Gangliosides influence EGFR/ErbB2 heterodimer stability but they do not modify EGF-dependent ErbB2 phosphorylation

Gangliosides are well-known regulators of cell differentiation through specific interactions with growth factor receptors. Previously, our group provided the first evidence about stable association of ganglioside GM₃ to EGFR/ErbB2 heterodimers in mammary epithelial cells. Goals of the present study were to better define the role of gangliosides in EGFR/ErbB2 heterodimerization and receptor phosphorylation events and to analyze their involvement in mammary cell differentiation. Experiments have been conducted using the ceramide analogue (+/−)-treo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP), which inhibits ceramide glucosyltransferase resulting in the endogenous ganglioside depletion, and the lactogenic hormone mix DIP (dexamethasone, insulin, prolactin), which induces cell differentiation and β-casein mRNA synthesis. In addition, treatments of ganglioside-depleted cells with exogenous GM₃ have been carried out to ascertain the specific involvement of this ganglioside. Results from co-immunoprecipitation and Western blot experiments have shown that the endogenous ganglioside depletion resulted in the disappearance of SDS-stable EGFR/ErbB2 heterodimers and in the appearance of tyrosine-phosphorylated EGFR also in the absence of EGF stimulation; exogenous GM₃ added in combination with [D]-PDMP reversed both these effects. In contrast, the tyrosine phosphorylation of ErbB2 in ganglioside-depleted cells occurred only after EGF stimulation. Moreover, when ganglioside-depleted cells were treated with DIP in absence of EGF, β-casein gene expression appeared strongly down-regulated, and β-casein mRNA levels were partially restored by exogenous GM₃ treatment. Altogether, although the involvement of other ganglioside species cannot be excluded, these findings sustain the ganglioside GM₃ as an essential molecule for EGFR/ErbB2 heterodimer stability and important regulator of EGFR tyrosine phosphorylation, but it is not crucial for tyrosine phosphorylation of the heterodimerization partner ErbB2. Moreover, modulation of EGFR phosphorylation may explain how gangliosides contribute to regulate the lactogenic hormone-induced mammary cell differentiation.     

MUC4 involvement in ErbB2/ErbB3 phosphorylation and signaling in response to airway cell mechanical injury

The receptor tyrosine kinases ErbB2 and ErbB3 are phosphorylated in response to injury of the airway epithelium. Since we have shown that the membrane mucin MUC4 can act as a ligand/modulator for ErbB2, affecting its localization in polarized epithelial cells and its phosphorylation, we questioned whether Muc4 was involved, along with ErbB2 and ErbB3, in the damage response of airway epithelia. To test this hypothesis, we first examined the localization of MUC4 in human airway samples. Both immunocytochemistry and immunofluorescence showed a co‐localization of MUC4 and ErbB2 at the airway luminal surface. Sequential immunoprecipitation and immunoblotting from airway cells demonstrated that the MUC4 and ErbB2 are present as a complex in airway epithelial cells. To assess the participation of MUC4 in the damage response, cultures of NCI‐H292 or airway cells were scratch‐wounded, then analyzed for association of phospho‐ErbB2 and ‐ErbB3 with MUC4 by sequential immunoprecipitation and immunoblotting. Wounded cultures exhibited increased phosphorylation of both receptors in complex with MUC4. Scratch wounding also increased activation of the downstream pathway through Akt, as predicted from our previous studies on Muc4 effects on ErbB2 and ErbB3. The participation of MUC4 in the phosphorylation response was also indicated by siRNA repression of MUC4 expression, which resulted in diminution of the phosphorylation of ErbB2 and ErbB3. These studies provide a new model for the airway epithelial damage response, in which the MUC4–ErbB2 complex is a key element in the sensor mechanism and phosphorylation of the receptors. J. Cell. Biochem. 107: 112–122, 2009. © 2009 Wiley‐Liss, Inc.     

Identification of the domain in ErbB2 that restricts ligand-induced degradation

Ligand-induced receptor degradation is an important process for down-regulation of plasma membrane receptors. While epidermal growth factor receptor (EGFR) is rapidly internalised and degraded upon ligand stimulation, ErbB2, the closest member to EGFR in ErbB receptor family, is resistant in ligand-induced degradation. To understand the molecular mechanisms underlying the impairment in ligand-induced degradation of ErbB2, we attempted to determine structural factor in ErbB2 that restricts the degradation. By analysis of ligand-induced degradation of EGFR/ErbB2 chimeras, we have identified a region between amino acid residues F1030 and L1075 in ErbB2 as the domain that restricts the ligand-induced degradation. We designated this domain as the Blocking ErbB2 Degradation or the BED domain. Replacement of the BED domain in an EGFR/ErbB2 chimera with the corresponding region of EGFR changed this chimera from a non-degradable to a degradable receptor, indicating that the BED domain is the factor restricting the ligand-induced degradation of ErbB2. In addition, we found that a non-degradable EGFR/ErbB2 chimera was not defective in tyrosine phosphorylation, ubiquitination and interaction with c-Cbl, rather, was defective in ligand-induced internalisation, suggesting that the endocytosis defect is the cause restricting the degradation of ErbB2, and that c-Cbl-catalysed mono-ubiquitination is not involved in the impairment in ligand-induced degradation of ErbB2.     

