EGF-dependent Phosphorylation Research Articles

APKCζ affects directed cell migration through the regulation of myosin light chain phosphorylation

ABSTRACTCell motility is an essential cellular process for a variety of biological events. It requires cross-talk between the signaling and the cytoskeletal systems. Despite the recognized importance of aPKCζ for cell motility, there is little understanding of the mechanism by which aPKCζ mediates extracellular signals to the cytoskeleton. In the present study, we report that aPKCζ is required for the cellular organization of acto-non-muscle myosin II (NMII) cytoskeleton, for proper cell adhesion and directed cell migration. We show that aPKCζ mediates EGF-dependent RhoA activation and recruitment to the cell membrane. We also show that aPKCζ mediates EGF-dependent myosin light chain (MRLC) phosphorylation that is carried out by Rho-associated protein kinase (ROCK), and that aPKCζ is required for EGF-dependent phosphorylation and inhibition of the myosin phosphatase targeting subunit (MYPT). Finally, we show that aPKCζ mediates the spatial organization of the acto-NMII cytoskeleton in response to EGF stimulation. Our data suggest that aPKCζ is an essential component regulator of acto-NMII cytoskeleton organization leading to directed cell migration, and is a mediator of the EGF signal to the cytoskeleton.

The Pleckstrin Homology Domain of Diacylglycerol Kinase η Strongly and Selectively Binds to Phosphatidylinositol 4,5-Bisphosphate

Type II diacylglycerol kinase (DGK) isozymes (δ, η, and κ) have a pleckstrin homology domain (PH) at their N termini. Here, we investigated the lipid binding properties of the PHs of type II DGK isozymes using protein-lipid overlay and liposome binding assays. The PH of DGKη showed the most pronounced binding activity to phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2) among the various glycero- and sphingolipids including PI 3,4,5-trisphosphate, PI 3,4-bisphosphate, PI 3-phosphate, PI 4-phosphate, and PI 5-phosphate. Moreover, the PI(4,5)P2binding activity of the DGKη-PH was significantly stronger than that of other type II DGK isozymes. Notably, compared with the PH of phospholipase C (PLC) δ1, which is generally utilized as a cellular PI(4,5)P2- probe, the DGKη-PH is equal to or superior than the PLCδ1-PH in terms of affinity and selectivity for PI(4,5)P2 Furthermore, in COS-7 cells, GFP-fused wild-type DGKη1 and its PH partly translocated from the cytoplasm to the plasma membrane where the PLCδ1-PH was co-localized in response to hyperosmotic stress in an inositol 5-phosphatase-sensitive manner, whereas a PH deletion mutant did not. Moreover, K74A and R85A mutants of DGKη-PH, which lack the conserved basic amino acids thought to ligate PI(4,5)P2, were indeed unable to bind to PI(4,5)P2and co-localize with the PLCδ1-PH even in osmotically shocked cells. Overexpression of wild-type DGKη1 enhanced EGF-dependent phosphorylation of ERK, whereas either K74A or R85A mutant did not. Taken together, these results indicate that the DGKη-PH preferentially interacts with PI(4,5)P2and has crucial roles in regulating the subcellular localization and physiological function of DGKη. Moreover, the DGKη-PH could serve as an excellent cellular sensor for PI(4,5)P2.

Epidermal Growth Factor Enhances Orthovanadate-Induced Contraction via Src and Myosin Phosphatase Target Subunit 1 in Rat Vascular Smooth Muscle

Inhibition of protein tyrosine phosphatase by orthovanadate induces vasoconstriction, which is mediated by the Rho kinase-dependent inactivation of myosin light chain phosphatase (MLCP) via signaling downstream of Src-induced activation of the epidermal growth factor (EGF) receptor. The present study investigated the potential role of EGF in orthovanadate (OVA)-dependent vaso-constriction. OVA-induced aortic contraction significantly increased in the presence of EGF, and was abolished by inhibitors of Rho kinase (Y27632), extracellular signal-regulated kinase 1 and 2 (Erk1/2) (FR180204), Erk1/2 kinase (PD98059), EGF receptor (AG1478), and Src (PP2). Treatment of the rat endothelium-denuded thoracic aorta with either EGF or OVA augmented the phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Thr-853 and of the EGF receptor at Tyr-1173. The phosphorylation of MYPT1 was further increased by co-stimulation with EGF and OVA. EGF receptor phosphorylation at Tyr-845 was also increased by EGF or OVA; this effect was augmented by co-stimulation with EGF and OVA, and was abolished by Src inhibition. In addition, Erk1/2 was phosphorylated by EGF or by co-treatment with EGF and OVA; this was abolished by an EGF receptor inhibitor, but not by Src inhibition. These results suggested that OVA-induced EGF-related contraction was mediated by the Rho kinase-dependent inactivation of MLCP via two different signaling cascades: Src-dependent phosphorylation of the EGF receptor at Tyr-845 and EGF-dependent phosphorylation of Erk1/2.

