Cholesterol depletion from the plasma membrane triggers ligand-independent activation of the epidermal growth factor receptor

We recently demonstrated that depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) caused activation of MAPK (Chen, X., and Resh, M. D. (2001) J. Biol. Chem. 276, 34617-34623). MAPK activation was phosphatidylinositol 3-kinase (PI3K)-dependent and involved increased tyrosine phosphorylation of the p85 subunit of PI3K. We next determined whether MbetaCD treatment induced tyrosine phosphorylation of other cellular proteins. Here we report that cholesterol depletion of serum-starved COS-1 cells with MbetaCD or filipin caused an increase in Tyr(P) levels of a 180-kDa protein that was identified as the epidermal growth factor receptor (EGFR). Cross-linking experiments showed that MbetaCD induced dimerization of EGFR, indicative of receptor activation. Reagents that block release of membrane-bound EGFR ligands did not affect MbetaCD-induced tyrosine phosphorylation of EGFR, indicating that MbetaCD activation of EGFR is ligand-independent. Moreover, MbetaCD treatment resulted in increased tyrosine phosphorylation of EGFR downstream targets and Ras activation. Incubation of cells with the specific EGFR inhibitor AG4178 blocked MbetaCD-induced phosphorylation of EGFR, SHC, phospholipase C-gamma, and Gab-1 as well as MAPK activation. We conclude that cholesterol depletion from the plasma membrane by MbetaCD causes ligand-independent activation of EGFR, resulting in MAPK activation by PI3K and Ras-dependent mechanisms. Moreover, these studies reveal a novel mode of action of MbetaCD, in addition to its ability to disrupt membrane rafts.     

Insulin-like and non-insulin-like selenium actions in 3T3-L1 adipocytes

In insulin-sensitive 3T3-L1 adipocytes, selenium stimulates glucose transport and antilipolysis and these actions of selenium, like insulin actions, are sensitive to wortmanin, an inhibitor of phosphatidylinositol-3-kinase (PI3K). Selenium stimulates PI3K activity that is sustained up to 24 h. Selenium after 5-10 min increases tyrosine phosphorylation of selective cellular proteins, but after 24 h overall tyrosine phosphorylation is increased. Tyrosine phosphorylation of insulin receptor substrate 1 is detected when enriched by immunoprecipitation with anti-PI3K antibody. Selenium, however, does not stimulate insulin receptor tyrosine kinase activity. Selenium also increases phosphorylation of other insulin signaling proteins, including Akt and extracellular signal regulated kinases. Selenium-stimulated glucose transport is accompanied by increases in glucose transporter-1 content in the plasma membrane. These data are consistent with similar selenium action in glucose transport in 3T3-L1 fibroblasts expressing mainly GLUT1. In chronic insulin-induced insulin resistant cells, selenium unlike insulin fully stimulates glucose transport. In summary, selenium stimulates glucose transport and antilipolysis in a PI3K-dependent manner, but independent of insulin receptor activation. Selenium exerts both insulin-like and non-insulin-like actions in cells.     

Effects of peroxynitrite-induced protein tyrosine nitration on insulin-stimulated tyrosine phosphorylation in HepG2 cells

Tyrosine nitration by peroxynitrite can affect signal transduction pathways involving tyrosine phosphorylation. The present study was undertaken to investigate the effects of peroxynitrite-induced protein tyrosine nitration on insulin-stimulated tyrosine phosphorylation in HepG2 cells. We show here that exposure of HepG2 cells to peroxynitrite led to a dose-dependent increase in tyrosine nitration of cellular proteins, mainly membrane and nuclear proteins. Furthermore, peroxynitrite induced differential responses in tyrosine phosphorylation of membrane proteins as well as cytosolic proteins according to peroxynitrite concentrations used. Our findings indicate at low concentrations peroxynitrite upregulates the insulin signaling and may operate as a signaling molecule, but at higher concentrations peroxynitrite downregulates the insulin signaling and may be involved in insulin resistance, suggesting peroxynitrite plays a dual role in regulation of the insulin signaling.     

Transformation-specific tyrosine phosphorylation of a novel cellular protein in chicken cells expressing oncogenic variants of the avian cellular src gene.

We used myristylated and nonmyristylated c-src-based variants and phosphotyrosine-specific antibodies to reevaluate the role of tyrosine phosphorylation in cellular transformation by pp60src. Prior methods used to detect tyrosine-phosphorylated proteins failed to discriminate predicted differences in tyrosine phosphorylation which are clearly observed with phosphotyrosine-specific antibodies and Western blotting (immunoblotting). Here we report the observation of a 120,000-Mr protein whose phosphorylation on tyrosine correlates with the induction of morphological transformation. p120 was not observed in cells overexpressing the regulated, nononcogenic pp60c-src, whereas phosphorylation of p120 was greatly enhanced in cells expressing activated, oncogenic pp60527F. Furthermore, phosphorylation of p120 was not induced by expression of the activated but nonmyristylated src variant pp602A/527F, which is transformation defective. p120 partitioned preferentially with cellular membranes, consistent with the observation that transforming src proteins are membrane associated. Although a number of additional putative substrates were identified and partially characterized with respect to intracellular localization, tyrosine phosphorylation of these proteins was not tightly linked to transformation.     

Tyrosine phosphorylation events during coxsackievirus B3 replication.

In order to study cellular and viral determinants of pathogenicity, interactions between coxsackievirus B3 (CVB3) replication and cellular protein tyrosine phosphorylation were investigated. During CVB3 infection of HeLa cells, distinct proteins become phosphorylated on tyrosine residues, as detected by the use of antiphosphotyrosine Western blotting. Two proteins of 48 and 200 kDa showed enhanced tyrosine phosphorylation 4 to 5 h postinfection (p.i.), although virus-induced inhibition of cellular protein synthesis had already occurred 3 to 4 h p.i. Subcellular fractionation experiments revealed distinct localization of tyrosine-phosphorylated proteins of 48 and 200 kDa in the cytosol and membrane fractions of infected cells, respectively. In addition, in Vero cells infected with CVB3, echovirus (EV)11, or EV12, increased tyrosine phosphorylation of a 200-kDa protein was detected 6 h p.i. Herbimycin A, a specific inhibitor of Src-like protein tyrosine kinases, was shown to inhibit virus-induced tyrosine phosphorylations and to reduce the production of progeny virions. In contrast, in cells treated with the inhibitors staurosporine and calphostin C, the synthesis of progeny virions was not affected. Immunoprecipitation experiments suggested that the tyrosine-phosphorylated 200-kDa protein in CVB3-infected cells is of cellular origin. In summary, these investigations have begun to unravel the effect of CVB3 as well as EV11 and EV12 replication on cellular tyrosine phosphorylation and support the importance of tyrosine phosphorylation events for effective virus replication. Such cellular phosphorylation events triggered in the course of enterovirus infection may enhance virus replication.     

Transactivation of ErbB2 and ErbB3 by tumor necrosis factor-alpha and anisomycin leads to impaired insulin signaling through serine/threonine phosphorylation of IRS proteins.

The cellular pathways involved in the impairment of insulin signaling by cellular stress, triggered by the inflammatory cytokine tumor necrosis factor-alpha (TNF) or by translational inhibitors like cycloheximide and anisomycin were studied. Similar to TNF, cycloheximide and anisomycin stimulated serine phosphorylation of IRS-1 and IRS-2, reduced their ability to interact with the insulin receptor, inhibited the insulin-induced tyrosine phosphorylation of IRS proteins, and diminished their association with phosphatidylinositol 3-kinase (PI3K). These defects were partially reversed by wortmannin and LY294002, indicating that a PI3K-regulated step is critical for the impairment of insulin signaling by cellular stress. Induction of cellular stress resulted in complex formation between PI3K and ErbB2/ErbB3 and enhanced PI3K activity, implicating ErbB proteins as downstream effectors of stress-induced insulin resistance. Indeed, stimulation of ErbB2/ErbB3 by NDFbeta1, the ErbB3 ligand, inhibited IRS protein tyrosine phosphorylation and recruitment of downstream effectors. Specific inhibitors of the ErbB2 tyrosine kinase abrogated the activation of ErbB2/ErbB3 and in parallel prevented the reduction in IRS protein functions. Taken together, our results suggest a novel mechanism by which cellular stress induces cross-talk between two different signaling pathways. Stress-dependent transactivation of ErbB2/ErbB3 receptors triggers a PI3K cascade that induces serine phosphorylation of IRS proteins culminating in insulin resistance.     