Cardiopulmonary baroreflexes do not modulate exercise-induced sympathoexcitation

The purpose of this study was to test the general hypothesis that sympathoinhibitory cardiopulmonary baroreflexes modulate sympathetic outflow during voluntary exercise in humans. Direct (microneurographic) measurements of postganglionic sympathetic nerve activity to noncontracting muscle (MSNA) were made from the right peroneal nerve in the leg, and arterial pressure (AP) and heart rate (HR) were recorded in 10 healthy subjects before (control) and for 2.5 min during each of five interventions: 1) lower-body negative pressure at -10 mmHg (LBNP) alone, 2 and 3) isometric handgrip exercise at 15 and 30% of maximal voluntary contraction (MVC) alone, and 4 and 5) handgrip at 15 and 30% MVC performed during LBNP. During LBNP alone, which should have reduced cardiopulmonary baroreflex sympathoinhibition, AP and HR did not change from control, but MSNA increased 93 +/- 24% (P less than 0.05). Handgrip elicited contraction intensity-dependent increases in AP and HR (P less than 0.05), but MSNA increased above control only at the 30% MVC level (165 +/- 30%, P less than 0.05). The HR, AP, and MSNA responses to either level of handgrip performed during LBNP were not different from the algebraic sums of the corresponding responses to handgrip and LBNP performed separately (P greater than 0.05). Since there was no facilitation of the MSNA response to handgrip when performed during LBNP compared with algebraic sums of the separate responses, our results do not support the hypothesis that cardiopulmonary baroreflexes modulate (inhibit) sympathetic outflow during exercise in humans.     

Immunoglobulin mu heavy chains do not mediate tyrosine phosphorylation of Ig alpha from the ER-cis-Golgi

Signals delivered by Ig receptors guide the development of functional B lymphocytes. For example, clonal expansion of early mu heavy chain ( mu HC)-positive pre-B cells requires the assembly of a signal-competent pre-B cell receptor complex (pre-BCR) consisting of a mu HC, a surrogate L chain, and the signal dimer Ig alpha beta. However, only a small fraction of the pre-BCR is transported to the cell surface, suggesting that pre-BCR signaling initiates already from an intracellular compartment, e.g., the endoplasmic reticulum (ER). The finding that differentiation of pre-B cells and allelic exclusion at the IgH locus take place in surrogate L chain-deficient mice further supports the presence of a mu HC-mediated intracellular signal pathway. To determine whether a signal-competent Ig complex can already be assembled in the ER, we analyzed the consequence of pervanadate on tyrosine phosphorylation of Ig alpha in J558L plasmacytoma and 38B9 pre-B cells transfected with either a transport-competent IgL chain-pairing or an ER-retained nonpairing micro HC. Flow cytometry, combined Western blot-immunoprecipitation-kinase assays, and confocal microscopy revealed that both the nonpairing and pairing mu HC assembled with the Ig alpha beta dimer; however, in contrast to a pairing mu HC, the nonpairing mu HC was retained in the ER-cis-Golgi compartment, and neither colocalized with the src kinase lyn nor induced tyrosine phosphorylation of Ig alpha after pervanadate treatment of cells. On the basis of these findings, we propose that a signal-competent Ig complex consisting of mu HC, Ig alpha beta, and associated kinases is assembled in a post-ER compartment, thereby supporting the idea that a pre-BCR must be transported to the cell surface to initiate pre-BCR signaling.     