Frank-ter Haar Syndrome Protein Tks4 Regulates Epidermal Growth Factor-dependent Cell Migration

Mutations in the SH3PXD2B gene coding for the Tks4 protein are responsible for the autosomal recessive Frank-ter Haar syndrome. Tks4, a substrate of Src tyrosine kinase, is implicated in the regulation of podosome formation. Here, we report a novel role for Tks4 in the EGF signaling pathway. In EGF-treated cells, Tks4 is tyrosine-phosphorylated and associated with the activated EGF receptor. This association is not direct but requires the presence of Src tyrosine kinase. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutations of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks4. Furthermore, a PX domain mutant (R43W) Tks4 carrying a reported point mutation in a Frank-ter Haar syndrome patient showed aberrant intracellular expression and reduced phosphoinositide binding. Finally, silencing of Tks4 was shown to markedly inhibit HeLa cell migration in a Boyden chamber assay in response to EGF or serum. Our results therefore reveal a new function for Tks4 in the regulation of growth factor-dependent cell migration.

Abstract 4971: WBP2: A novel phospho-oncoprotein in estrogen receptor-positive breast cancer

Abstract WBP2 (WW-domain Binding Protein 2) was first implicated in breast cancer through our previous phosphoproteomics screen on the MCF10AT xenograft-derived isogenic cell line model of human breast cancer progression, where WBP2 was found to be differentially phosphorylated during breast cancer development. We had also shown it to be an authentic tyrosine phosphorylation target of EGFR signaling. In a follow up study, we found that WBP2 expression was low or undetectable in normal breast epithelial cells but overexpressed in breast cancer progression model as well as multiple breast cancer cells of various subtypes. Therefore, we aimed to investigate the oncogenic role of WBP2 in the growth and progression of human breast cancer through its protein overexpression and tyrosine phosphorylation. We first mapped the EGF-dependent tyrosine phosphorylation sites on WBP2 and identified a member of the Src family of tyrosine kinases as the putative upstream kinase of WBP2. Next, we found that Estrogen (E2) stimulation could similarly induce tyrosine phosphorylation of WBP2 via a crosstalk between EGFR and ER pathways. Subcellular fractionation study revealed a phosphorylation-dependent nuclear entry of WBP2 upon E2 or EGF stimulation. Consistent with the reported role of WBP2 as a coactivator for nuclear hormone receptor (ERα/PR), abrogation of tyrosine phosphorylation on WBP2 significantly impaired the E2-induced ER transactivation through reduced E2/EGF-stimulated nuclear entry of WBP2 and in vivo E2-dependent WBP2-ER interaction. Stable overexpression of wild type and phospho-mimic mutant of WBP2 in ER-positive MCF7 breast cancer cell enhanced its cell proliferation, anchorage-independent growth in soft agar, cell migration and cell invasion in the E2-independent and/or E2-dependent manner. These cellular processes could be inhibited, at least in part, by the phosphorylation-defective mutant of WBP2. Potential mechanisms of WBP2-mediated oncogenic transformation will be presented. Collectively, our findings have uncovered the potential of WBP2 as a novel phospho-oncoprotein in ER+ breast cancer. It is conceivable that WBP2 and/or its tyrosine phosphorylated form could serve as a potential prognostic marker or therapeutic target for human breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4971.

