Association with membrane protrusions makes ErbB2 an internalization-resistant receptor

In contrast to the epidermal growth factor (EGF) receptor, ErbB2 is known to remain at the plasma membrane after ligand binding and dimerization. However, why ErbB2 is not efficiently down-regulated has remained elusive. Basically, two possibilities exist: ErbB2 is internalization resistant or it is efficiently recycled after internalization. By a combination of confocal microscopy, immunogold labeling electron microscopy, and biochemical techniques we show that ErbB2 is preferentially associated with membrane protrusions. Moreover, it is efficiently excluded from clathrin-coated pits and is not seen in transferrin receptor-containing endosomes. This pattern is not changed after binding of EGF, heregulin, or herceptin. The exclusion from coated pits is so pronounced that it cannot just be explained by lack of an internalization signal. Although ErbB2 is a raft-associated protein, the localization of ErbB2 to protrusions is not a result of raft binding. Also, an intact actin cytoskeleton is not required for keeping ErbB2 away from coated pits. However, after efficient cross-linking, ErbB2 is removed from protrusions to occur on the bulk membrane, in coated pits, and in endosomes. These data show that ErbB2 is a remarkably internalization-resistant receptor and suggest that the mechanism underlying the firm association of ErbB2 with protrusions also is the reason for this resistance.     

Heregulin-mediated ErbB2-ERK signaling activates hyaluronan synthases leading to CD44-dependent ovarian tumor cell growth and migration

Heregulin (HRG)-induced cell responses are mediated by the ErbB family of tyrosine kinase receptors. In this study we have investigated HRG activation of ErbB2, extracellular signal-regulated kinase (ERK) signaling, and their role in regulating hyaluronan synthase (HAS) activity in human ovarian tumor cells (SK-OV-3.ipl cells). Immunological and biochemical analyses indicate that ErbB2, ErbB3, and ErbB4 are all expressed in SK-OV-3.ipl cells and that ErbB4 (but not ErbB3) is physically linked to ErbB2 following HRG stimulation. Furthermore, our data indicate that the HRG-induced ErbB2.ErbB4 complexes stimulate ErbB2 tyrosine kinase, which induces both ERK phosphorylation and kinase activity. The activated ERK then increases the phosphorylation of HAS1, HAS2, and HAS3. Consequently, all three HAS isozymes are activated resulting in hyaluronan (HA) production. Because HRG-mediated HAS isozyme phosphorylation/activation can be effectively blocked by either AG825 (an ErbB2 inhibitor) or thiazolidinedione compound (an ERK blocker), we conclude that ErbB2-ERK signaling and HAS isozyme phosphorylation/HA production are functionally coupled in SK-OV-3.ipl cells. HRG also promotes HA- and CD44-dependent oncogenic events (e.g. CD44-Cdc42 association, p21-activated kinase 1 activation, and p21-activated kinase 1-filamin complex formation) and tumor cell-specific behaviors in an ErbB2-ERK signaling-dependent manner. Finally, we have found that the down-regulation of HAS isozyme expression (by transfecting cells with HAS1/HAS2/HAS3-specific small interfering RNAs) not only inhibits HRG-mediated HAS phosphorylation/activation and HA production but also impairs CD44-specific Cdc42-PAK1/filamin signaling, cytoskeleton activation and tumor cell behaviors. Taken together, these findings clearly indicate that HRG activation of ErbB2-ERK signaling modulates HAS phosphorylation/activation and HA production leading to CD44-mediated oncogenic events and ovarian cancer progression.     

