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

Following binding of platelet-derived growth factor (PDGF), the PDGF alpha receptor (alphaPDGFR) becomes tyrosine phosphorylated and associates with a number of signal transduction molecules, including phospholipase Cgamma-1 (PLCgamma-1), phosphatidylinositol 3-kinase (PI3K), the phosphotyrosine phosphatase SHP-2, Grb2, and Src. Here, we present data identifying a novel phosphorylation site in the kinase insert domain of the alphaPDGFR at tyrosine (Y) 720. We replaced this residue with phenylalanine and expressed the mutated receptor (F720) in Patch fibroblasts that do not express the alphaPDGFR. Characterization of the F720 mutant indicated that binding of two proteins, SHP-2 and Grb2, was severely impaired, whereas PLCgamma-1 and PI3K associated to wild-type levels. In addition, mutating Y720 to phenylalanine dramatically reduced PDGF-dependent tyrosine phosphorylation of SHP-2. Since Y720 was required for recruitment of two proteins, we investigated the mechanism by which these two proteins associated with the alphaPDGFR. SHP-2 bound the alphaPDGFR directly, whereas Grb2 associated indirectly, most probably via SHP-2, as Grb2 and SHP-2 coimmunoprecipitated when SHP-2 was tyrosine phosphorylated. We also compared the ability of the wild-type and F720 alphaPDGFRs to mediate a number of downstream events. Preventing the alphaPDGFR from recruiting SHP-2 and Grb2 did not compromise PDGF-AA-induced activation of Ras, initiation of DNA synthesis, or growth of cells in soft agar. We conclude that phosphorylation of the alphaPDGFR at Y720 is required for association of SHP-2 and Grb2 and tyrosine phosphorylation of SHP-2; however, these events are not required for the alphaPDGFR to activate Ras or initiate a proliferative response. In addition, these findings reveal that while SHP-2 binds to both of the receptors, it binds in different locations: to the carboxy terminus of the betaPDGFR but to the kinase insert of the alphaPDGFR.
Mol Cell Biol 1996 Dec
PMID:Phosphorylation of tyrosine 720 in the platelet-derived growth factor alpha receptor is required for binding of Grb2 and SHP-2 but not for activation of Ras or cell proliferation. 894 48

The mechanisms governing neuronal differentiation, including the signals underlying the induction of voltage-dependent sodium (Na+) channel expression by neurotrophic factors, which occurs independent of Ras activity, are not well understood. Therefore, Na+ channel induction was analyzed in sublines of PC12 cells stably expressing platelet-derived growth factor (PDGF) beta receptors with mutations that eliminate activation of specific signalling molecules. Mutations eliminating activation of phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLC gamma), the GTPase-activating protein (GAP), and Syp phosphatase failed to diminish the induction of type II Na+ channel alpha-subunit mRNA and functional Na+ channel expression by PDGF, as determined by RNase protection assays and whole-cell patch clamp recording. However, mutation of juxtamembrane tyrosines that bind members of the Src family of kinases upon receptor activation inhibited the induction of functional Na+ channels while leaving the induction of type II alpha-subunit mRNA intact. Mutation of juxtamembrane tyrosines in combination with mutations eliminating activation of PI3K, PLC gamma, GAP, and Syp abolished the induction of type II alpha-subunit mRNA, suggesting that at least partially redundant signaling mechanisms mediate this induction. The differential effects of the receptor mutations on Na+ channel expression did not reflect global changes in receptor signaling capabilities, as in all of the mutant receptors analyzed, the induction of c-fos and transin mRNAs still occurred. The results reveal an important role for the Src family in the induction of Na+ channel expression and highlight the multiplicity and combinatorial nature of the signaling mechanisms governing neuronal differentiation.
Mol Cell Biol 1997 Jan
PMID:Analysis of mutant platelet-derived growth factor receptors expressed in PC12 cells identifies signals governing sodium channel induction during neuronal differentiation. 897 89

