Role of a tyrosine phosphorylation of SMG-9 in binding of SMG-9 to IQGAP and the NMD complex

SMG-9 is a component of the NMD complex, a heterotetramer that also includes SMG-1 and SMG-8 in the complex. SMG-9 was also originally identified as a tyrosine-phosphorylated protein but the role of the phosphorylation is not yet known. In this study, we determined that IQGAP protein, an actin cytoskeleton modifier acts as a binding partner with SMG-9 and this binding is regulated by phosphorylation of SMG-9 at Tyr-41. SMG-9 is co-localized with IQGAP1 as a part of the process of actin enrichment in non-stimulated cells, but not in the EGF-stimulated cells. Furthermore, an increase in the ability of SMG-9 to bind to SMG-8 occurs in response to EGF stimulation. These results suggest that tyrosine phosphorylation of SMG-9 may play a role in the formation of the NMD complex in the cells stimulated by the growth factor.     

Induction of a Distinct Morphology and Signal Transduction in TrkB/PC12 Cells by Nerve Growth Factor and Brain-Derived Neurotrophic Factor

Abstract: A clonal cell line stably expressing trkB (TrkB/PC12) was established from rat pheochromocytoma PC12 cells. Brain-derived neurotrophic factor (BDNF), as well as nerve growth factor (NGF), stimulates neurite outgrowth in TrkB/PC12 cells. However, the morphology of BDNF-differentiated cells was clearly different from NGF-differentiated cells. BDNF treatment brought about longer and thicker neurites and induced a flattened soma and an increase in somatic size. This is not explained enough by the quantitative difference in the strength between TrkA and TrkB stimulation, because the level of BDNF-stimulated tyrosine phosphorylation of TrkB was similar to that of TrkA stimulated with NGF in PC12/TrkB cells. There was no difference in major tyrosine phosphorylated proteins induced by NGF and BDNF. Signal proteins such as phosphatidylinositol 3-kinase, phospholipase C-γ1, Shc, and mitogen-activated protein kinase seem to be involved in both TrkA- and TrkB-mediated signaling pathways. However, a tyrosine-phosphorylated 38-kDa protein (pp38) was detected in anti-pan-Trk immunoprecipitation only after NGF stimulation. Immunoprecipitation using three distinct anti-pan-Trk antibodies suggests that pp38 is not a fragment of TrkA. These data indicate that TrkA has a unique signal transduction pathway that is not stimulated through TrkB in TrkB/PC12 cells and suggest distinct functions among neurotrophin receptors.     

Dephosphorylation of Tctex2-related dynein light chain by type 2A protein phosphatase

Maspin is a 42kDa tumor suppressor protein that belongs to the serine protease inhibitor (serpin) family. It inhibits cell motility and invasion in vitro, and tumor growth and metastasis in nude mice; however, maspin's molecular mechanism of action has remained elusive. Maspin contains several tyrosine residues and we hypothesized that phosphorylation of maspin could play a role in its biological function. Our study reveals that maspin is phosphorylated on tyrosine moiety(ies) in normal mammary epithelial cells endogenously expressing maspin. In addition, transfection of the maspin gene, using either a stable or inducible system into maspin-deficient breast cancer cell lines, yields a protein product that is phosphorylated on tyrosine residue(s). Furthermore, recombinant maspin protein can be tyrosine-phosphorylated by the kinase domain from the epidermal growth factor receptor in vitro. These novel observations suggest that maspin, which deviates from the classical serpin, may be an important signal transduction molecule in its phosphorylated form.     

Transient Ischemia Differentially Increases Tyrosine Phosphorylation of NMDA Receptor Subunits 2A and 2B