Bovine skeletal growth factor stimulates protein phosphorylation of chicken bone cells in vitro

1. Bovine skeletal growth factor (SGF), a potent bone cell mitogen, stimulated protein phosphorylation in cultured chicken calvarial cells. 2. SDS-PAGE followed by autoradiographic analysis of the cellular proteins indicated that [32P] incorporation was enhanced in several proteins in response to 10 ng/ml of SGF (the maximum stimulatory mitogenic dose for these cells). 3. Under conditions favoring tyrosine kinases, SGF stimulated phosphorylation of at least 6 proteins in crude calvarial cell membrane fraction, and caused a time-dependent stimulation of phosphorylation of angiotensin II by crude calvarial cell membrane fractions. 4. Thus, our data demonstrate that SGF stimulates protein phosphorylation in bone cells, and suggest that at least some of the protein phosphorylation involves tyrosine residues.     

Glycoprotein tyrosine phosphorylation in Rous sarcoma virus-transformed chicken embryo fibroblasts.

The level of tyrosine phosphorylation of cellular glycoproteins isolated by wheat germ agglutinin chromatography in cells infected with a variety of kinase-positive/transformation-defective src mutants was examined in an effort to identify cellular membrane proteins whose phosphorylation correlates with phenotypic transformation. We have identified two glycoproteins, with molecular masses of 95 and 135 kilodaltons, whose phosphorylation correlates with morphological transformation, growth in soft agar, and an increase in the rate of 2-deoxyglucose uptake. The strong correlation obtained between transformation and phosphorylation of these proteins suggests that they may be substrates for pp60src which are important in the process of transformation.     

Two-dimensional analysis of interleukin 2-regulated tyrosine kinase activation mediated by the p70-75 beta subunit of the interleukin 2 receptor

The proliferation of activated T lymphocytes is dependent on the interaction of the polypeptide growth factor interleukin 2 (IL 2) with its heterodimeric receptor, which consists of a p55 alpha subunit and a p70-75 beta subunit. Previously, it was shown that IL 2 stimulates rapid serine phosphorylation of several membrane and cytysolic proteins. Here, using anti-phosphotyrosine antibodies to purify phosphotyrosyl proteins and two-dimensional gel analysis, we show that IL 2 stimulates rapid tyrosine phosphorylation of a variety of cellular proteins, including pp180, pp92, and pp42 in activated human T lymphocytes. In addition, we have examined IL 2-induced tyrosine phosphorylation in the human cell line YT2C2 which expresses mostly the beta subunit of the IL 2 receptor and the gibbon cell line MLA-144 which expresses only the beta subunit. In both of these cell lines, IL 2 induced tyrosine phosphorylation of the same proteins phosphorylated in normal human T lymphocytes in response to IL 2. We conclude that the beta subunit is sufficient to induce tyrosine phosphorylation of the normal cellular target substrates involved in signal transduction.     

Tyrosine decaging leads to substantial membrane trafficking during modulation of an inward rectifier potassium channel