The inhibitory function of CTLA-4 does not require its tyrosine phosphorylation

CTLA-4 is a negative regulator of T cell responses. Sequence analysis of this molecule reveals the presence of two cytoplasmic tyrosine residues at positions 165 and 182 that are potential Src homology (SH)-2 domain binding sites. The role of phosphorylation of these residues in CTLA-4-mediated signaling is unknown. Here, we show that sole TCR ligation induces zeta-associated protein (ZAP)-70-dependent tyrosine phosphorylation of CTLA-4 that is important for cell surface retention of this molecule. However, CTLA-4 tyrosine phosphorylation is not required for down-regulation of T cell activation following CD3-CTLA-4 coengagement. Specifically, inhibition of extracellular signal-regulated kinase (ERK) activation and of IL-2 production by CTLA-4-mediated signaling occurs in T cells expressing mutant CTLA-4 molecules lacking the cytoplasmic tyrosine residues, and in lck-deficient or ZAP-70-deficient T cells. Therefore, CTLA-4 function involves interplay between two different levels of regulation: phosphotyrosine-dependent cell surface retention and phosphotyrosine-independent association with signaling molecules.     

Recycling of EGFR and ErbB2 is associated with impaired Hrs tyrosine phosphorylation and decreased deubiquitination by AMSH

ErbB receptors play an important role in normal cellular growth, differentiation and development, but overexpression or poor downregulation can result in enhanced signaling and cancerous growth. ErbB signaling is terminated by clathrin-dependent receptor-mediated endocytosis, followed by incorporation in multi-vesicular bodies and subsequent degradation in lysosomes. In contrast to EGFR, ErbB2 displays poor ligand-induced downregulation and enhanced recycling, but the molecular mechanisms underlying this difference are poorly understood. Given our previous observation that both EGFR and an EGFR-ErbB2 chimera undergo Cbl-mediated K63-polyubiquitination, we investigated in the present study whether activation of the EGFR and the EGFR-ErbB2 chimera is associated with tyrosine phosphorylation of the ESCRT-0 complex subunit Hrs and AMSH-mediated deubiquitination. EGF stimulation of the EGFR resulted in efficient Hrs tyrosine phosphorylation and deubiquitination by the K63-polyubiquitin chain-specific deubiquitinating enzyme AMSH. In contrast, EGF activation of EGFR-ErbB2 showed significantly decreased Hrs tyrosine phosphorylation and deubiquitination by AMSH. To test whether this phenotype is the result of endosomal recycling, we induced recycling of the EGFR by stimulation with TGFα. Indeed, even though TGFα-stimulation of EGFR is associated with efficient ligand-stimulated K63-polyubiquitination, we observed that Hrs tyrosine phosphorylation as well as AMSH-mediated deubiquitination is significantly reduced under these conditions. Using various EGFR-ErbB2 chimeras, we demonstrate that enhanced recycling, decreased Hrs tyrosine phosphorylation and decreased AMSH mediated deubiquitination of EGFR-ErbB2 chimeras is primarily due to the presence of ErbB2 sequences or the absence of EGFR sequences C-terminal to the Cbl binding site. We conclude that endosomal recycling of the EGFR and ErbB2 receptors is associated with significantly impaired tyrosine phosphorylation of the ESCRT-0 subunit Hrs as well as decreased deubiquitination by AMSH, which is consistent with the finding that recycling receptors are not efficiently incorporated in the MVB pathway.     

Hyposmolarity-induced ErbB4 phosphorylation and its influence on the non-receptor tyrosine kinase network response in cultured cerebellar granule neurons

Exposure of cultured cerebellar granule neurons (24 h serum-starved) during 3 min to 30% hyposmotic medium activated the tyrosine kinase receptor ErbB4 in the absence of its ligand. Hyposmolarity also activated the non-receptor tyrosine kinases, Src, focal adhesion kinase (FAK), extracellular signal-regulated protein kinase (ERK)1/2, and the tyrosine kinase target phosphatidyl-inositol-3-kinase (PI3K). The hyposmotic-induced activation of these kinases required the prior phosphorylation of ErbB4 as shown by the effect of ErbB4 blockade with AG213 reducing by 85-95% the phosphorylation of FAK and ERK1/2, by 74% and 36% that of PI3K and Src, respectively. These results suggest a key role of ErbB4 as a signal integrator of events associated with hyposmolarity. PI3K seems to be an important connecting element in the signaling network evoked by the hyposmolarity/ErbB4 activation as: (i) the p85 regulatory subunit of PI3K co-immunoprecipitates with ErbB4 and with FAK; (ii) PI3K blockade with wortmannin reduced the hyposmotic activation of FAK (90%) and ERK1/2 (84-91%). Inhibition of Src with PP2 reduced ErbB4 phosphorylation and inhibited the subsequent cytosolic kinase activation with the same potency as ErbB4 blockade. These results point to Src and ErbB4 and as early targets of the hyposmotic stimulus and osmosignaling. The functional significance for cell volume regulation of the ErbB4-Src-PI3K signaling cascade is indicated by the 48-66% decrease of the hyposmotic taurine efflux observed by inhibition of these kinases.     