Abstract: Activation of the N -methyl- d -aspartate (NMDA) receptor has been implicated in the events leading to ischemia-induced neuronal cell death. Recent studies have indicated that the properties of the NMDA receptor channel may be regulated by tyrosine phosphorylation. We have therefore examined the effects of transient cerebral ischemia on the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in different regions of the rat brain. Transient (15 min) global ischemia was produced by the four-vessel occlusion procedure. The tyrosine phosphorylation of NR2A and NR2B subunits was examined by immunoprecipitation with anti-tyrosine phosphate antibodies followed by immunoblotting with antibodies specific for NR2A or NR2B, and by immunoprecipitation with subunit-specific antibodies followed by immunoblotting with anti-phosphotyrosine antibodies. Transient ischemia followed by reperfusion induced large (23–29-fold relative to sham-operated controls), rapid (within 15 min of reperfusion), and sustained (for at least 24 h) increases in the tyrosine phosphorylation of NR2A and smaller increases in that of NR2B in the hippocampus. Ischemia-induced tyrosine phosphorylation of NR2 subunits in the hippocampus was higher than that of cortical and striatal NR2 subunits. The enhanced tyrosine phosphorylation of NR2A or NR2B may contribute to alterations in NMDA receptor function or in signaling pathways in the postischemic brain and may be related to pathogenic events leading to neuronal death.     

Thrombin-stimulated Pyk2 phosphorylation in human endothelium is dependent on intracellular calcium and independent of protein kinase C and Src kinases

G-protein-coupled receptor agonists (GPCAs) cause functional responses in endothelial cells including secretion, proliferation, and altering monolayer permeability. These events are mediated in part by activation of the p42/44 mitogen-activated protein kinase (MAPK) cascade. The cytosolic tyrosine kinase Pyk2 is postulated to link GPCA-induced changes in intracellular calcium to activation of the MAP kinase cascade. We have investigated the regulation of Pyk2 in human umbilical vein endothelial cells in response to GPCAs and show that (1) thrombin, a PAR-1 peptide, and histamine cause rapid concentration- and time-dependent phosphorylation on tyrosines 402 (Src kinase binding site), 881 (Grb2 binding site), and 580 (an autophosphorylation site), (2) thrombin-stimulated phosphorylation is dependent on intracellular calcium and independent of PKC and PI-3 kinase, and (3) inhibition of Src kinases has no significant effect on thrombin-stimulated phosphorylation, implying that tyrosine phosphorylation of Pyk2 is independent of Src binding.     

Thrombin Receptor Activation Stimulates Astrocyte Proliferation and Reversal of Stellation by Distinct Pathways: Involvement of Tyrosine Phosphorylation

Abstract: Treatment of cultured type-1 astrocytes with thrombin leads to cell proliferation and reversal of stellation. The half-maximal concentrations of thrombin required for each response are 500 and 2 p M , respectively. To test whether they might be mediated by different receptors, we examined the contribution of the G protein-coupled thrombin receptor to these responses in purified rat astrocytes by using the agonist peptide SFLLRNP. In the absence of added growth factors, SFLLRNP fully mimicked the effects of thrombin at half-maximal concentrations of 30 µ M for an increase in cell number and DNA synthesis and 100 n M for the reversal of stellation. The role of protein tyrosine phosphorylation in these events was investigated using antiphosphotyrosine antibodies. Thrombin and SFLLRNP at concentrations at least 10-fold greater than those required for half-maximal reversal of stellation but below those required for mitogenesis induced an identical pattern of tyrosine phosphorylation on several proteins of 55–65, 106, 110–115, and 120–130 kDa. The response was rapid (<1 min) and transient with a peak response after ∼2 min. The specific tyrosine kinase inhibitor herbimycin A did not affect thrombin- or SFLLRNP-mediated reversal of stellation at concentrations of up to 1 µ M . In contrast, 1 µ M herbimycin fully inhibited the ability of thrombin and SFLLRNP to increase cell number and stimulate DNA synthesis. Furthermore, this inhibition by 1 µ M herbimycin A corresponded to inhibition of receptor-induced tyrosine phosphorylation. Thus, cell proliferation but not reversal of stellation is dependent on thrombin receptor-activated tyrosine kinase activity. These observations support the hypotheses that the thrombin receptor mediates the actions of thrombin in these cells and that activation of the thrombin receptor leads to multiple second messages that stimulate distinct cellular responses.     

Altered signaling through IL-12 receptor in children with very high serum IgE levels

An alteration of Th1/Th2 homeostasis may lead to diseases in humans. In this study, we investigated whether an impaired IL-12R signaling occurred in children with elevated serum IgE levels divided on the basis of the IgE levels (group A: >2000 kU/l; group B: <2000 kU/l). We evaluated the integrity of the IL-12R signaling through the analysis of phosphorylation/activation of STAT4, and mRNA expression and membrane assembly of the receptor chains. At a functional level, a proliferative defect of lymphocytes from group A patients was observed. In these patients, an abnormal IL-12R signaling was documented, and this finding was associated with abnormal expression of the IL-12Rβ2 chain. Our data indicate that in patients with very high IgE levels the generation of Th1 response is impaired, and that this abnormality associates with abnormal IL-12R signaling.     