Tyrosine side chains participate in several distinct signaling pathways, including phosphorylation and membrane trafficking. A nonsense suppression procedure was used to incorporate a caged tyrosine residue in place of the natural tyrosine at position 242 of the inward rectifier channel Kir2.1 expressed in Xenopus oocytes. When tyrosine kinases were active, flash decaging led both to decreased K(+) currents and also to substantial (15-26%) decreases in capacitance, implying net membrane endocytosis. A dominant negative dynamin mutant completely blocked the decaging-induced endocytosis and partially blocked the decaging-induced K(+) channel inhibition. Thus, decaging of a single tyrosine residue in a single species of membrane protein leads to massive clathrin-mediated endocytosis; in fact, membrane area equivalent to many clathrin-coated vesicles is withdrawn from the oocyte surface for each Kir2.1 channel inhibited. Oocyte membrane proteins were also labeled with the thiol-reactive fluorophore tetramethylrhodamine-5-maleimide, and manipulations that decreased capacitance also decreased surface membrane fluorescence, confirming the net endocytosis. In single-channel studies, tyrosine kinase activation decreased the membrane density of active Kir2.1 channels per patch but did not change channel conductance or open probability, in agreement with the hypothesis that tyrosine phosphorylation results in endocytosis of Kir2.1 channels. Despite the Kir2.1 inhibition and endocytosis stimulated by tyrosine kinase activation, neither Western blotting nor (32)P labeling produced evidence for direct tyrosine phosphorylation of Kir2.1. Therefore, it is likely that tyrosine phosphorylation affects Kir2.1 function indirectly, via interactions between clathrin adaptor proteins and a tyrosine-based sorting motif on Kir2.1 that is revealed by decaging the tyrosine side chain. These interactions inhibit a fraction of the Kir2.1 channels, possibly via direct occlusion of the conduction pathway, and also lead to endocytosis, which further decreases Kir2.1 currents. These data establish that side chain decaging can provide valuable time-resolved data about intracellular signaling systems.     

Receptor tyrosine kinases.

A major process through which environmental information is transmitted into cells is via activation of protein tyrosine kinases. Receptor tyrosine kinases contain extracellular ligand recognition, single membrane spanning, and cytoplasmic protein tyrosine kinase domains. The cytoplasmic kinase core is flanked by regulatory segments, which in some family members are also inserted into the core kinase domain. Ligand binding initiates receptor signaling from the cell surface. Activated receptors autophosphorylate to remove alternate substrate/inhibitory constraints and to provide loci for assembly of proteins that contain SRC homology regions. Information is transmitted and diffused by tyrosine phosphorylation of the assembled proteins and of cellular substrates that include protein kinases with specificity for serine/threonine residues. Signaling, which is strictly ligand-dependent, is attenuated by down-regulation of receptors and by feed-back inhibitory loops that involve receptor phosphorylation by cellular kinases. The tyrosine kinase receptors are essential for normal growth, development, and reparative processes. Mutations that remove normal regulatory constraints on the approximately 290 amino acid kinase core of these large proteins result in constitutive function and cell transformation.     

Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells.

Protein tyrosine phosphorylation has not been considered to be important for cellular activation by phospholipase C-linked vasoactive peptides. We found that endothelin, angiotensin II, and vasopressin (AVP), peptides that signal via phospholipase C activation, rapidly enhanced tyrosine phosphorylation of proteins of approximate molecular mass 225, 190, 135, 120, and 70 kDa in rat renal mesangial cells. The phosphorylated proteins were cytosolic or membrane-associated, and none were integral to the membrane, suggesting that the peptide receptors are not phosphorylated on tyrosine. Epidermal growth factor (EGF), which does not activate phospholipase C in these cells, induced the tyrosine phosphorylation of its own 175-kDa receptor, in addition to five proteins of identical molecular mass to those phosphorylated in response to endothelin, AVP, and angiotensin II. This suggests that in mesangial cells there is a common signaling pathway for phospholipase C-coupled agonists and agonists classically assumed to signal via receptor tyrosine kinase pathways, such as EGF. The phorbol ester, phorbol 12-myristate 13-acetate, and the synthetic diacylglycerol, oleoyl acetylglycerol, stimulated the tyrosine phosphorylation of proteins identical to those phosphorylated by the phospholipase C-linked peptides, suggesting that protein kinase C (PKC) activation is sufficient to active tyrosine phosphorylation. However, the PKC inhibitor, staurosporine, and down-regulation of PKC activity by prolonged exposure to phorbol esters completely inhibited tyrosine phosphorylation in response to PMA but not to endothelin, AVP, or EGF. In conclusion, endothelin, angiotensin II, and AVP enhances protein tyrosine phosphorylation via at least two pathways, PKC-dependent and PKC-independent. Although activation of PKC may be sufficient to enhance protein tyrosine phosphorylation, PKC is not necessary and may not be the primary route by which these agents act. At least one of these pathways is shared with the growth factor EGF, suggesting not only common intermediates in the signaling pathways for growth factors and vasoactive peptides but also perhaps common cellular tyrosine kinases which phosphorylate these intermediates.     

Inhibition of the cellular actions of nerve growth factor by staurosporine and K252A results from the attenuation of the activity of the trk tyrosine kinase.

The protein kinase inhibitors staurosporine and K252A inhibit some of the cellular actions of nerve growth factor (NGF). To explore the molecular mechanisms involved, we test the ability of these agents to block one of the earliest cellular responses to NGF, protein tyrosine phosphorylation. Concentrations of 10-100 nM staurosporine and K252A inhibit NGF-dependent tyrosine phosphorylation in PC12 cells and inhibit trk oncogene-dependent tyrosine phosphorylation in trk-transformed NIH3T3 (trk-3T3 cells). In contrast, these compounds are without effect on epidermal growth factor (EGF)-stimulated tyrosine phosphorylation in PC12 cells. NGF-stimulated tyrosine phosphorylation of the pp140c-trk NGF receptor and tyrosine phosphorylation of pp70trk are also inhibited by similar concentrations of staurosporine and K252A, whereas tyrosine phosphorylation of the EGF receptor, insulin receptor, and v-src is not affected. Both staurosporine and K252A inhibit the autophosphorylation of pp70trk on tyrosine residues in an in vitro immune complex kinase reaction. Incubation of trk-3T3 cells with 10 nM staurosporine causes rounded transformed cells to revert to a normal flattened phenotype, whereas src-transformed cells are unaffected by this agent. These data suggest that staurosporine and K252A specifically inhibit the trk tyrosine kinase activity through a direct mechanism, probably accounting for the attenuation by these agents of the cellular actions of NGF.     

EFFECTS OF 50 Hz MAGNETIC FIELD EXPOSURE ON PROTEIN TYROSINE PHOSPHORYLATION IN CULTURED CELLS

Protein phosphorylation is an extremely important and widely used mechanism of cellular regulation. Here, the effects of 50 Hz magnetic fields (MFs) on tyrosine phosphorylation were studied. A Chinese hamster lung (CHL) cell line was exposed to 50 Hz magnetic fields at two intensities (0.4 mT and 0.8 mT) for different exposure durations, and western blot analysis was used to measure the degree of tyrosine phosphorylation of cellular proteins. Results showed that both 0.4 mT and 0.8 mT 50 Hz magnetic fields could affect the protein tyrosine phosphorylation in cultured cells. Both intensities could affect the tyrosine phosphorylation of 38 and 97.4 kDa proteins. In addition, 0.4 mT could affect tyrosine phosphorylation of 61.7, 105, and 112 kDa proteins, and 0.8 mT affected the tyrosine phosphorylation of 79 and 150 kDa proteins. Moreover, all the tyrosine phosphorylation changes of these proteins were time-dependent. The findings from this study demonstrated that under these experimental conditions, there was evidence that protein tyrosine phosphorylation was a possible process for ELF-EMF producing bioeffects.     

CD98-dependent homotypic aggregation is associated with translocation of protein kinase Cdelta and activation of mitogen-activated protein kinases

CD98 is a protein found on the surface of many activated cell types, and is implicated in the regulation of cellular differentiation, adhesion, growth, and apoptosis. Despite many studies addressing CD98 function, there is little information on the intracellular signalling pathways that mediate its activity. In this study, we examine protein kinase pathways that are activated following ligation by the CD98 antibody AHN-18, an antibody that induces U937 homotypic aggregation and inhibits antigen presenting activity and T-cell activation. Ligation by CD98 antibody AHN-18 induces tyrosine kinase activity, but inhibition of this activity does not affect U937 aggregation. Ligation also induces membrane translocation of the serine/threonine kinase novel PKCdelta, but not other members of the PKC family. Translocation is blocked by rottlerin, and this inhibitor also blocks aggregation. PKCdelta activation in turn mediates activation of ERK1/2 and p38, as well as tyrosine phosphorylation of multiple proteins, and MAPK activation is essential for cellular aggregation. One of the targets of CD98-induced tyrosine phosphorylation is itself PKCdelta, suggesting that this phosphorylation may act as a negative feedback to limit the overall activation of the CD98 pathway.     

Capacitation induces tyrosine phosphorylation of proteins in the boar sperm plasma membrane.

Capacitation (activation) of mammalian spermatozoa is accompanied by protein phosphorylation, elevation of the intracellular calcium concentration and an increased plasma membrane fluidity. The subcellular localization of tyrosine phosphorylation during capacitation have not yet been elucidated. The aim of this study was to investigate whether boar sperm capacitation induces tyrosine phosphorylation of plasma membrane proteins. Capacitation induced tyrosine phosphorylation of 3 proteins (27, 37, and 40 kDa), which coincided with an increase in the plasma membrane fluidity. The importance of the induced tyrosine phosphorylation in sperm binding to the zona pellucida and the induction of the acrosome reaction is discussed.     

Depolarization-Dependent Tyrosine Phosphorylation in Rat Brain Synaptosomes

Synaptosomes from rat forebrain were analyzed for the presence of phosphotyrosine-containing proteins by immunoblotting with antiphosphotyrosine antibodies. Using this technique, 10-11 phosphotyrosine-containing proteins were detected. Depolarization of synaptosomes by transfer to a high (41 mM) K+ medium resulted in increases in the phosphotyrosine content of several synaptosomal proteins, the most pronounced increase being associated with a membrane protein of M(r) 117,000 (ptp117). Additional proteins exhibiting depolarization-dependent increases in phosphotyrosine content had molecular weights of 39,000, 104,000, 135,000, and 160,000. The depolarization-dependent increase in the phosphotyrosine content of ptp117 was apparent within 30 s of the onset of depolarization, reached a maximum between 3 and 5 min, and then decreased to near control values by 30 min. The increase in tyrosine phosphorylation of ptp117 was dependent on the concentration of K+ in the depolarizing medium and was maximal with [K+] in excess of 50 mM. It was also calcium dependent and did not occur in the absence of extracellular calcium. The addition of veratridine to the incubation medium also resulted in an increase in the tyrosine phosphorylation of ptp117. The results suggest that the phosphorylation of synaptic proteins on tyrosine residues may be involved in the regulation or modulation of synaptic activity.     

Association between B-lymphocyte membrane immunoglobulin and multiple members of the Src family of protein tyrosine kinases.

Treatment of B lymphocytes with antibodies to membrane immunoglobulin (Ig) stimulates protein tyrosine phosphorylation. We have examined the phosphorylation in vitro of proteins associated with membrane Ig. The Src family protein tyrosine kinases p53/56lyn, p59fyn, and p56lck are associated with membrane Ig in spleen B cells and B-cell lines and undergo phosphorylation in vitro. The pattern of expression of Src family protein tyrosine kinases in B cells varied. Our studies suggest that multiple kinases can potentially interact with membrane Ig and that within any one B-cell type, all of the Src family kinases expressed can be found in association with membrane Ig. We also observed that the Ig-associated Ig alpha protein, multiple forms of Ig beta, and proteins of 100 and 25 kDa were tyrosine phosphorylated in vitro. The 100- and 25-kDa proteins remain unidentified.     

Evidence for a role of the nerve growth factor receptor TrkA in tyrosine phosphorylation and processing of beta-APP

The cytoplasmic tail of the beta-amyloid precursor protein (APP) contains a Y(682)ENPTY(687) sequence through which APP associates with phosphotyrosine binding (PTB) domain containing proteins in a tyrosine phosphorylation-independent manner. We have recently found that tyrosine phosphorylation of APP-Y(682) promotes docking of Shc proteins that modulate growth factor signaling to the ERK and PI3K/Akt pathways. We have also shown that APP is phosphorylated on Y(682) in cells that overexpress a constitutively active form of the tyrosine kinase abl. Here we present evidence that the nerve growth factor receptor TrkA may also promote phosphorylation of APP. Overexpression of TrkA, but not of mutated, kinase inactive TrkA resulted in tyrosine phosphorylation of APP. Site-directed mutagenesis studies showed that TrkA overexpression was associated with phosphorylation of APP-Y(682). Moreover, overexpression of TrkA also affected APP processing reducing the generation of the APP intracellular domain (AID). Thus, tyrosine phosphorylation of APP may functionally link APP processing and neurotrophic signaling to intracellular pathways associated with cellular differentiation and survival.