Insulin is a key hormone regulating glucose homeostasis and has many cellular effects on metabolism, growth, and differentiation. Insulin action is mediated through a specific cell-surface receptor. The first step following insulin binding consists in receptor autophosphorylation and stimulation of its tyrosine kinase activity. Among the multiple substrates, the insulin receptor substrate-1 (IRS-1) is the major cytoplasmic substrate for insulin. IRS-1 binds several Src homology 2 (SH2) proteins through its multiple tyrosine phosphorylation sites: phosphatidylinositol 3-kinase (PI 3-kinase), the Ras guanine-nucleotide-releasing complex Grb2-SOS, the tyrosine phosphatase Syp, and the adapter protein Nck. IRS-1 is essential for many, but not all of the insulin's biological responses. Recently, a primary alternative substrate, i.e. IRS-2, was purified and cloned. Numerous biochemical abnormalities of the insulin signaling system lead to insulin resistance. No doubt, the recent data about the molecular mechanisms of insulin action will provide new insights into the pathophysiology and therapy of diabetes and other insulin resistant states.
Mol Med (Sofia) 1996
PMID:[The insulin transduction system]. 898 14

Mice made insulin receptor substrate 1 (IRS-1) deficient by targeted gene knockout exhibit growth retardation and abnormal glucose metabolism due to resistance to the actions of insulin-like growth factor 1 (IGF-1) and insulin (E. Araki et al., Nature 372:186-190, 1994; H. Tamemoto et al., Nature 372:182-186, 1994). Embryonic fibroblasts and 3T3 cell lines derived from IRS-1-deficient embryos exhibit no IGF-1-stimulated IRS-1 phosphorylation or IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity but exhibit normal phosphorylation of IRS-2 and Shc and normal IRS-2-associated PI 3-kinase activity. IRS-1 deficiency results in a 70 to 80% reduction in IGF-1-stimulated cell growth and parallel decreases in IGF-1-stimulated S-phase entry, PI 3-kinase activity, and induction of the immediate-early genes c-fos and egr-1 but unaltered activation of the mitogen-activated protein kinases ERK 1 and ERK 2. Expression of IRS-1 in IRS-1-deficient cells by retroviral gene transduction restores IGF-1-stimulated mitogenesis, PI 3-kinase activation, and c-fos and egr-1 induction in proportion to the level of reconstitution. Increasing the level of IRS-2 in these cells by using a retrovirus reconstitutes IGF-1 activation of PI 3-kinase and immediate-early gene expression to the same degree as expression of IRS-1; however, IRS-2 overexpression has only a minor effect on IGF-1 stimulation of cell cycle progression. These results indicate that IRS-1 is not necessary for activation of ERK 1 and ERK 2 and that activation of ERK 1 and ERK 2 is not sufficient for IGF-1-stimulated activation of c-fos and egr-1. These data also provide evidence that IRS-1 and IRS-2 are not functionally interchangeable signaling intermediates for stimulation of mitogenesis despite their highly conserved structure and many common functions such as activating PI 3-kinase and early gene expression.
Mol Cell Biol 1997 Mar
PMID:Differential signaling by insulin receptor substrate 1 (IRS-1) and IRS-2 in IRS-1-deficient cells. 903 79

We have found that insulin-like growth factor I (IGF-I) can protect fibroblasts from apoptosis induced by UV-B light. Antiapoptotic signalling by the IGF-I receptor depended on receptor kinase activity, as cells overexpressing kinase-defective receptor mutants could not be protected by IGF-I. Overexpression of a kinase-defective receptor which contained a mutation in the ATP binding loop functioned as a dominant negative and sensitized cells to apoptosis. The antiapoptotic capacity of the IGF-I receptor was not shared by other growth factors tested, including epidermal growth factor (EGF) and thrombin, although the cells expressed functional receptors for all the agonists. However, EGF was antiapoptotic for cells overexpressing the EGF receptor, and expression of activated pp60v-src also was protective. There was no correlation between protection from apoptosis and activation of mitogen-activated protein kinase, p38/HOG1, or p70S6 kinase. On the other hand, protection by any of the tyrosine kinases against UV-induced apoptosis was blocked by wortmannin, implying a role for phosphatidylinositol 3-kinase (PI3 kinase). To test this, we transiently expressed constitutively active or kinase-dead PI3 kinase and found that overexpression of activated phosphatidylinositol 3-kinase (PI3 kinase) was sufficient to provide protection against apoptosis. Because Akt/PKB is believed to be a downstream effector for PI3 kinase, we also examined the role of this serine/threonine protein kinase in antiapoptotic signalling. We found that membrane-targeted Akt was sufficient to protect against apoptosis but that kinase-dead Akt was not. We conclude that the endogenous IGF-I receptor has a specific antiapoptotic signalling capacity, that overexpression of other tyrosine kinases can allow them also to be antiapoptotic, and that activation of PI3 kinase and Akt is sufficient for antiapoptotic signalling.
Mol Cell Biol 1997 Mar
PMID:Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. 903 87

The human proto-oncogene product c-Cbl and a similar protein in Caenorhabditis elegans (Sli-1) contain a proline-rich COOH-terminal region that binds Src homology 3 (SH3) domains of proteins such as the adapter Grb2. Cb1-Grb2 complexes can be recruited to tyrosine-phosphorylated epidermal growth factor (EGF) receptors through the SH2 domain of Grb2. Here we identify by molecular cloning a Drosophila cDNA encoding a protein (Drosophila Cbl [D-Cbl]) that shows high sequence similarity to the N-terminal region of human c-Cbl but lacks proline-rich sequences and fails to bind Grb2. Nonetheless, in COS-1 cells, expression of hemagglutinin epitope-tagged D-Cbl results in its coimmunoprecipitation with EGF receptors in response to EGF. EGF also caused tyrosine phosphorylation of D-Cbl in such cells, but no association of phosphatidylinositol 3-kinase was detected in assays using anti-p85 antibody. A point mutation in D-Cbl (G305E) that suppresses the negative regulation of LET-23 by the Cbl homolog Sli-1 in C. elegans prevented tyrosine phosphorylation of D-Cbl as well as binding to the liganded EGF receptor in COS-1 cells. Colocalization of EGF receptors with both endogenous c-Cbl or expressed D-Cbl in endosomes of EGF-treated COS-1 cells is also demonstrated by immunofluorescence microscopy. In lysates of adult transgenic Drosophila melanogaster, GST-DCbl binds to the tyrosine-phosphorylated 150-kDa torso-DER chimeric receptor. Expression of D-Cbl directed by the sevenless enhancer in intact Drosophila compromises severely the development of the R7 photoreceptor neuron. These data suggest that despite the lack of Grb2 binding sites, D-Cbl functions as a negative regulator of receptor tyrosine kinase signaling in the Drosophila eye by a mechanism that involves its association with EGF receptors or other tyrosine kinases.
Mol Cell Biol 1997 Apr
PMID:Interactions of Drosophila Cbl with epidermal growth factor receptors and role of Cbl in R7 photoreceptor cell development. 912 72

In the present study we have examined the role of phosphatidylinositol 3-kinase (PI 3-kinase) in the insulin-like growth factor I (IGF-I)-signaling pathways involved in differentiation and in mitogenesis in fetal rat brown adipocytes. Activation of PI 3-kinase in response to IGF-I was markedly inhibited by two PI 3-kinase inhibitors (wortmannin and LY294002) in a dose-dependent manner. IGF-I-stimulated glucose uptake was also inhibited by both compounds. The expression of adipogenic-related genes such as fatty acid synthase, malic enzyme, glycerol 3-phosphate dehydrogenase, and acetylcoenzyme A carboxylase induced by IGF-I was totally prevented in the presence of IGF-I and any of those inhibitors, resulting in a marked decrease of the cytoplasmic lipid content. Moreover, the expression of the thermogenic marker uncoupling protein induced by IGF-I was also down-regulated in the presence of wortmannin/LY294002. IGF-I-induced adipogenic- and thermogenic-related gene expression was only partly inhibited by the p70S6k inhibitor rapamycin. In addition, pretreatment of brown adipocytes with either wortmannin or LY294002, but not with rapamycin, blocked protein kinase C zeta activation by IGF-I. In contrast, IGF-I-induced fetal brown adipocyte proliferation was PI 3-kinase-independent. Our results show for the first time an essential requirement of PI 3-kinase in the IGF-I-signaling pathways leading to fetal brown adipocyte differentiation, but not leading to mitogenesis. In addition, protein kinase C zeta seems to be a signaling molecule also involved in the IGF-I differentiation pathways downstream from PI 3-kinase.
Mol Endocrinol 1997 May
PMID:Phosphatidylinositol 3-kinase is a requirement for insulin-like growth factor I-induced differentiation, but not for mitogenesis, in fetal brown adipocytes. 913 3

Gas6 is a secreted protein previously identified as the ligand of the Axl receptor tyrosine kinase. We have shown that Gas6 is able to induce cell cycle reentry of serum-starved NIH 3T3 cells and to efficiently prevent apoptosis after complete growth factor removal, a survival effect uncoupled from Gas6-induced mitogenesis. Here we report that the mitogenic effect of Gas6 requires phosphatidylinositol 3-kinase (PI3K) activity since it is abrogated both by the specific inhibitor wortmannin and by overexpression of the dominant negative P13K p85 subunit. Consistently, Gas6 activates the P13K downstream targets S6K and Akt, whose activation is abrogated by addition of wortmannin. Moreover, rapamycin treatment blocks Gas6-induced entry into the S phase of serum-starved NIH 3T3 cells. We also demonstrate the requirement of Src tyrosine kinase for Gas6 signalling since stable or transient expression of a catalytically inactive form of Src significantly inhibited Gas6-stimulated entry into the S phase. Accordingly, Gas6 addition to serum-starved NIH 3T3 cells causes activation of the intrinsic Src kinase activity. When specifically analyzed in a survival assay, these elements were found to be required for the survival effect of Gas6. Taken together, the evidence presented here identifies elements involved in the Gas6 transduction pathway that are responsible for its antiapoptotic effect and suggests that Src is involved in the events regulating cell survival.
Mol Cell Biol 1997 Aug
PMID:Requirement of phosphatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl mitogenic and survival activities in NIH 3T3 fibroblasts. 923 2

Mannose 6-phosphate receptors carry newly synthesized lysosomal hydrolases from the trans-Golgi network to endosomes, then return to the trans-Golgi network for another round of enzyme delivery. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, interferes with the delivery of newly synthesized lysosomal enzymes to lysosomes. We used two independent assays of mannose 6-phosphate receptor trafficking to determine the precise step that is blocked by wortmannin. Using an assay that monitors resialylation of desialylated cell surface 300-kDa mannose 6-phosphate receptors, we found that receptor endocytosis and transport to the trans-Golgi network were not inhibited by 2 microM wortmannin. In addition, this concentration of drug had no effect on the transport of the mannose 6-phosphate receptor from late endosomes to the trans-Golgi network using a system that reconstitutes this transport process in cell extracts. Under the same conditions, wortmannin significantly inhibited the generation of mature cathepsin D. In addition, the structurally unrelated phosphatidylinositol 3-kinase inhibitor, LY294002, was also without effect when added to in vitro endosome-trans-Golgi network transport reactions. These experiments demonstrate that the interruption in lysosomal enzyme targeting is most likely due to a wortmannin-sensitive process required for the export of these receptors from the trans-Golgi network, consistent with the established role of phosphatidylinositol 3-kinase in the equivalent transport process in Saccharomyces cerevisiae.
Mol Biol Cell 1997 Apr
PMID:Phosphatidylinositol 3-kinase is not required for recycling of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network. 924 39

Stimulation of Nb2 cells with PRL results in the rapid phosphorylation of a 120-kDa protein identified as the adapter protein cbl on tyrosine residues. Maximal phosphorylation of cbl occurs at 20 min after PRL stimulation and declines thereafter. Stimulation with as little as 5 nM PRL resulted in the phosphorylation of cbl; increasing the concentration of PRL to 100 nM had only a minimal effect upon the phosphorylation of cbl. The cbl protein appears to be constitutively associated with grb2 and the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase). The constitutive association of cbl with the p85 subunit of PI 3-kinase was observed in Nb2 cells as well as in 32Dcl3 cells transfected with either the rat Nb2 (intermediate) form of the PRL receptor or the long form of the human PRL receptor. A glutathione S-transferase fusion protein encoding the SH3 domain of the p85 subunit of PI 3-kinase bound to cbl in lysates of both unstimulated and PRL-stimulated Nb2 cells; however, neither of the SH2 domains of p85 bound to cbl under the same conditions. PRL stimulation increased the cbl-associated PI kinase activity. The majority of PI kinase activity appeared to be cbl-associated after PRL stimulation. These results suggest that cbl may function as an adapter protein in PRL-mediated signaling events and regulate activation of PI 3-kinase. Our model suggests that the p85 subunit of PI 3-kinase is constitutively associated with cbl through binding of the p85 SH3 domain to a proline-rich sequence in cbl. After the tyrosine phosphorylation of cbl, an SH2 domain(s) of p85 binds to a specific phosphorylation site(s) in cbl, leading to the activation of PI 3-kinase.
Mol Endocrinol 1997 Aug
PMID:Phosphorylation of cbl after stimulation of Nb2 cells with prolactin and its association with phosphatidylinositol 3-kinase. 925 13


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