Disruption of spectrin-like cytoskeleton in differentiating keratinocytes by PKCδ activation is associated with phosphorylated adducin

Spectrin is a central component of the cytoskeletal protein network in a variety of erythroid and non-erythroid cells. In keratinocytes, this protein has been shown to be pericytoplasmic and plasma membrane associated, but its characteristics and function have not been established in these cells. Here we demonstrate that spectrin increases dramatically in amount and is assembled into the cytoskeleton during differentiation in mouse and human keratinocytes. The spectrin-like cytoskeleton was predominantly organized in the granular and cornified layers of the epidermis and disrupted by actin filament inhibitors, but not by anti-mitotic drugs. When the cytoskeleton was disrupted PKCδ was activated by phosphorylation on Thr505. Specific inhibition of PKCδ(Thr505) activation with rottlerin prevented disruption of the spectrin-like cytoskeleton and the associated morphological changes that accompany differentiation. Rottlerin also inhibited specific phosphorylation of the PKCδ substrate adducin, a cytoskeletal protein. Furthermore, knock-down of endogenous adducin affected not only expression of adducin, but also spectrin and PKCδ, and severely disrupted organization of the spectrin-like cytoskeleton and cytoskeletal distribution of both adducin and PKCδ. These results demonstrate that organization of a spectrin-like cytoskeleton is associated with keratinocytes differentiation, and disruption of this cytoskeleton is mediated by either PKCδ(Thr505) phosphorylation associated with phosphorylated adducin or due to reduction of endogenous adducin, which normally connects and stabilizes the spectrin-actin complex.     

Autocrine extracellular signal-regulated kinase (ERK) activation in normal human keratinocytes: metalloproteinase-mediated release of amphiregulin triggers signaling from ErbB1 to ERK

ErbB signaling through extracellular signal-regulated kinase (ERK) has been implicated in regulating the expression of ErbB ligands in hyperproliferative skin disorders and wound healing. Here, we characterize the process of autocrine ERK activation in cultured normal human keratinocytes (NHKs) subjected to growth factor (GF) deprivation. Basal ERK phosphorylation was lower after 48 h than after 24 h of GF deprivation, and lowest at 30-60 min after an additional medium change. ERK phosphorylation was markedly increased by low concentrations of epidermal growth factor (EGF) (0.2-1 ng/ml) that provoked only a limited increase in ErbB1 tyrosine phosphorylation and internalization. Basal ErbB tyrosine phosphorylation and ERK phosphorylation were inhibited by two different ErbB receptor tyrosine kinase inhibitors, by the ErbB1-specific neutralizing monoclonal antibody 225 IgG, by two different metalloproteinase inhibitors, and by neutralizing antibodies against amphiregulin (AR). In contrast, these responses were unaffected by neutralizing antibodies against other ErbB1 ligands or the ErbB2 inhibitors geldanamycin and AG825. The time course of autocrine ERK phosphorylation correlated with the appearance of soluble AR, and two different metalloproteinase inhibitors blocked AR release. These results define an amphiregulin- and ErbB1-dependent mechanism by which autocrine ERK activation is maintained in NHKs, even when ErbB1 autophosphorylation and internalization are limited.     

Heregulin-dependent activation of phosphoinositide 3-kinase and Akt via the ErbB2/ErbB3 co-receptor

The ErbB2/ErbB3 heregulin co-receptor has been shown to couple to phosphoinositide (PI) 3-kinase in a heregulin-dependent manner. The recruitment and activation of PI 3-kinase by this co-receptor is presumed to occur via its interaction with phosphorylated Tyr-Xaa-Xaa-Met (YXXM) motifs occurring in the ErbB3 C terminus. In this study, mutant ErbB3 receptor proteins expressed in COS7 cells were used to investigate PI 3-kinase-dependent signaling pathways activated by the ErbB2/ErbB3 co-receptor. We observed that a mutant ErbB3 protein with each of its six YXXM motifs containing a Tyr --> Phe substitution was unable to bind either the p85 regulatory or p110 catalytic subunit of PI 3-kinase. However, restoration of a single YXXM motif was sufficient to mediate association with the PI 3-kinase holoenzyme, although at a lower level than wild-type ErbB3. When ErbB3 YXXM motifs were restored in pairs, evidence for cooperativity between two, those incorporating Tyr-1273 and Tyr-1286, was observed. Interestingly, we have shown that an apparent association of PI 3-kinase activity with ErbB2/Neu was due to the residual presence of ErbB3 in ErbB2 immunoprecipitates. The necessity of ErbB3 association with PI 3-kinase for downstream signaling to the effector kinase Akt was also investigated. Here, the heregulin-dependent translocation of Akt to the plasma membrane and its subsequent activation was observed in intact NIH-3T3 fibroblasts. Recruitment of PI 3-kinase to ErbB3 was required for both activities, and it appeared that ErbB2 activation alone was not sufficient to activate PI 3-kinase signaling in these cells.     