Dual silencing of insulin-like growth factor-I receptor and epidermal growth factor receptor in colorectal cancer cells is associated with decreased proliferation and enhanced apoptosis

Overexpression and activation of tyrosine kinase receptors are common features of colorectal cancer. Using the human colorectal cancer cell lines DLD-1 and Caco-2, we evaluated the role of the insulin-like growth factor-I (IGF-I) receptor (IGF-IR) and epidermal growth factor receptor (EGFR) in cellular functions of these cells. We used the small interfering RNA (siRNA) technology to specifically down-regulate IGF-IR and EGFR expression. Knockdown of IGF-IR and EGFR resulted in inhibition of cell proliferation of DLD-1 and Caco-2 cells. An increased rate of apoptosis was associated with siRNA-mediated silencing of IGF-IR and EGFR as assessed by activation of caspase-3/caspase-7. The combined knockdown of both EGFR and IGF-IR decreased cell proliferation and induced cell apoptosis more effectively than did silencing of either receptor alone. Comparable effects on cell proliferation and apoptosis were observed after single and combinational treatment of cells by the IGF-IR tyrosine kinase inhibitor NVP-AEW541 and/or the EGFR tyrosine kinase inhibitor erlotinib. Combined IGF-IR and EGFR silencing by either siRNAs or tyrosine kinase inhibitors diminished the phosphorylation of downstream signaling pathways AKT and extracellular signal-regulated kinase (ERK)-1/2 more effectively than did the single receptor knockdown. Single IGF-IR knockdown inhibited IGF-I-dependent phosphorylation of AKT but had no effect on IGF-I- or EGF-dependent phosphorylation of ERK1/2, indicating a role of EGFR in ligand-dependent ERK1/2 phosphorylation. The present data show that inhibition of the IGF-IR transduction cascade augments the antipoliferative and proapoptotic effects of EGFR inhibition in colorectal cancer cells. A clinical application of combination therapy targeting both EGFR and IGF-IR could be a promising therapeutic strategy.

Cdk5 phosphorylates PLD2 to mediate EGF-dependent insulin secretion

Insulin secretion from pancreatic β-cells is an important process that affects the regulation of glucose level in the blood. In our previous study, we suggested that epidermal growth factor (EGF) stimulates insulin secretion by activating phospholipase D2 (PLD2) [H.Y. Lee, K. Yea, J. Kim, B.D. Lee, Y.C. Chae, H.S. Kim, D.W. Lee, S.H. Kim, J.H. Cho, C.J. Jin, D.S. Koh, K.S. Park, P.G. Suh, S.H. Ryu, 2007. Epidermal Growth Factor Increases Insulin Secretion and Lowers Blood Glucose in Diabetic Mice. J. Cell. Mol. Med. 5:5]. However, the specific mechanism by which PLD2 activation leads to insulin secretion was not determined. In this study, we suggest that the phosphorylation and activation of PLD2 by cyclin-dependent kinase 5 (Cdk5) is critical for EGF-dependent insulin secretion. We found that a Cdk5 inhibitor, roscovitine, and dominant-negative Cdk5 inhibited EGF-dependent PLD2 activation and insulin secretion. EGF stimulation activated Cdk5 activity in rat insulinoma RINm5F cells, and PLD2 phosphorylation by Cdk5 was observed in vitro and in cells. The kinetics of PLD2 phosphorylation correlates with the interaction between PLD2 and Cdk5 and its effect on EGF signaling. We determined that the phosphorylation site of PLD2 was located at Ser 134. PLD2-S134A did not show EGF-dependent phosphorylation and activation by Cdk5. Furthermore, this mutant was unable to mediate EGF-dependent insulin secretion in pancreatic β cell lines, suggesting that the phosphorylation of PLD2 at Ser 134 by Cdk5 is critical for this process. The study results suggest that PLD2 is a new substrate of Cdk5 and that the phosphorylation of PLD2 by Cdk5 is involved in EGF-dependent insulin secretion.

Epidermal Growth Factor Receptor Tyrosine Phosphorylation and Signaling Controlled by a Nuclear Receptor Coactivator, Amplified in Breast Cancer 1