Src-dependent tyrosine phosphorylation regulates dynamin self-assembly and ligand-induced endocytosis of the epidermal growth factor receptor

Endocytosis of ligand-activated receptors requires dynamin-mediated GTP hydrolysis, which is regulated by dynamin self-assembly. Here, we demonstrate that phosphorylation of dynamin I by c-Src induces its self-assembly and increases its GTPase activity. Electron microscopic analyses reveal that tyrosine-phosphorylated dynamin I spontaneously self-assembles into large stacks of rings. Tyrosine 597 was identified as being phosphorylated both in vitro and in cultured cells following epidermal growth factor receptor stimulation. The replacement of tyrosine 597 with phenylalanine impairs Src kinase-induced dynamin I self-assembly and GTPase activity in vitro. Expression of Y597F dynamin I in cells attenuates agonist-driven epidermal growth factor receptor internalization. Thus, c-Src-mediated tyrosine phosphorylation is required for the function of dynamin in ligand-induced signaling receptor internalization.     

Tyrosine phosphorylation of ErbB4 is enhanced by PSD95 and repressed by protein tyrosine phosphatase receptor type Z

Protein tyrosine phosphatase receptor type Z (Ptprz/PTPzeta/RPTPbeta) is a receptor-like protein tyrosine phosphatase (RPTP) preferentially expressed in the brain. ErbB4 is a member of the ErbB-family tyrosine kinases known as a neuregulin (NRG) receptor. Both are known to bind to postsynaptic density-95 (PSD95) on the second and the first/second PDZ (PSD95/Disc large/zona occludens1) domains, respectively, through the PDZ-binding motif of their carboxyl termini. Here we report a functional interaction between Ptprz and ErbB4. An intracellular carboxyl-terminal region of Ptprz pulled-down PSD95 and ErbB4 from an adult rat synaptosomal preparation. ErbB4 and Ptprz showed co-localization in cell bodies and apical dendrites of neurons in the prefrontal cortex. In HEK293T cells, phosphorylation of ErbB4 was raised by co-expression of PSD95, which was repressed by additional expression of Ptprz. In vitro experiments using the whole intracellular region (ICR) of ErbB4 also showed that PSD95 stimulates the autophosphorylation of ErbB4, and that the ICR of Ptprz dephosphorylates ErbB4 independent of the presence of PSD95. Taken together with the finding that the tyrosine phosphorylation level of ErbB4 was increased in Ptprz-deficient mice, these results suggest that Ptprz has a role in suppressing the autoactivation of ErbB4 by PSD95 at the postsynaptic density in the adult brain.     

Thromboxane A2 and prostacyclin do not modulate pulmonary hemodynamics during exercise in sheep

The purpose of this study was to determine the role of thromboxane and prostacyclin in modulating pulmonary hemodynamics during maximal cardiopulmonary stress in the healthy lung. We studied 11 yearling sheep in paired studies during progressive maximal treadmill exercise with and without meclofenamate (n = 5), ibuprofen (n = 6), or UK38485 (n = 2). We also studied five sheep during hypoxia and hypoxic exercise, and six sheep during prolonged steady-state treadmill exercise for 45-60 min with and without drug treatment. We measured the metabolites of thromboxane A2 (thromboxane B2, TxB2) and prostacyclin (6-ketoprostaglandin F1 alpha, 6-keto-PGF1 alpha) in blood plasma and lung lymph in each protocol. We found that progressive exercise significantly reduced pulmonary vascular resistance but that cyclooxygenase or thromboxane synthesis blockade did not alter the change. Plasma TxB2 rose minimally but significantly during maximal exercise, but 6-keto-PGF1 alpha did not change. During continuous hypoxia, exercise reduced pulmonary vascular resistance nearly to base-line levels, but the degree of reduction was also unchanged by drug treatment. There were also no significant changes in lymph or plasma TxB2 or 6-keto-PGF1 alpha during 45-60 min of continuous moderate exercise. We conclude that neither TxB2 nor prostacyclin modulate pulmonary hemodynamics in the normal lung during maximal exercise, prolonged moderate exercise, or exercise-induced reductions in vascular resistance during hypoxia.     

