent phosphorylation of Raf-1 on serine residues. Hyperphosphorylated Raf-1 has lower mobility on sodium dodecyl sulfate gels and has sixfold-increased activity in immunocomplex kinase assay with histone H1 or Raf-1 sequence-derived peptide as a substrate. Raf-1 activation requires kinase-active EGF receptor; a point mutant lacking tyrosine kinase activity in inactive in Raf-1 coupling and association. It is noteworthy that tyrosine phosphorylation of c-Raf-1 induced by EGF was not detected in these cells. These observations suggest that Raf-1 kinase may act as an important downstream effector of EGF signal transduction.
Mol Cell Biol 1991 Feb
PMID:Epidermal growth factor (EGF) stimulates association and kinase activity of Raf-1 with the EGF receptor. 199 Feb 91


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