Mass spectrometric characterization of oat phytochrome A: isoforms and posttranslational modifications.

At least four mRNAs for oat phytochrome A (phyA) are present in etiolated oat tissue. The complete amino acid sequences of two phyA isoforms (A3 and A4) and the N-terminal amino acid sequence of a third isoform (A5) were deduced from cDNA sequencing (Hershey et al., 1985). In the present study, heterogeneity of phyA on a protein level was studied by tryptic mapping using electrospray ionization mass-spectrometry (ESIMS). The total tryptic digest of iodoacetamide-modified phyA was fractionated by gel filtration chromatography followed by reversed-phase high-performance liquid chromatography. ESIMS was used to identify peptides. Amino acid sequences of the peptides were confirmed or determined by collision-induced dissociation mass spectrometry (CID MS), MS/MS, or by subdigestion of the tryptic peptides followed by ESIMS analysis. More than 97% of the phyA3 sequence (1,128 amino acid residues) was determined in the present study. Mass-spectrometric analysis of peptides unique to each form showed that phyA purified from etiolated oat seedling is represented by three isoforms A5, A3, and A4, with ratio 3.4:2.3:1.0. Possible light-induced changes in phytochrome in vivo phosphorylation site at Ser7 (Lapko VN et al., 1997, Biochemistry 36:10595-10599) as well at Ser17 and Ser598 (known as in vitro phosphorylation sites) were also analyzed. The extent of phosphorylation at Ser7 appears to be the same for phyA isolated from dark-grown and red-light illuminated seedlings. In addition to Ser7, Ser598 was identified as an in vivo phosphorylation site in oat phyA. Ser598 phosphorylation was found only in phyA from the red light-treated seedlings, suggesting that the protein phosphorylation plays a functional role in the phytochrome A-mediated light-signal transduction.     

Distinct roles for ADAM10 and ADAM17 in ectodomain shedding of six EGFR ligands

All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are released from the membrane by proteases. In several instances, ectodomain release is critical for activation of EGFR ligands, highlighting the importance of identifying EGFR ligand sheddases. Here, we uncovered the sheddases for six EGFR ligands using mouse embryonic cells lacking candidate-releasing enzymes (a disintegrin and metalloprotease [ADAM] 9, 10, 12, 15, 17, and 19). ADAM10 emerged as the main sheddase of EGF and betacellulin, and ADAM17 as the major convertase of epiregulin, transforming growth factor alpha, amphiregulin, and heparin-binding EGF-like growth factor in these cells. Analysis of adam9/12/15/17-/- knockout mice corroborated the essential role of adam17-/- in activating the EGFR in vivo. This comprehensive evaluation of EGFR ligand shedding in a defined experimental system demonstrates that ADAMs have critical roles in releasing all EGFR ligands tested here. Identification of EGFR ligand sheddases is a crucial step toward understanding the mechanism underlying ectodomain release, and has implications for designing novel inhibitors of EGFR-dependent tumors.     

Prediction of possible sites for posttranslational modifications in human gamma crystallins: effect of glycation on the structure of human gamma-B-crystallin as analyzed by molecular modeling

Crystallins are recognized as one of the long-lived proteins of lens tissue that might serve as the target for several posttranslational modifications leading to cataract development. We have studied several such sites present in the human gamma-crystallins based either on PROSITE pattern search results or earlier experimental evidences. Their probabilities were examined on the basis of the database analysis of the gamma-crystallin sequences and on their specific locations in the constructed homology models. An N-glycosylation site in human gammaD-crystallin and several phosphorylation sites in all four human gamma-crystallins were predicted by the PROSITE search. Some of these sites were found to be strongly conserved in the gamma-crystallin sequences from different sources. An extensive analysis of these sites was performed to predict their probabilities as potential sites for protein modifications. Glycation studies were performed separately by attaching sugars to the human gammaB-crystallin model, and the effect of binding was analyzed. The studies showed that the major effect of alphaD-glucose (alphaD-G) and alphaD-glucose-6-phosphate (alphaD-G6P) binding was the disruption of charges not only at the surface but also within the molecule. Only a minor alteration in the distances of sulfhydryl groups of cysteines and on their positions in the three-dimensional models were observed, leading us to assume that glycation alone is not responsible for intra- and intermolecular disulfide bond formation.     

Caveolin-1 phosphorylation in human squamous and epidermoid carcinoma cells: dependence on ErbB1 expression and Src activation

Previous studies have shown that EGF can induce the tyrosine phosphorylation of caveolin-1 in murine fibroblasts following ErbB1 (EGF receptor) mutation or overexpression, but the cell signaling events linking EGF action with caveolin phosphorylation are not fully established. In this regard, we examined multiple human carcinoma cell lines that express various ErbB family members, including A431 epidermoid carcinoma cells and several squamous carcinoma cell lines. In all cases, EGF treatment induced the tyrosine phosphorylation of caveolin-1 in a time- and EGF dose-dependent manner, and immunoblotting analysis revealed that this phosphorylation occurred at tyrosine-14. The EGF-dependent phosphorylation of caveolin-1 was observed at low temperatures (4 degrees C) and was enhanced by caveolae-disrupting agents (cyclodextrin), suggesting that this EGF-dependent system is in a low temperature-stable arrangement that allows for their interaction under conditions where mobility in the membrane is altered. To further assess the events linking EGF action with caveolin phosphorylation, we evaluated the ligand specificity of these responses and their dependence on known effectors of EGF receptor function. We observed that EGF and HB-EGF, but not heregulin, promoted caveolin-1 phosphorylation in A431 cells, suggesting that these responses are linked to EGF receptor activation and not solely occurring via the activation of other endogenous ErbB family members. In addition, the EGF-induced phosphorylation of caveolin-1 in A431 cells was blocked by the Src kinase antagonists PP1 and PP2, but not by the MEK inhibitor PD98059, the phosphoinositide 3-kinase inhibitors LY294002 and wortmannin, or cytoskeleton-disrupting agents, such as cytochalasin D, colchicine, and nocadazole. Altogether, these data indicate that multiple human carcinoma cells exhibit an EGF receptor-dependent tyrosine phosphorylation of caveolin-1 and that this process is sensitive to Src family kinase inhibitors. These observations support a role for caveolin tyrosine phosphorylation in the profile of cellular responses by which Src potentiates cancer progression following EGF receptor overexpression.     

Phosphorylation of IRS1 at serine 307 and serine 312 in response to insulin in human adipocytes

Feedback control in insulin signaling involves serine phosphorylation of insulin receptor substrate-1 (IRS1). By analyzing the insulin-induced phosphorylation of IRS1 at serine 307, serine 312, and tyrosine in the same primary human adipocytes, we now report that negative feedback phosphorylation of serine 312 (corresponding to murine serine 307) required relatively high concentrations of insulin (EC(50)=3 nM) for a long time (t(1/2) ca. 30 min) and reduced the steady-state tyrosine phosphorylation, without affecting the cellular concentration, of IRS1. In contrast, positive feedback phosphorylation of serine 307 was a rapid (t(1/2) ca. 2 min) event at physiological concentrations of insulin (EC(50)=0.2 nM).     

IGF-I induces caveolin 1 tyrosine phosphorylation and translocation in the lipid rafts

Caveolin 1, a component of caveolae, regulates signalling pathways compartmentalization interacting with tyrosine kinase receptors and their substrates. The role of caveolin 1 in the Insulin Receptor (IR) signalling has been well investigated. On the contrary, the functional link between caveolin 1 and IGF-I Receptor (IGF-IR) remains largely unknown. Here we show that (1) IGF-IR colocalizes with caveolin 1 in the lipid rafts enriched fractions on plasmamembrane in R-IGF-IR(WT) cells, (2) IGF-I induces caveolin 1 phosphorylation at the level of tyrosine 14, (3) this effect is rapid and results in the translocation of caveolin 1 and in the formation of membrane patches on cell surface. These actions are IGF-I specific since we did not detect caveolin 1 redistribution in insulin stimulated R(-) cells overexpressing IRs.     