Constitutive loss and acute pharmacological manipulation of ErbB4 signaling do not affect attention and inhibitory control in mice

The receptor tyrosine kinase ErbB4 and its ligand trophic factors of the neuregulin (NRG) family have been associated with schizophrenia and other mental disorders in human genetic studies. In vivo studies in mice have shown how abnormal Nrg–ErbB4 signaling leads to deviant behaviors relevant to distinct aspects of schizophrenia, including hyperactivity, sensory gating deficits, working and spatial memory deficits and impaired social behavior. However, so far little is known on the role of ErbB4 in attention and inhibitory control, two aspects of executive functions that are impaired in schizophrenia. Here we investigated the effects of constitutive loss of ErbB4 in the central nervous system of mice on performance in a 5‐choice serial reaction time task (5CSRTT) assessing attention and inhibitory control. In this task, ErbB4^?/? mice did not show deficits in various parameters of attention, and premature responses as measure of inhibitory control. Nonetheless, ErbB4^?/? mice recapitulated a specific set of behavioral phenotypes associated with schizophrenia, including a deficit in spatial learning and memory in the Barnes Maze and in contextual fear learning, and a trend for a deficit in sensorimotor gating. Furthermore, we investigated the effect of acute pharmacological inhibition of ErbB tyrosine kinase receptor using the pan‐ErbB kinase inhibitor JNJ‐28871063 (JNJ), in an automated version of the 5CSRTT. JNJ did not affect attention and inhibitory control. In conclusion, our data suggest no direct involvement of a classical Nrg‐ErbB4 pathway in attention and inhibitory control in mice, while it confirms the involvement of this pathway in other domains relevant to schizophrenia.     

Ligand-independent regulation of ErbB4 receptor phosphorylation by activated Ras

The ErbB family of receptor tyrosine kinases regulates cell growth, differentiation and survival. Activation of the receptors is induced by specific growth factors in an autocrine, paracrine or juxtacrine manner. The activated ErbB receptors turn on a large variety of signaling cascades, including the prominent Ras-dependent signaling pathways. The activated Ras can induce secretion of growth factors such as EGF and neuregulin, which activate their respective receptors. In the present study, we demonstrate for the first time that activated Ras can activate ErbB4 receptor in a ligand-independent manner. Expression of constitutively active H-Ras(12V), K-Ras(12V) or N-Ras(13V) in PC12-ErbB4 cells induced ErbB4-receptor phosphorylation, indicating that each of the most abundant Ras isoforms can induce receptor activation. NRG-induced phosphorylation of ErbB4 receptor was blocked by the soluble ErbB4 receptor, which had no effect on the Ras-induced receptor phosphorylation. Moreover, conditioned medium from H-Ras(12V)-transfected PC12-ErbB4 cells had no effect on receptor phosphorylation. It thus indicates that Ras induces ErbB4 phosphorylation in a ligand-independent manner. Each of the Ras effector domain mutants, H-Ras(12V)S35, H-Ras(12V)C40, and H-Ras(12V)G37, which respectively activate Raf1, PI3K, and RalGEF, induced a small but significant receptor phosphorylation. The PI3K inhibitor LY294002 and the MEK inhibitor PD98059 caused a partial inhibition of the Ras-induced ErbB4 receptor phosphorylation. Using a mutant ErbB4 receptor, which lacks kinase activity, we demonstrated that the Ras-mediated ErbB4 phosphorylation depends on the kinase activity of the receptor and facilitates ligand-independent neurite outgrowth in PC12-ErbB4 cells. These experiments demonstrate a novel mechanism controlling ErbB receptor activation. Ras induces ErbB4 receptor phosphorylation in a non-autocrine manner and this activation depends on multiple Ras effector pathways and on ErbB4 kinase activity.     

The transmembrane domains of ErbB receptors do not dimerize strongly in micelles

The epidermal growth factor receptors (erbB) constitute an important class of single pass transmembrane receptors involved in the transduction of signals important for cell proliferation and differentiation. Receptor association is a key event in the signal transduction process, but the molecular basis of this interaction is not fully understood. Previous biochemical and genetic studies have suggested that the single transmembrane helices of these receptor proteins might play a role in stabilizing the receptor complexes. To determine if the erbB transmembrane domains could provide a driving force to stabilize the receptor dimers, we carried out a thermodynamic study of these domains expressed as C-terminal fusion proteins with staphylococcal nuclease. Similar fusion constructs have been used successfully to investigate the oligomerization and association thermodynamics of a number of transmembrane sequences, including that of glycophorin A. Using SDS-PAGE analysis and sedimentation equilibrium analytical ultracentrifugation, we do not find strong, specific homo or hetero-interactions between the transmembrane domains of the erbB receptors in micellar solutions. Our results indicate that any preferential interactions between these domains in micellar solutions are extremely modest, of the order of 1 kcal mol(-1) or less. We applied a thermodynamic formalism to assess the effect of weakly interacting TM segments on the behavior of a covalently attached soluble domain. In the case of the ligand-bound EGFR ectodomain, we find that restriction of the ectodomain to the micellar phase by a hydrophobic TM, even in the absence of strong specific interactions, is largely sufficient to account for the previously reported increase in dimerization affinity.     