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.     

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.     

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.     

Activation of ErbB2 by 2-methyl-1,4-naphthoquinone (menadione) in human keratinocytes: role of EGFR and protein tyrosine phosphatases

Activation of ErbB receptor tyrosine kinases triggers multiple signaling pathways that regulate cellular proliferation and survival. We here demonstrate that ErbB2 is activated via the epidermal growth factor receptor (EGFR) upon exposure of cultured human keratinocytes to 2-methyl-1,4-naphthoquinone (menadione). Both ErbB2 and EGFR are shown to be regulated by protein tyrosine phosphatases that are inhibited by menadione, giving rise to the hypothesis that phosphatase inhibition by menadione may result in a net activation of EGFR and an enhanced ErbB2 phosphorylation. Isolated PTP-1B, a protein tyrosine phosphatase known to be associated with ErbB receptors, is demonstrated to be inhibited by menadione.     

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.     

Activation of ErbB2 receptor tyrosine kinase supports invasion of endothelial cells by Neisseria meningitidis

ErbB2 is a receptor tyrosine kinase belonging to the family of epidermal growth factor (EGF) receptors which is generally involved in cell differentiation, proliferation, and tumor growth, and activated by heterodimerization with the other members of the family. We show here that type IV pilus-mediated adhesion of Neisseria meningitidis onto endothelial cells induces tyrosyl phosphorylation and massive recruitment of ErbB2 underneath the bacterial colonies. However, neither the phosphorylation status nor the cellular localization of the EGF receptors, ErbB3 or ErbB4, were affected in infected cells. ErbB2 phosphorylation induced by N. meningitidis provides docking sites for the kinase src and leads to its subsequent activation. Specific inhibition of either ErbB2 and/or src activity reduces bacterial internalization into endothelial cells without affecting bacteria-induced actin cytoskeleton reorganization or ErbB2 recruitment. Moreover, inhibition of both actin polymerization and the ErbB2/src pathway totally prevents bacterial entry. Altogether, our results provide new insight into ErbB2 function by bringing evidence of a bacteria-induced ErbB2 clustering leading to src kinase phosphorylation and activation. This pathway, in cooperation with the bacteria-induced reorganization of the actin cytoskeleton, is required for the efficient internalization of N. meningitidis into endothelial cells, an essential process enabling this pathogen to cross host cell barriers.     

Mutational analysis of ErbB2 intracellular localization

In the present study, the sub-cellular localization of ErbB2 and its mutants expressed as GFP-tagged proteins in MCF-7 cells or endogenous ErbB2 in SKBR3 cells was examined. The data presented here demonstrate that the full-length ErbB2 was localized at the cytoplasmic membrane and ErbB2 ICD localized in the nucleus predominantly. The sequence of ErbB2 ICD contains the information supporting its nuclear translocation and cytoplasmic retention. A region (residues 721–970) harboring an arginine triplet is essential for the cytoplasmic trafficking of ErbB2. The results indicate that differential sub-cellular localization of ErbB2 ICD and the full-length ErbB2 is dependent on their structural determinants. The present results give initial clues for further analysis of the mechanism of ErbB2 intracellular localization.     

Structure-Function Relationships of ErbB RTKs in the Plasma Membrane of Living Cells

We review the states of the ErbB family of receptor tyrosine kinases (RTKs), primarily the EGF receptor (EGFR, ErbB1, HER1) and the orphan receptor ErbB2 as they exist in living mammalian cells, focusing on four main aspects: (1) aggregation state and distribution in the plasma membrane; (2) conformational features of the receptors situated in the plasma membrane, compared to the crystallographic structures of the isolated extracellular domains; (3) coupling of receptor disposition on filopodia with the transduction of signaling ligand gradients; and (4) ligand-independent receptor activation by application of a magnetic field.This review deals exclusively with the disposition and function of the human ErbB (HER) family of receptor tyrosine kinases (RTKs) in the plasma membrane of living cells. We have divided the material into four main topics: (1) distribution and aggregation state of ErbB family members; (2) the 3D structure of the ErbB1 and ErbB2 receptors; (3) the role of receptors located on extensions (filopodia) of the cell body; and (4) the phenomenon and implications of nonligand-dependent activation of the receptors.     