Epidermal growth factor receptors on PC12 cells: Alteration of binding properties by lectins

The PC12 cell line displays cell surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). It has been previously shown that the lectin wheat germ agglutinin (WGA) alters the properties of NGF receptors on these cells. We now report that preincubations with either WGA or concanavalin A (Con A) decrease the binding of ¹²⁵I-EGF to PC12 cells by greater than 50%. The inhibition of binding occurred at 37°C and 4°C and could be blocked or reversed by the addition of sugars which bind specifically to WGA or Con A. Scatchard analysis revealed that these lectins decreased binding primarily by lowering the affinity of the receptor and to a lesser extent by decreasing receptor number. Succinylalion of Con A (sCon A) produced a derivative that was less effective than the native lectin in decreasing EGF binding; however, addition of an antibody against Con A restored the ability of sCon A to decrease binding. Similar to results obtained with ¹²⁵I-NGF binding, WGA but not Con A was found to increase, by scveralfold; the proportion of ¹²⁵I-EGF binding that is resistant to solubilization by Triton X-100 detergent. A potential association of the EGF receptor with cytoskeletal elements is discussed which could account for such results.     

Co-localization of cortactin and phosphotyrosine identifies active invadopodia in human breast cancer cells

Invadopodia are filopodia-like projections possessing protease activity that participate in tumor cell invasion. We demonstrate that co-localization of cortactin and phosphotyrosine identifies a subset of cortactin puncta termed "invadopodial complexes" that we find to be closely associated with the plasma membrane at active sites of focal degradation of the extracellular matrix in MDA-MB-231 breast cancer cells. Manipulation of c-Src activity in cells by transfection with kinase activated c-Src(527) or kinase inactive c-Src(295) results in a dramatic increase or decrease, respectively, in the number of these structures associated with changes in the number of sites of active matrix degradation. Overexpression of kinase-inactive c-Src(295) does not prevent localization of cortactin at the membrane; however, co-localized phosphotyrosine staining is decreased. Thus, elevated phosphotyrosine at invadopodial complexes is specifically associated with the proteolytic activity of invadopodia. Further, invadopodial complexes are spatially, morphologically and compositionally distinct from focal adhesions as determined by localization of focal adhesion kinase (FAK), which is not present in invadopodial complexes. Expression of kinase-inactive c-Src(295) blocks invadopodia activity, but does not block filopodia formation. Thus, invadopodia, but not filopodia, are highly correlated with matrix invasion, and sites of invadopodial activity can be identified by the formation of invadopodial complexes.     

雌激素拮抗剂对胎盘EGF受体及其基因表达的影响

中期孕鼠在他莫昔芬作用下,其颌下腺,血清中ＥＧＦ含量下降,胎盘中ＥＧＦ受体结合位点数下降以及它的ｍＲＮＡ表达受到抑制,再次证实了他莫昔芬抑制雌激素诱导ＥＧＦ受体ｍＲＮＡ的表达。从而使ＥＧＦ受体结合位点数减少,因此,他莫昔芬对孕鼠胚胎生长发育有不可忽视的影响。     

Fyn-induced phosphorylation of beta-adducin at tyrosine 489 and its role in their subcellular localization

Fyn is a Src-family tyrosine kinase involved in neuronal development, transmission, and plasticity in mammalian central nervous system. We have previously reported that Fyn binds to a cytoskeletal protein, beta-adducin, in a phosphorylation-dependent manner. In the present report, we show that Fyn phosphorylates beta-adducin at tyrosine 489 located in its C-terminal tail domain. Phosphorylation of beta-adducin at Y489 was required for its association with the Fyn-SH2 domain. An antibody specific to the phosphorylated form of beta-adducin was raised in rabbits and showed that Y489 of beta-adducin was phosphorylated in wild type, but not in Fyn(-/-) mice, suggesting that Y489 of beta-adducin is phosphorylated downstream of Fyn in vivo. After phosphorylation at Y489, beta-adducin was translocated to the cell periphery, and colocalized with Fyn. These results suggest that Fyn phosphorylates and binds to beta-adducin at Y489, resulting in translocation of beta-adducin to the Fyn-enriched regions in the plasma membrane.     

The N-Methyl-d-Aspartate Receptor Subunits NR2A and NR2B Bind to the SH2 Domains of Phospholipase C-γ

Abstract: The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-γ (PLC-γ). A glutathione S -transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-γ was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-γ and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.