Lysophospholipids do not directly modulate Na+-H+ exchange

Lysophosphatidylcholine (LPC) has been reported to stimulate Na⁺-H⁺ exchange in rat cardiomyocytes. This action may be important in pathological conditions like ischemic injury where LPC is generated and Na⁺-H⁺ exchange activation is an important determinant of cardiac damage and dysfunction. It is unclear, however, if this stimulation of Na⁺-H⁺ exchange by LPC occurs through a direct action on the exchanger or through stimulation of a second messenger pathway. The purpose of the present investigation was to determine if lysolipids could directly affect Na⁺-H⁺ exchange. Purified cardiac sarcolemmal membranes were isolated and Na⁺-H⁺ exchange was measured by radioisotopic methods following addition of LPC. There were no effects of LPC on Na⁺-H⁺ exchange at LPC concentrations of 100 M at all reaction times examined. Lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI) and lysoplasmenylcholine (LP_EC) also did not alter Na⁺-H⁺ exchange at all concentrations and reaction times examined. We conclude that any stimulatory effects of lysolipids on Na⁺-H⁺ exchange do not occur through a direct action on the exchanger or its membrane lipid environment and must occur through a second messenger pathway.     

Oxidized LDL and 4-hydroxynonenal modulate tyrosine kinase receptor activity

Among the diverse risk factors involved in atherosclerosis, LDL are thought to become atherogenic after undergoing oxidative modifications, characterized by oxidized lipid formation and structural alterations of apoB. Oxidized LDL alter various signaling pathways and exhibit a broad range of biological responses including inflammation, gene expression, cell proliferation or apoptosis. The biological effects of oxidized LDL are related to the presence of peroxidation products such as hydroperoxides, lysophosphatidylcholines, oxysterols and aldehydes.4-Hydroxynonenal (HNE) is one of the most abundant aldehydes formed during the oxidation of polyunsaturated fatty acids in LDL and in membranes. It is able to react with thiols and free amino group residues of proteins. HNE is involved in apoB modifications that alter LDL metabolism and cell protein-adduct formation which may mediate in part the biological effects of oxidized LDL. We report here that HNE delivered to cells by oxidized LDL reacts with cellular proteins, for instance with tyrosine kinase receptors (RTK) such as EGFR and PDGFR. HNE induces in vitro derivatization and tyrosine phosphorylation of RTK (the fine molecular mechanism and conformational changes remain to be elucidated). In intact living cells, oxidized LDL (and pure HNE) trigger HNE-adduct formation and activation of PDGFR and EGFR, through an antioxidant-insensitive and reactive oxygen species independent mechanism. The presence of HNE-PDGFR adducts in atherosclerotic areas lead one to hypothesize that oxidized lipids may also react in vivo with membrane RTK, thereby disturbing their cellular functions.     

The mucin Muc4 potentiates neuregulin signaling by increasing the cell-surface populations of ErbB2 and ErbB3

Mucins provide a protective barrier for epithelial surfaces, and their overexpression in tumors has been implicated in malignancy. We have previously demonstrated that Muc4, a transmembrane mucin that promotes tumor growth and metastasis, physically interacts with the ErbB2 receptor tyrosine kinase and augments receptor tyrosine phosphorylation in response to the neuregulin-1beta (NRG1beta) growth factor. In the present study we demonstrate that Muc4 expression in A375 human melanoma cells, as well as MCF7 and T47D human breast cancer cells, enhances NRG1beta signaling through the phosphatidylinositol 3-kinase pathway. In examining the mechanism underlying Muc4-potentiated ErbB2 signaling, we found that Muc4 expression markedly augments NRG1beta binding to A375 cells without altering the total quantity of receptors expressed by the cells. Cell-surface protein biotinylation experiments and immunofluorescence studies suggest that Muc4 induces the relocalization of the ErbB2 and ErbB3 receptors from intracellular compartments to the plasma membrane. Moreover, Muc4 interferes with the accumulation of surface receptors within internal compartments following NRG1beta treatment by suppressing the efficiency of receptor internalization. These observations suggest that transmembrane mucins can modulate receptor tyrosine kinase signaling by influencing receptor localization and trafficking and contribute to our understanding of the mechanisms by which mucins contribute to tumor growth and progression.     

Silencing of ErbB3/ErbB2 Signaling by Immunoglobulin-like Necl-2

ErbB2 and ErbB3, members of the EGF receptor/ErbB family, form a heterodimer upon binding of a ligand, inducing the activation of Rac small G protein and Akt protein kinase for cell movement and survival, respectively. The enhanced ErbB3/ErbB2 signaling causes tumorigenesis, invasion, and metastasis. We found here that the ErbB3/ErbB2 signaling is regulated by immunoglobulin-like Necl-2, which is down-regulated in various cancer cells and serves as a tumor suppressor. The extracellular region of ErbB3, but not ErbB2, interacted in cis with that of Necl-2. This interaction reduced the ligand-induced, ErbB2-catalyzed tyrosine phosphorylation of ErbB3 and inhibited the consequent ErbB3-mediated activation of Rac and Akt, resulting in the inhibition of cancer cell movement and survival. These inhibitory effects of Necl-2 were mediated by the protein-tyrosine phosphatase PTPN13 which interacted with the cytoplasmic tail of Necl-2. We describe here this novel mechanism for silencing of the ErbB3/ErbB2 signaling by Necl-2.ErbB2 and ErbB3 are members of the EGF receptor/ErbB family, which has ErbB1 and ErbB4 as additional members (1). ErbB2 and ErbB3 are also known as HER2/Neu and HER3, respectively. No ligands binding directly to ErbB2 have been identified yet, whereas heregulin (HRG)³-α and -β, also known as neuregulin-1 and -2, respectively, directly bind to ErbB3. ErbB2 and ErbB3 have kinase domains in their cytoplasmic tails, but that of ErbB3 lacks kinase activity. Therefore, the homodimer of ErbB3 formed by binding of HRG does not transduce any intracellular signaling. By contrast, ErbB2 heterophilically interacts in cis with HRG-occupied ErbB3 and phosphorylates nine tyrosine residues of ErbB3, causing recruitment and activation of the p85 subunit of phosphoinositide 3-kinase (PI3K) and the subsequent activation of Rac small G protein and Akt protein kinase (2). The activation of Rac enhances cell movement and that of Akt prevents cell apoptosis (3).ErbB2 serves as an oncogenic protein (4), and amplification of the ErbB2 gene is observed in many types of cancers. For instance, it is amplified in ∼3% of lung cancers, ∼30% of breast cancers, ∼20% of gastric cancers, and ∼60% of ovarian cancers (5). Moreover, mutation of the ErbB2 gene is found in many types of cancers, namely, ∼10% of lung cancers, ∼4% of breast cancers, ∼5% of gastric cancers, and ∼3% of colorectal cancers (6). This gene amplification or mutation causes enhanced signaling for cell movement and survival, eventually resulting in tumorigenesis, invasiveness, and metastasis. On the basis of these properties of ErbB2, it has been recognized as a good target for cancer therapy; indeed, ErbB2-targeting drugs have already been developed and used clinically (7, 8). However, it remains unknown whether ErbB2 is involved in oncogenesis in cancers in which its gene is not amplified or mutated. In addition, it was recently reported that overexpression of ErbB3 is also involved in tumor malignancy (9), but it remains unknown how ErbB3 serves as an oncogenic protein in cancers in which it is not overexpressed.The nectin-like molecule (Necl) family consists of five members, Necl-1, Necl-2, Necl-3, Necl-4, and Necl-5, and comprises a superfamily with the nectin family, which consists of four members, nectin-1, nectin-2, nectin-3, and nectin-4 (10). All members of this superfamily have similar domain structures: they have one extracellular region with three Ig-like loops, one transmembrane segment, and one cytoplasmic tail. We recently found that the extracellular region of Necl-5 directly interacts in cis with that of the platelet-derived growth factor (PDGF) receptor and that this interaction enhances the PDGF-induced cell proliferation and movement (11–14). Necl-5 is up-regulated in many types of cancer cells and causes at least partly enhanced movement and proliferation of cancer cells (11, 12). These earlier findings prompted us to study the potential interaction of other Necls with other growth factor receptors. Consequently, we found here that the extracellular region of Necl-2 directly interacts in cis with that of ErbB3, but not ErbB2, and reduces the HRG-induced signaling pathways of the ErbB3/ErbB2 heterodimer for cell movement and survival.Necl-2 is known by many names: IgSF4a, RA175, SgIGSF, TSLC1, and SynCAM1 (15–19). Necl-2 was directly reported in GenBank^TM in 1998; IgSF4a was identified as a candidate for a tumor suppressor gene in the loss of heterozygosity region of chromosome 11q23.2 (16); RA175 was identified as a gene highly expressed during the neuronal differentiation of embryonic carcinoma cells (19); SgIGSF was identified as a gene expressed in spermatogenic cells during the early stages of spermatogenesis (18); TSLC1 was identified as a tumor suppressor in human non-small cell lung cancer (17); and SynCAM1 was identified as a brain-specific synaptic adhesion molecule (15). In this study, we use the name “Necl-2,” because it was first reported.Necl-2 shows Ca²⁺-independent homophilic cell-cell adhesion activity and Ca²⁺-independent heterophilic cell-cell adhesion activity with other members of the nectin and Necl families, Necl-1 and nectin-3, and another Ig-like molecule with two Ig-like loops, CRTAM (20–22). These cell-cell adhesion activities are mediated by their extracellular regions. Necl-2 is associated with many peripheral membrane proteins through its cytoplasmic tail. The juxtamembrane region of the cytoplasmic tail contains a band 4.1-binding motif and binds the tumor suppressor DAL-1, a band 4.1 family member, which connects Necl-2 to the actin cytoskeleton (23). In addition, the cytoplasmic tail contains a PDZ domain-binding motif in its C-terminal region and binds Pals2, Dlg3/MPP3, and CASK, which are MAGUK subfamily members having an L27 domain (15, 20, 24, 25). However, the exact roles of the binding of these molecules to Necl-2 remain unknown.Necl-2 is widely expressed in various tissues and organs, and abundantly expressed in epithelial cells (20, 26). Its expression is down-regulated in many types of cancer cells owing to hypermethylation of the Necl-2 gene promoter and/or loss of heterozygosity of 11q23.2 (26). Its expression is also undetectable in fibroblasts, such as NIH3T3, Swiss3T3, and L cells (20). Necl-2 has been shown to be a tumor suppressor in human non-small cell lung cancer (17), but it remains unknown how it fulfills this role. The relationship between Necl-2 and the ErbB family remains unknown, either. In addition, the heterophilic interaction of Necl-2 with CRTAM enhances the cytotoxicity of NK cells and the secretion of γ-interferon from CD8⁺ T cells to attack the Necl-2-expressing cells (22, 27). Studies using Necl-2-deficient mice have revealed that Necl-2 in Sertoli cells is an important cell adhesion molecule for Sertoli-spermatid junctions during spermatogenesis (28–30). In the seminiferous tubules of Necl-2-deficient mice, round and elongating spermatids with a distorted shape are generated owing to failure of contact with Sertoli cells, resulting in male-specific infertility. In the present study, we focused on the role of Necl-2 as a tumor suppressor and clarified its mode of action.     

ErbB2 is necessary for induction of carcinoma cell invasion by ErbB family receptor tyrosine kinases

The epidermal growth factor (EGF) family of tyrosine kinase receptors (ErbB1, -2, -3, and -4) and their ligands are involved in cell differentiation, proliferation, migration, and carcinogenesis. However, it has proven difficult to link a given ErbB receptor to a specific biological process since most cells express multiple ErbB members that heterodimerize, leading to receptor cross-activation. In this study, we utilize carcinoma cells depleted of ErbB2, but not other ErbB receptor members, to specifically examine the role of ErbB2 in carcinoma cell migration and invasion. Cells stimulated with EGF-related peptides show increased invasion of the extracellular matrix, whereas cells devoid of functional ErbB2 receptors do not. ErbB2 facilitates cell invasion through extracellular regulated kinase (ERK) activation and coupling of the adaptor proteins, p130CAS and c-CrkII, which regulate the actin-myosin cytoskeleton of migratory cells. Overexpression of ErbB2 in cells devoid of other ErbB receptor members is sufficient to promote ERK activation and CAS/Crk coupling, leading to cell migration. Thus, ErbB2 serves as a critical component that couples ErbB receptor tyrosine kinases to the migration/invasion machinery of carcinoma cells.     

Constitutively active ErbB4 and ErbB2 mutants exhibit distinct biological activities.

ErbB4 is a member of the epidermal growth factor receptor(EGFR) family of tyrosine kinases, which includes EGFR/ErbB1, ErbB2/HER2/Neu, and ErbB3/HER3. These receptors play important roles both in normal development and in neoplasia. For example, deregulated signaling by ErbB1 and ErbB2 is observed in many human malignancies. In contrast, the roles that ErbB4 plays in tumorigenesis and normal biological processes have not been clearly defined. To identify the biological responses that are coupled to ErbB4, we have constructed three constitutively active ErbB4 mutants. Unlike a constitutively active ErbB2 mutant, the ErbB4 mutants are not coupled to increased cell proliferation, loss of contact inhibition, or anchorage independence in a rodent fibroblast cell line. This suggests that ErbB2 and ErbB4 may play distinct roles in tumorigenesis in vivo.