The steroid receptor coactivator amplified in breast cancer 1 (AIB1) as well as epidermal growth factor receptor (EGFR) family members are frequently overexpressed in epithelial tumors, and their expression is associated with poor prognosis. However, a direct role of AIB1 in EGF signaling has not been determined. To address this, we reduced endogenous AIB1 levels using RNA interference in lung, breast, and pancreatic cancer cell lines. We found that a knockdown of AIB1 levels resulted in a loss of the growth response of these cell lines to EGF. Further analysis revealed that the depletion of AIB1 reduced tyrosine phosphorylation of EGFR at multiple residues both at autophosphorylation and Src kinase phosphorylation sites. AIB1 knockdown did not affect tyrosine phosphorylation of the receptor tyrosine kinases, platelet-derived growth factor receptor and HER3, or overall tyrosine phosphorylation of cellular proteins. However, EGF-dependent phosphorylation of HER2 was decreased. EGFR levels and membrane trafficking were not changed by AIB1 depletion, but there was less recruitment of Src homology 2 domain-containing proteins to the EGFR. This led to a substantial reduction in EGF-induced phosphorylation of signal transducers and activators of transcription 5 and c-Jun NH(2)-terminal kinase but no significant change in the activation of AKT. Vanadate treatment of cells revealed that the reduction in EGFR tyrosine phosphorylation is dependent in part on changes in cellular phosphatase activity. We propose that a portion of the oncogenic effect of AIB1 could be through control of EGFR and HER2 activity and subsequent modulation of cellular signaling pathways.

Myosin-IIA Heavy-Chain Phosphorylation Regulates the Motility of MDA-MB-231 Carcinoma Cells

In mammalian nonmuscle cells, the mechanisms controlling the localized formation of myosin-II filaments are not well defined. To investigate the mechanisms mediating filament assembly and disassembly during generalized motility and chemotaxis, we examined the EGF-dependent phosphorylation of the myosin-IIA heavy chain in human breast cancer cells. EGF stimulation of MDA-MB-231 cells resulted in transient increases in both the assembly and phosphorylation of the myosin-IIA heavy chains. In EGF-stimulated cells, the myosin-IIA heavy chain is phosphorylated on the casein kinase 2 site (S1943). Cells expressing green fluorescent protein-myosin-IIA heavy-chain S1943E and S1943D mutants displayed increased migration into a wound and enhanced EGF-stimulated lamellipod extension compared with cells expressing wild-type myosin-IIA. In contrast, cells expressing the S1943A mutant exhibited reduced migration and lamellipod extension. These observations support a direct role for myosin-IIA heavy-chain phosphorylation in mediating motility and chemotaxis.

Cool-1 functions as an essential regulatory node for EGFreceptor- and Src-mediated cell growth

Cool-1 (cloned-out of library 1) has a key role in regulating epidermal growth factor receptor (EGFR) degradation. Here, we show that Cool-1 performs this function by functioning as both an upstream activator and downstream target for Cdc42. EGF-dependent phosphorylation of Cool-1 enables it to act as a nucleotide exchange factor for Cdc42 and to form a complex with the E3 ligase Cbl, thus regulating Cbl-catalysed EGFR degradation. The EGF-dependent phosphorylation is normally transient; however, Cool-1 phosphorylation is sustained in cells expressing v-Src and is essential for cellular transformation, as well as for v-Src-induced tumour formation in mice. These findings demonstrate that the regulated phosphorylation of Cool-1 is necessary to maintain the balance between normal signalling by EGFR and Src versus aberrant growth and transformation.

Dok-R Mediates Attenuation of Epidermal Growth Factor-Dependent Mitogen-Activated Protein Kinase and Akt Activation through Processive Recruitment of c-Src and Csk

Dok-R has previously been shown to associate with the epidermal growth factor receptor (EGFR) and become tyrosine phosphorylated in response to EGF stimulation. The recruitment of Dok-R to the EGFR, which is mediated through its phosphotyrosine binding (PTB) domain, results in attenuation of mitogen-activated protein kinase (MAPK) activation. Dok-R's ability to attenuate EGF-driven MAPK activation is independent of its ability to recruit rasGAP, a known attenuator of MAPK activity, suggesting an alternate Dok-R-mediated pathway. Herein, we have determined the structural determinants within Dok-R that are required for its ability to attenuate EGF signaling and to associate with c-Src and with the Src family kinase (SFK)-inhibitory kinase, Csk. We demonstrate that Dok-R associates constitutively with c-Src through an SH3-dependent interaction and that this association is essential to Dok-R's ability to attenuate c-Src activity and diminish MAPK and Akt/PKB activity. We further illustrate that EGF-dependent phosphorylation of Dok-R requires SFK activity and, more specifically, that SFK-dependent phosphorylation of tyrosine 402 on Dok-R facilitates the inducible recruitment of Csk. We propose that recruitment of Csk to Dok-R serves to bring Csk to c-Src and down-regulate its activity, resulting in a concomitant attenuation of MAPK and Akt/PKB activity. Furthermore, we demonstrate that Dok-R can abrogate c-Src's ability to protect the breast cancer cell line SKBR3 from anoikis and that an association with c-Src and Csk is required for this activity. Collectively these results demonstrate that Dok-R acts as an EGFR-recruited scaffolding molecule that processively assembles c-Src and Csk to attenuate signaling from the EGFR.

Mechanisms of resistance to Erbitux (anti–epidermal growth factor receptor) combination therapy in pancreatic adenocarcinoma cells

We previously demonstrated that pancreatic adenocarcinoma BxPC-3 xenografts display resistance to treatment with Erbitux, gemcitabine, and radiation, whereas MIA PaCa-2 xenografts are highly sensitive to the same therapy. Here, we elucidate in vitro mechanisms that may explain the observed differential response of epidermal growth factor receptor (EGFR) expressing pancreatic adenocarcinoma xenografts to Erbitux-based combination therapy in vivo. MIA PaCa-2 and BxPC-3 protein lysates were probed with antibodies to EGFR, ErbB2, ErbB3, and ErbB4. Constitutive ErbB3 activity was visualized by immunoblot analysis using anti-phosphotyrosine antibodies and receptor-specific immunoprecipitates. erbB2 and erbB3 gene expression in both cell lines was quantified with real-time polymerase chain reaction. Erbitux-induced internalization of EGFR was determined by flow cytometry following Erbitux treatment for different incubation times at 0 degrees C and 37 degrees C. MIA PaCa-2 and BxPC-3 protein extracts were also probed with anti-phospho-mitogen-activated protein kinase antibody after stimulation with EGF and in the presence of Erbitux. Although both cell lines expressed EGFR and ErbB2 protein, ErbB3 protein was selectively expressed by BxPC-3 cells, where it also showed evidence of constitutive phosphorylation. There was a 10-fold increase of erbB3 transcript levels in BxPC-3 cells compared with MIA PaCa-2. ErbB4 protein was not detectable in either cell line. Erbitux mediated EGFR internalization in MIA PaCa-2 cells after 2 hours of incubation, whereas it did not promote EGFR internalization in BxPC-3 cells. Likewise, EGF-dependent phosphorylation of MAPK p44/42 was blocked by Erbitux treatment in MIA PaCa-2 but not BxPC-3 cells. Erbitux selectively interfered with EGF-induced MAPK activation in MIA PaCa-2 but not BxPC-3 cells. Persistent MAPK activation and impaired in vitro internalization of EGFR by BxPC-3 pancreatic cancer cells may be due to constitutive ErbB3 signaling, facilitated by heterodimerization with EGFR, which may explain resistance to Erbitux-based combination therapy in vivo.

Epidermal Growth Factor-dependent Regulation of Cdc42 Is Mediated by the Src Tyrosine Kinase

Treatment of cells with epidermal growth factor (EGF) promotes the activation of the small GTP-binding protein Cdc42, as well as its phosphorylation in cells. The EGF-dependent phosphorylation of Cdc42 occurs at tyrosine 64 in the Switch II domain and appears to be mediated through the Src tyrosine kinase, because both the expression of a dominant-negative Src mutant (mouse Src(K297R)) and treatment of cells with the Src kinase inhibitor PP2 blocks the EGF-stimulated phosphorylation of Cdc42, whereas expression of an activated Src mutant (Src(Y529F)) promotes phosphorylation in the absence of EGF treatment. The EGF-stimulated phosphorylation of Cdc42 is not required for its activation, nor does it directly affect the interactions of activated Cdc42 with target/effector proteins including PAK, ACK, WASP, or IQGAP. However, the EGF-stimulated phosphorylation of Cdc42 is accompanied by an enhancement in the interaction of Cdc42 with the Rho-GDP dissociation inhibitor (RhoGDI). The EGF-stimulated activation of Cdc42 does require activated Src, as well as the Vav2 protein, a member of the Dbl family of guanine nucleotide exchange factors. Src catalyzes the tyrosine phosphorylation of Vav2, and overexpression of Vav2 together with activated Src (Src(Y529F)) can completely bypass the need for EGF to promote the activation of Cdc42. Thus, EGF signaling through Src appears to have dual regulatory effects on Cdc42: 1). it leads to the activation of Cdc42 as mediated by the Vav2 guanine nucleotide exchange factor, and 2). it results in the phosphorylation of Cdc42, which stimulates the binding of RhoGDI, perhaps to direct the movement of Cdc42 to a specific cellular site to trigger a signaling response, because Cdc42-RhoGDI interactions are essential for Cdc42-induced cellular transformation.