Activation of the neuregulin/ErbB system during physiological ventricular remodeling in pregnancy

The neuregulin-1 (NRG1)/ErbB system has emerged as a paracrine endothelium-controlled system in the heart, which preserves left ventricular (LV) performance in pathophysiological conditions. Here, we analyze the activity and function of this system in pregnancy, which imparts a physiological condition of LV hemodynamic overload. NRG1 expression and ErbB receptor activation were studied by Western blot analyses in rats and mice at different stages of pregnancy. LV performance was evaluated by transthoracic echocardiography, and myocardial performance was assessed from twitches of isolated papillary muscles. NRG1/ErbB signaling was inhibited by oral treatment of animals with the dual ErbB1/ErbB2 tyrosine kinase inhibitor lapatinib. Analyses of LV tissue revealed that protein expression of different NRG1 isoforms and levels of phosphorylated ErbB2 and ErbB4 significantly increased after 1-2 wk of pregnancy. Lapatinib prevented phosphorylation of ErbB2 and ERK1/2, but not of ErbB4 and protein kinase B (Akt), revealing that lapatinib only partially inhibited NRG1/ErbB signaling in the LV. Lapatinib did not prevent pregnancy-induced changes in LV mass and did not cause apoptotic cell death or fibrosis in the LV. Nevertheless, lapatinib led to premature maternal death of ～25% during pregnancy and it accentuated pregnancy-induced LV dilatation, significantly reduced LV fractional shortening, and induced abnormalities of twitch relaxation (but not twitch amplitude) of isolated papillary muscles. This is the first study showing that the NRG1/ErbB system is activated, and plays a modulatory role, during physiological hemodynamic overload associated with pregnancy. Inhibiting this system during physiological overload may cause LV dysfunction in the absence of myocardial cell death.     

Heat Shock Factor Hsf1 Cooperates with ErbB2 (Her2/Neu) Protein to Promote Mammary Tumorigenesis and Metastasis

ErbB2/Neu oncogene is overexpressed in 25% of invasive/metastatic breast cancers. We have found that deletion of heat shock factor Hsf1 in mice overexpressing ErbB2/Neu significantly reduces mammary tumorigenesis and metastasis. Hsf1^+/−ErbB2/Neu⁺ tumors exhibit reduced cellular proliferative and invasive properties associated with reduced activated ERK1/2 and reduced epithelial-mesenchymal transition (EMT). Hsf1^+/+Neu⁺ mammary epithelial cells exposed to TGFβ show high levels of ERK1/2 activity and EMT; this is associated with reduced expression of E-cadherin and increased expression of Slug and vimentin, a mesenchymal marker. In contrast, Hsf1^−/−Neu⁺ or Hsf1^+/+Neu⁺ cells do not exhibit activated ERK1/2 and show reduced EMT in the presence of TGFβ. The ineffective activation of the RAS/RAF/MEK/ERK1/2 signaling pathway in cells with reduced levels of HSF1 is due to the low levels of HSP90 in complex with RAF1 that are required for RAF1 stability and maturation. These results indicate a powerful inhibitory effect conferred by HSF1 downstream target genes in the inhibition of ErbB2-induced breast cancers in the absence of the Hsf1 gene.     