Epidermal Growth Factor-mediated Transient Phosphorylation and Membrane Localization of Myosin II-B Are Required for Efficient Chemotaxis

Epidermal growth factor (EGF) stimulation of prostate metastatic tumor cells results in transient phosphorylation and cellular localization of non-muscle myosin heavy chain II-B (NMHC II-B) with kinetics similar to those seen in chemotaxis. We demonstrate that expression of 18- and 72-kDa fragments derived from the NMHC II-B C terminus that contain EGF-dependent NMHC II-B phosphorylation sites serve as dominant-negative mutations for EGF-dependent NMHC II-B phosphorylation and localization. Both fragments inhibited the EGF-dependent phosphorylation by competing with NMHC II-B on the myosin heavy chain kinase. However, only expression of the 72-kDa fragment resulted in cells with abnormalities in cell shape, focal adhesions, and chemotaxis. We found that the 72-kDa (but not 18-kDa) fragment is capable of self-assembly. To our knowledge, these results provide the first strong evidence that EGF-dependent NMHC II-B phosphorylation is required for the cellular localization of NMHC II-B and that NMHC II-B is required for normal cell attachment and for chemotactic response.

The UIM domain of Hrs couples receptor sorting to vesicle formation.

Hepatocyte growth factor regulated tyrosine kinase substrate (Hrs), a main component of the 'bilayered' clathrin coat on sorting endosomes, was originally identified as a substrate of activated tyrosine kinase receptors. We have analysed Hrs phosphorylation in response to epidermal growth factor (EGF) stimulation and show that the evolutionary conserved tyrosines Y329 and Y334 provide the principal phosphorylation sites. Hrs is proposed to concentrate ubiquitinated receptors within clathrin-coated regions via direct interaction with its UIM (ubiquitin interaction motif) domain. We show that the same UIM domain is necessary for EGF-stimulated tyrosine phosphorylation of Hrs. Over-expression of wild-type Hrs or a double mutant, Y329/334F, defective in EGF-dependent phosphorylation, both substantially retard EGF receptor (EGFR) degradation by inhibiting internal vesicle formation and thereby preventing EGFR incorporation into lumenal vesicles of the multivesicular bodies. In contrast, mutation or deletion of the Hrs-UIM domain strongly suppresses this effect. In addition the UIM-deletion and point mutants are also observed on internal membranes, indicating a failure to dissociate from the endosomal membrane prior to incorporation of the receptor complex into lumenal vesicles. Our data suggest a role for the UIM-domain of Hrs in actively retaining EGFR at the limiting membrane of endosomes as a prelude to lumenal vesicle formation.

Caveolin-1 Phosphorylation in Human Squamous and Epidermoid Carcinoma Cells: Dependence on ErbB1 Expression and Src Activation

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

Plasma membrane phospholipid scramblase 1 is enriched in lipid rafts and interacts with the epidermal growth factor receptor.

We have identified physical and functional interactions between the epidermal growth factor (EGF) receptor and phospholipid scramblase 1 (PLSCR1), an endofacial plasma membrane protein proposed to affect phospholipid organization. PLSCR1, a palmitoylated protein, was found to partition with the EGF receptor in membrane lipid rafts. Cell stimulation with EGF transiently elevated Tyr-phosphorylation of PLSCR1, peaking at 5 min. Although PLSCR1 is a known substrate of c-Abl [Sun, J., et al. (2001) J.Biol. Chem. 276, 28984-28990], the Abl inhibitor STI571 did not substantially affect its EGF-dependent phosphorylation, suggesting PLSCR1 is a substrate of the EGF receptor kinase, or another EGF-activated kinase. Coinciding with phosphorylation, there was a transient increase in physical association of PLSCR1 with both the EGF receptor and the adapter protein Shc, as determined by immunoprecipitation and Western blotting. Confocal immunofluorescence analysis revealed that EGF initiates rapid internalization of both the EGF receptor and PLSCR1, with trafficking into both distinct and common endosomal pools. These data also suggested that whereas the EGF receptor is ultimately degraded, much of the endocytosed PLSCR1 is recycled to the cell surface within 3 h after EGF treatment. Consistent with this interpretation, Western blotting revealed neither ubiquitination nor proteolysis of PLSCR1 under these conditions, whereas the ubiquitination and degradation of the EGF receptor were readily confirmed. Finally, stimulation with EGF was also found to markedly increase the total cellular expression of PLSCR1, suggesting that in addition to its initial interactions with activated EGF receptor, PLSCR1 may also contribute to posttranscriptional effector pathway(s) mediating the cellular response to EGF.

Heterologous desensitization of EGF receptors and PDGF receptors by sequestration in caveolae.

Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors have been reported to signal via caveolin-containing membranes called caveolae. In contrast, others report that EGF and PDGF receptors are exclusively associated with caveolin-devoid membranes called rafts. Our subcellular fractionation and coimmunoprecipitation studies demonstrate that, in the absence of ligand, EGF and PDGF receptors are associated with rafts. However, in the presence of ligand, EGF and PDGF receptors transiently associate with caveolae. Surprisingly, pretreatment of cells with EGF prevents PDGF-dependent phosphorylation of PDGF receptors and extracellular signal-regulated kinase (ERK) 1/2 kinase activation. Furthermore, cells pretreated with PDGF prevent EGF-dependent phosphorylation of EGF receptors and ERK1/2 kinase activation. Radioligand binding studies demonstrate that incubation of cells with EGF or PDGF causes both EGF and PDGF receptors to be reversibly sequestered from the extracellular space. Experiments with methyl-beta-cyclodextrin, filipin, and antisense caveolin-1 demonstrate that sequestration of the receptors is dependent on cholesterol and caveolin-1. We conclude that ligand-induced stimulation of EGF or PDGF receptors can cause the heterologous desensitization of the other receptor by sequestration in cholesterol-rich, caveolin-containing membranes or caveolae.

Myosin II heavy chain isoforms are phosphorylated in an EGF-dependent manner

To explore the involvement and regulation of the nonmuscle myosin II heavy chains isoforms, MHC-A and MHC-B in the chemotaxis of metastatic tumor cells, we analyzed the changes in phosphorylation and cellular localization of these isoforms upon stimulation of prostate tumor cells with epidermal growth factor (EGF). EGF stimulation of prostate tumor cells resulted in transient increases in MHC-A and MHC-B phosphorylation and subcellular localization with quite different kinetics. Furthermore, the kinetics of subcellular localization correlated with the in vivo kinetics of MHC-B phosphorylation but not of MHC-A phosphorylation, suggesting different modes of regulation for these myosin II isoforms. We further showed that protein kinase C (PKC) is involved in the EGF-dependent phosphorylation of MHC-A and MHC-B. To our knowledge, this is the first report demonstrating that MHC phosphorylation might regulate its subcellular localization and that the EGF signal is transmitted to MHC-A and MHC-B via PKC. The correlation between MHC-B phosphorylation and localization in response to EGF stimulation might suggest that MHC-B is the myosin II isoform that is involved in chemotaxis.

EGF stimulates gastrin promoter through activation of Sp1 kinase activity.

Epidermal growth factor (EGF) receptor activation stimulates gastrin gene expression through a GC-rich element called gastrin EGF response element (gERE). This element is bound by Sp1 family members and is a target of the ras-extracellular signal-regulated kinase (Erk) signal transduction cascade. This raised the possibility that Sp1 may be phosphorylated by kinases of this signaling pathway. Erk is capable of phosphorylating other mitogen-inducible transcription factors, e.g., Elk and Sap, suggesting that Erk may also mediate EGF-dependent phosphorylation of Sp1. This possibility was tested by studying Sp1-dependent kinase activity in extracts prepared from EGF-activated AGS cells by use of solid-phase kinase assays and immunoprecipitation of metabolically labeled Sp1. The results revealed that Sp1 kinase activity (like gastrin promoter activation) is inhibited by PD-98059 and, therefore, is dependent on mitogen-activated protein kinase kinase 1 (Mek 1). However, EGF-dependent activation of endogenous Erk did not account for most of the Sp1 kinase activity, since Erk and additional Sp1 kinase activity analyzed in a solid-phase kinase assay eluted from an ion-exchange column in different fractions. Phosphoamino acid analysis of in vivo radiolabeled Sp1 demonstrated that the kinase phosphorylates Sp1 on Ser and Thr in response to EGF. Therefore, most EGF-stimulated Sp1 kinase activity is Mek 1 dependent and distinct from Erk.