Differential localization of ErbB2 in different tissues of the rat female reproductive tract: implications for the use of specific antibodies for ErbB2 analysis

ErbB2 has been implicated in numerous functions, including normal and aberrant development of a variety of tissues. Although no soluble ligand has been identified for ErbB2, we have recently shown that ASGP-2, the transmembrane subunit of the cell surface glycoprotein Muc4 (also called sialomucin complex, SMC), can act as an intramembrane ligand for ErbB2 and modulate its activity. Muc4/SMC is abundantly expressed at the apical surface of most epithelia of the rat female reproductive tract. Since Muc4/SMC can interact with ErbB2 when they are expressed in the same cell and membrane, we investigated whether these two proteins are co-expressed and co-localized in tissues of the female reproductive tract. Using an anti-ErbB2 antibody from Dako, we found moderate staining at the basolateral surface of the oviduct and also around the cell membrane of the most superficial and medial layers of the stratified epithelia of the vagina. In contrast, Neomarkers neu Ab1 antibody intensely stained the apical surface of the epithelium of the oviduct and the medial and basal layers of the stratified epithelia of the vagina, substantially overlapping the distribution of Muc4/SMC. Furthermore, Muc4/SMC and ErbB2 association in different tissues of the female reproductive tract was demonstrated by co-immunoprecipitation analysis. Interestingly, phosphorylated ErbB2 detected by anti-phospho-ErbB2 is primarily present at the apical surface of the oviduct. Thus, our results show that differentially localized forms of ErbB2 are recognized by different antibodies and raise interesting questions about the nature of the different forms of ErbB2, the mechanism for differential localization, and possible functions of ErbB2 in the female reproductive tract. They also raise a cautionary note about the use of different ErbB2 antibodies for expression and localization studies.     

ErbB2, but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini

Both ErbB1 and ErbB2 are overexpressed or amplified in breast tumours. To examine the effects of activating ErbB receptors in a context that mimics polarized epithelial cells in vivo, we activated ErbB1 and ErbB2 homodimers in preformed, growth-arrested mammary acini cultured in three-dimensional basement membrane gels. Activation of ErbB2, but not that of ErbB1, led to a reinitiation of cell proliferation and altered the properties of mammary acinar structures. These altered structures share several properties with early-stage tumours, including a loss of proliferative suppression, an absence of lumen, retention of the basement membrane and a lack of invasive properties. ErbB2 activation also disrupted tight junctions and the cell polarity of polarized epithelia, whereas ErbB1 activation did not have any effect. Our results indicate that ErbB receptors differ in their ability to induce early stages of mammary carcinogenesis in vitro and this three-dimensional model system can reveal biological activities of oncogenes that cannot be examined in vitro in standard transformation assays.     

A phosphoproteomic analysis of the ErbB2 receptor tyrosine kinase signaling pathways

Overexpression of the ErbB2 receptor tyrosine kinase is common in human cancers and is associated with an increased level of metastasis. To better understand the cellular signaling networks activated by ErbB2, a phosphoproteomic analysis of tyrosine-phosphorylated proteins was carried out in ErbB2-overexpressing breast and ovarian cancer cell lines. A total of 153 phosphorylation sites were assigned on 78 proteins. Treatment of cells with Herceptin, a monoclonal antibody that inhibits ErbB2 activity, significantly reduced the number of detectable protein phosphorylation sites, suggesting that many of these proteins participate in ErbB2-driven cell signaling. Of the 71 proteins that were differentially phosphorylated, only 13 were previously reported to directly associate with ErbB2. The differentially phosphorylated proteins included kinases, adaptor/docking proteins, proteins involved in cell proliferation and migration, and several uncharacterized RNA binding proteins. Selective depletion of some of these proteins, including RNA binding proteins SRRM2, SFRS1, SFRS9, and SFRS10, by siRNAs reduced the rate of migration of ErbB2-overexpressing ovarian cancer cells.     

Differential subcellular membrane recruitment of Src may specify its downstream signalling

Most Src family members are diacylated and constitutively associate with membrane "lipid rafts" that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at "rafts" remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to "lipid rafts"; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 degrees C and floated into sucrose density gradients like caveolin-1 and flotillin-2, i.e. "lipid rafts". By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe ( approximately 70%) cholesterol extraction with methyl-beta-cyclodextrin (MbetaCD) did not abolish "rafts" floatation, but strongly decreased Src association with floating "rafts" and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to MbetaCD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at "non-raft" domains on endosomes, then via PI3-kinase-Akt on a distinct set of "rafts" at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined.