Syndecan-1 and Syndecan-4 Capture Epidermal Growth Factor Receptor Family Members and the α3β1 Integrin Via Binding Sites in Their Ectodomains: NOVEL SYNSTATINS PREVENT KINASE CAPTURE AND INHIBIT α6β4-INTEGRIN-DEPENDENT EPITHELIAL CELL MOTILITY*

The α6β4 integrin is known to associate with receptor tyrosine kinases when engaged in epithelial wound healing and in carcinoma invasion and survival. Prior work has shown that HER2 associates with α6β4 integrin and syndecan-1 (Sdc1), in which Sdc1 engages the cytoplasmic domain of the β4 integrin subunit allowing HER2-dependent motility and carcinoma cell survival. In contrast, EGFR associates with Sdc4 and the α6β4 integrin, and EGFR-dependent motility depends on cytoplasmic engagement of β4 integrin with Sdc4. However, how HER2 and EGFR assimilate into a complex with the syndecans and integrin, and why kinase capture is syndecan-specific has remained unknown. In the present study, we demonstrate that HER2 is captured via a site, comprised of amino acids 210–240, in the extracellular domain of human Sdc1, and EGFR is captured via an extracellular site comprised of amino acids 87–131 in human Sdc4. Binding assays using purified recombinant proteins demonstrate that the interaction between the EGFR family members and the syndecans is direct. The α3β1 integrin, which is responsible for the motility of the cells, is captured at these sites as well. Peptides based on the interaction motifs in Sdc1 and Sdc4, called synstatins (SSTN_210–240 and SSTN_87–131) competitively displace the receptor tyrosine kinase and α3β1 integrin from the syndecan with an IC₅₀ of 100–300 nm. The syndecans remain anchored to the α6β4 integrin via its cytoplasmic domain, but the activation of cell motility is disrupted. These novel SSTN peptides are potential therapeutics for carcinomas that depend on these HER2- and EGFR-coupled mechanisms for their invasion and survival.     

Cytoplasmic Domain Interactions of Syndecan-1 and Syndecan-4 with α6β4 Integrin Mediate Human Epidermal Growth Factor Receptor (HER1 and HER2)-dependent Motility and Survival

Epithelial cells are highly dependent during wound healing and tumorigenesis on the α6β4 integrin and its association with receptor tyrosine kinases. Previous work showed that phosphorylation of the β4 subunit upon matrix engagement depends on the matrix receptor syndecan (Sdc)-1 engaging the cytoplasmic domain of the β4 integrin and coupling of the integrin to human epidermal growth factor receptor-2 (HER2). In this study, HER2-dependent migration activated by matrix engagement is compared with migration stimulated by EGF. We find that whereas HER2-dependent migration depends on Sdc1, EGF-dependent migration depends on a complex consisting of human epidermal growth factor receptor-1 (HER1, commonly known as EGFR), α6β4, and Sdc4. The two syndecans recognize distinct sites at the extreme C terminus of the β4 integrin cytoplasmic domain. The binding motif in Sdc1 is QEEXYX, composed in part by its syndecan-specific variable (V) region and in part by the second conserved (C2) region that it shares with other syndecans. A cell-penetrating peptide containing this sequence competes for HER2-dependent epithelial migration and carcinoma survival, although it is without effect on the EGFR-stimulated mechanism. β4 mutants bearing mutations specific for Sdc1 and Sdc4 recognition act as dominant negative mutants to block cell spreading or cell migration that depends on HER2 or EGFR, respectively. The interaction of the α6β4 integrin with the syndecans appears critical for it to be utilized as a signaling platform; migration depends on α3β1 integrin binding to laminin 332 (LN332; also known as laminin 5), whereas antibodies that block α6β4 binding are without effect. These findings indicate that specific syndecan family members are likely to have key roles in α6β4 integrin activation by receptor tyrosine kinases.     

Structural Basis for the Binding Specificity of Human Recepteur d'Origine Nantais (RON) Receptor Tyrosine Kinase to Macrophage-stimulating Protein

Recepteur d''origine nantais (RON) receptor tyrosine kinase and its ligand, serum macrophage-stimulating protein (MSP), play important roles in inflammation, cell growth, migration, and epithelial to mesenchymal transition during tumor development. The binding of mature MSPαβ (disulfide-linked α- and β-chains) to RON ectodomain modulates receptor dimerization, followed by autophosphorylation of tyrosines in the cytoplasmic receptor kinase domains. Receptor recognition is mediated by binding of MSP β-chain (MSPβ) to the RON Sema. Here we report the structure of RON Sema-PSI-IPT₁ (SPI₁) domains in complex with MSPβ at 3.0 Å resolution. The MSPβ serine protease-like β-barrel uses the degenerate serine protease active site to recognize blades 2, 3, and 4 of the β-propeller fold of RON Sema. Despite the sequence homology between RON and MET receptor tyrosine kinase and between MSP and hepatocyte growth factor, it is well established that there is no cross-reactivity between the two receptor-ligand systems. Comparison of the structure of RON SPI₁ in complex with MSPβ and that of MET receptor tyrosine kinase Sema-PSI in complex with hepatocyte growth factor β-chain reveals the receptor-ligand selectivity determinants. Analytical ultracentrifugation studies of the SPI₁-MSPβ interaction confirm the formation of a 1:1 complex. SPI₁ and MSPαβ also associate primarily as a 1:1 complex with a binding affinity similar to that of SPI₁-MSPβ. In addition, the SPI₁-MSPαβ ultracentrifuge studies reveal a low abundance 2:2 complex with ∼10-fold lower binding affinity compared with the 1:1 species. These results support the hypothesis that the α-chain of MSPαβ mediates RON dimerization.     

Overexpression of transforming growth factor type?III receptor restores TGF-β1 sensitivity in human tongue squamous cell carcinoma cells

The transforming growth factor type III receptor (TβRIII), also known as β-glycan, is a multi-functional sensor that regulates growth, migration and apoptosis in most cancer cells. We hereby investigated the expression of TβRIII in clinical specimens of tongue squamous cell carcinoma (TSCC) and the underlying mechanism that TβRIII inhibits the growth of CAL-27 human oral squamous cells. The TSCC tissues showed a significant decrease in TβRIII protein expression as detected by immunohistochemistry (IHC) and western blot analysis. Transfection of TβRIII-containing plasmid DNA dramatically promoted TGF-β1 (10 ng/ml)-induced decrease in cell viability, apoptosis and cell arrest at the G₀-/G₁-phase. Moreover, transient overexpression of TβRIII enhanced the TGF-β1-induced cyclin-dependent kinase inhibitor 2b (CDKN2b) and p38 protein activity, but did not affect the activities of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2 (JNK1/2) in CAL-27 cells. These results suggest overexpression of TβRIII receptor restored TGF-β1 sensitivity in CAL-27 cells, which may provide some new insights on exploiting this molecule therapeutically.     

Syndecan-1 Is Required to Maintain Intradermal Fat and Prevent Cold Stress

Homeostatic temperature regulation is fundamental to mammalian physiology and is controlled by acute and chronic responses of local, endocrine and nervous regulators. Here, we report that loss of the heparan sulfate proteoglycan, syndecan-1, causes a profoundly depleted intradermal fat layer, which provides crucial thermogenic insulation for mammals. Mice without syndecan-1 enter torpor upon fasting and show multiple indicators of cold stress, including activation of the stress checkpoint p38α in brown adipose tissue, liver and lung. The metabolic phenotype in mutant mice, including reduced liver glycogen, is rescued by housing at thermoneutrality, suggesting that reduced insulation in cool temperatures underlies the observed phenotypes. We find that syndecan-1, which functions as a facultative lipoprotein uptake receptor, is required for adipocyte differentiation in vitro. Intradermal fat shows highly dynamic differentiation, continuously expanding and involuting in response to hair cycle and ambient temperature. This physiology probably confers a unique role for Sdc1 in this adipocyte sub-type. The PPARγ agonist rosiglitazone rescues Sdc1−/− intradermal adipose tissue, placing PPARγ downstream of Sdc1 in triggering adipocyte differentiation. Our study indicates that disruption of intradermal adipose tissue development results in cold stress and complex metabolic pathology.     

Design,synthesis, and anti-cancer evaluation of new pyrido[2,3-d]pyrimidin-4(3H)-one derivatives as potential EGFRWT and EGFRT790M inhibitors and apoptosis inducers

A new series of pyrido[2,3-d]pyrimidin-4(3H)-one derivatives having the essential pharmacophoric features of EGFR inhibitors has been designed and synthesised. Cell viability screening was performed for these compounds against A-549, PC-3, HCT-116, and MCF-7 cell lines at a dose of 100 μM. The highest active derivatives (8a, 8 b, 8d, 9a, and 12b) were selected for IC₅₀ screening. Compounds 8a, 8 b, and 9a showed the highest cytotoxic activities and were further investigated for wild EGFR^WT and mutant EGFR^T790M inhibitory activities. Compound 8a showed the highest inhibitory activities against EGFR^WT and EGFR^T790M with IC₅₀ values of 0.099 and 0.123 µM, respectively. In addition, it arrested the cell cycle at pre-G1 phase and induced a significant apoptotic effect in PC-3 cells. Furthermore, compound 8a induced a 5.3-fold increase in the level of caspase-3 in PC-3 cells. Finally, docking studies were carried out to examine the binding mode of the synthesised compounds against both EGFR^WT and EGFR^T790M.     

EGFR-Targeted TRAIL and a Smac Mimetic Synergize to Overcome Apoptosis Resistance in KRAS Mutant Colorectal Cancer Cells

TRAIL is a death receptor ligand that induces cell death preferentially in tumor cells. Recombinant soluble TRAIL, however, performs poorly as an anti-cancer therapeutic because oligomerization is required for potent biological activity. We previously generated a diabody format of tumor-targeted TRAIL termed Db_αEGFR-scTRAIL, comprising single-stranded TRAIL molecules (scTRAIL) and the variable domains of a humanized variant of the EGFR blocking antibody Cetuximab. Here we define the bioactivity of Db_αEGFR-scTRAIL with regard to both EGFR inhibition and TRAIL receptor activation in 3D cultures of Caco-2 colorectal cancer cells, which express wild-type K-Ras. Compared with conventional 2D cultures, Caco-2 cells displayed strongly enhanced sensitivity toward Db_αEGFR-scTRAIL in these 3D cultures. We show that the antibody moiety of Db_αEGFR-scTRAIL not only efficiently competed with ligand-induced EGFR function, but also determined the apoptotic response by specifically directing Db_αEGFR-scTRAIL to EGFR-positive cells. To address how aberrantly activated K-Ras, which leads to Cetuximab resistance, affects Db_αEGFR-scTRAIL sensitivity, we generated stable Caco-2tet cells inducibly expressing oncogenic K-Ras^G12V. In the presence of doxycycline, these cells showed increased resistance to Db_αEGFR-scTRAIL, associated with the elevated expression of the anti-apoptotic proteins cIAP2, Bcl-xL and Flip_S. Co-treatment of cells with the Smac mimetic SM83 restored the Db_αEGFR-scTRAIL-induced apoptotic response. Importantly, this synergy between Db_αEGFR-scTRAIL and SM83 also translated to 3D cultures of oncogenic K-Ras expressing HCT-116 and LoVo colorectal cancer cells. Our findings thus support the notion that Db_αEGFR-scTRAIL therapy in combination with apoptosis-sensitizing agents may be promising for the treatment of EGFR-positive colorectal cancers, independently of their KRAS status.     

Syndecan-1-Induced ECM Fiber Alignment Requires Integrin αvβ3 and Syndecan-1 Ectodomain and Heparan Sulfate Chains

Expression of the cell surface proteoglycan syndecan-1 (Sdc1) is frequently induced in stromal fibroblasts of invasive breast carcinomas. We have recently identified a correlation between stromal Sdc1 expression and extracellular matrix (ECM) fiber alignment, both in vitro and in vivo. ECMs derived from Sdc1-positive human mammary fibroblasts (HMF) showed an aligned fiber architecture, which contrasted markedly with the more random fiber arrangement in the ECM produced by Sdc1-negative HMFs. We further demonstrated that aligned fiber architecture promotes the directional migration and invasion of breast carcinoma cells. To decipher the molecular mechanisms governing the formation of an aligned, invasion-permissive ECM, a series of Sdc1 mutants was introduced into HMF. We found that both the ectodomain and heparan sulfate chains of Sdc1 were required for full activity of Sdc1 in regulating ECM alignment, while transmembrane and cytoplasmic domains were dispensable. Sdc1 regulates the activities of several integrins via its ectodomain. Integrins are key players in the assembly of fibronectin-rich ECM. In addition, integrins are capable of regulating cell morphology and cell shape and orientation may affect ECM architecture. Therefore, we investigated the role of integrins in Sdc1-mediated ECM fiber alignment. Sdc1-overexpressing HMF gained an enhanced spindle-shaped morphology when cultured in an overconfluent state under conditions permissive for ECM production, which was partially reversed by siRNA-mediated silencing of β3 integrin expression. Moreover, suppression of αvβ3 integrin activity by a function-blocking antibody or β3 knockdown largely abolished the aligned ECM fiber architecture and consequently the invasion-permissive properties of the ECM induced by Sdc1. The results suggest that Sdc1 may modulate fibronectin fibrillogenesis and/or alter cell morphology during ECM production through αvβ3 integrin, thereby mediating ECM fiber alignment. Understanding the mechanisms governing ECM organization may lead to the development of novel stroma-targeted therapy for breast cancer, aiming at converting an invasion-permissive to an invasion-restrictive microenvironment.     

Identification and Characterization of a G Protein-binding Cluster in α7 Nicotinic Acetylcholine Receptors

α7 nicotinic acetylcholine receptors (nAChRs) play an important role in synaptic transmission and inflammation. In response to ligands, this receptor channel opens to conduct cations into the cell but desensitizes rapidly. In recent studies we show that α7 nAChRs bind signaling proteins such as heterotrimeric GTP-binding proteins (G proteins). Here, we demonstrate that direct coupling of α7 nAChRs to G proteins enables a downstream calcium signaling response that can persist beyond the expected time course of channel activation. This process depends on a G protein-binding cluster (GPBC) in the M3-M4 loop of the receptor. A mutation of the GPBC in the α7 nAChR (α7_345–348A) abolishes interaction with Gα_q as well as Gβγ while having no effect on receptor synthesis, cell-surface trafficking, or α-bungarotoxin binding. Expression of α7_345–348A, however, did significantly attenuate the α7 nAChR-induced Gα_q calcium signaling response as evidenced by a decrease in PLC-β activation and IP₃R-mediated calcium store release in the presence of the α7 selective agonist choline. Taken together, the data provides new evidence for the existence of a GPBC in nAChRs serving to promote intracellular signaling.     

Nuclear Translocation of Calcium/Calmodulin-dependent Protein Kinase IIδ3 Promoted by Protein Phosphatase-1 Enhances Brain-derived Neurotrophic Factor Expression in Dopaminergic Neurons

We have reported previously that dopamine D₂ receptor stimulation activates calcium/calmodulin-dependent protein kinase II (CaMKII) δ3, a CaMKII nuclear isoform, increasing BDNF gene expression. However, the mechanisms underlying that activity remained unclear. Here we report that CaMKIIδ3 is dephosphorylated at Ser³³² by protein phosphatase 1 (PP1), promoting CaMKIIδ3 nuclear translocation. Neuro-2a cells transfected with CaMKIIδ3 showed cytoplasmic and nuclear staining, but the staining was predominantly nuclear when CaMKIIδ3 was coexpressed with PP1. Indeed, PP1 and CaMKIIδ3 coexpression significantly increased nuclear CaMKII activity and enhanced BDNF expression. In support of this idea, chronic administration of the dopamine D₂ receptor partial agonist aripiprazole increased PP1 activity and promoted nuclear CaMKIIδ3 translocation and BDNF expression in the rat brain substantia nigra. Moreover, aripiprazole treatment enhanced neurite extension and inhibited cell death in cultured dopaminergic neurons, effects blocked by PP1γ knockdown. Taken together, nuclear translocation of CaMKIIδ3 following dephosphorylation at Ser³³² by PP1 likely accounts for BDNF expression and subsequent neurite extension and survival of dopaminergic neurons.     

The syndecan-1 ectodomain regulates alphavbeta3 integrin activity in human mammary carcinoma cells

The α_vβ₃ integrin participates in cell morphogenesis, growth factor signaling, and cell survival. Activation of the integrin is central to these processes and is influenced by specific ECM components, which engage both integrins and syndecans. This paper demonstrates that the α_vβ₃ integrin and syndecan-1 (S1) are functionally coupled. The integrin is dependent on the syndecan to become activated and to mediate signals required for MDA-MB-231 and MDA-MB-435 human mammary carcinoma cell spreading on vitronectin or S1-specific antibody. Coupling of the syndecan to α_vβ₃ requires the S1 ectodomain (ED), as ectopic expression of glycosylphosphatidylinositol-linked S1ED enhances α_vβ₃ recognition of vitronectin; and treatments that target this domain, including competition with recombinant S1ED protein or anti-S1ED antibodies, mutation of the S1ED, or down-regulation of S1 expression by small-interfering RNAs, disrupt α_vβ₃-dependent cell spreading and migration. Thus, S1 is likely to be a critical regulator of many cellular behaviors that depend on activated α_vβ₃ integrins.     

Structure and Function of the Hypertension Variant A486V of G Protein-coupled Receptor Kinase 4

G-protein-coupled receptor (GPCR) kinases (GRKs) bind to and phosphorylate GPCRs, initiating the process of GPCR desensitization and internalization. GRK4 is implicated in the regulation of blood pressure, and three GRK4 polymorphisms (R65L, A142V, and A486V) are associated with hypertension. Here, we describe the 2.6 Å structure of human GRK4α A486V crystallized in the presence of 5′-adenylyl β,γ-imidodiphosphate. The structure of GRK4α is similar to other GRKs, although slight differences exist within the RGS homology (RH) bundle subdomain, substrate-binding site, and kinase C-tail. The RH bundle subdomain and kinase C-terminal lobe form a strikingly acidic surface, whereas the kinase N-terminal lobe and RH terminal subdomain surfaces are much more basic. In this respect, GRK4α is more similar to GRK2 than GRK6. A fully ordered kinase C-tail reveals interactions linking the C-tail with important determinants of kinase activity, including the αB helix, αD helix, and the P-loop. Autophosphorylation of wild-type GRK4α is required for full kinase activity, as indicated by a lag in phosphorylation of a peptide from the dopamine D₁ receptor without ATP preincubation. In contrast, this lag is not observed in GRK4α A486V. Phosphopeptide mapping by mass spectrometry indicates an increased rate of autophosphorylation of a number of residues in GRK4α A486V relative to wild-type GRK4α, including Ser-485 in the kinase C-tail.     

Design,synthesis and biological evaluation of a series of dianilinopyrimidines as EGFR inhibitors

This paper described our efforts to develop dianilinopyrimidines as novel EGFR inhibitors. All the target compounds were tested for inhibitory effects against wild type EGFR (EGFR^wt) and three tumour cells, including A549, PC-3, and HepG2. Some of the compounds performed well in antitumor activities. Especially, compound 4c 2-((2-((4-(3-fluorobenzamido)phenyl)amino)-5-(trifluoromethyl) pyrimidin-4-yl)amino)-N-methylthiophene-3-carboxamide showed higher anti-tumour activities than Gefitinib. The IC₅₀ values of compound 4c against A549, PC-3, and HepG2. reached 0.56 μM, 2.46 μM, and 2.21 μM, respectively. In addition, further studies indicated that compound 4c could induce apoptosis against A549 cells and arrest A549 cells in the G2/M phase. Molecular docking studies showed that compound 4c could closely interact with EGFR. Generally, compound 4c was the potential for developing into an anti-tumour drug.     

The Interaction of Protein-tyrosine Phosphatase α (PTPα) and RACK1 Protein Enables Insulin-like Growth Factor 1 (IGF-1)-stimulated Abl-dependent and -independent Tyrosine Phosphorylation of PTPα

Protein tyrosine phosphatase α (PTPα) promotes integrin-stimulated cell migration in part through the role of Src-phosphorylated PTPα-Tyr(P)-789 in recruiting and localizing p130Cas to focal adhesions. The growth factor IGF-1 also stimulates PTPα-Tyr-789 phosphorylation to positively regulate cell movement. This is in contrast to integrin-induced PTPα phosphorylation, that induced by IGF-1 can occur in cells lacking Src family kinases (SFKs), indicating that an unknown kinase distinct from SFKs can target PTPα. We show that this IGF-1-stimulated tyrosine kinase is Abl. We found that PTPα binds to the scaffold protein RACK1 and that RACK1 coordinates the IGF-1 receptor, PTPα, and Abl in a complex to enable IGF-1-stimulated and Abl-dependent PTPα-Tyr-789 phosphorylation. In cells expressing SFKs, IGF-1-stimulated phosphorylation of PTPα is mediated by RACK1 but is Abl-independent. Furthermore, expressing the SFKs Src and Fyn in SFK-deficient cells switches IGF-1-induced PTPα phosphorylation to occur in an Abl-independent manner, suggesting that SFK activity dominantly regulates IGF-1/IGF-1 receptor signaling to PTPα. RACK1 is a molecular scaffold that integrates growth factor and integrin signaling, and our identification of PTPα as a RACK1 binding protein suggests that RACK1 may coordinate PTPα-Tyr-789 phosphorylation in these signaling networks to promote cell migration.     

Stimulation of β1-Integrin Function by Epidermal Growth Factor and Heregulin-β Has Distinct Requirements for erbB2 but a Similar Dependence on Phosphoinositide 3-OH Kinase

Integrins and growth factor receptors are important participants in cellular adhesion and migration. The EGF receptor (EGFR) family of tyrosine kinases and the β1-integrin adhesion receptors are of particular interest, given the implication for their involvement in the initiation and progression of tumorigenesis. We used adhesion and chemotaxis assays to further elucidate the relationship between these two families of transmembrane signaling molecules. Specifically, we examined integrin-mediated adhesive and migratory characteristics of the metastatic breast carcinoma cell line MDA-MB-435 in response to stimulation with growth factors that bind to and activate the EGFR or erbB3 in these cells. Although ligand engagement of the EGFR stimulated modest β1-dependent increases in cell adhesion and motility, heregulin-β (HRGβ) binding to the erbB3 receptor initiated rapid and potent induction of breast carcinoma cell adhesion and migration and required dimerization of erbB3 with erbB2. Pharmacologic inhibitors of phosphoinositide 3-OH kinase (PI 3-K) or transient expression of dominant negative forms of PI 3-K inhibited both EGF- and HRGβ-mediated adhesion and potently blocked HRGβ- and EGF-induced cell motility. Our results illustrate the critical role of PI 3-K activity in signaling pathways initiated by the EGFR or erbB3 to up-regulate β1-integrin function.     

ST6Gal-I–mediated sialylation of the epidermal growth factor receptor modulates cell mechanics and enhances invasion

Heterogeneity within the glycocalyx influences cell adhesion mechanics and signaling. However, the role of specific glycosylation subtypes in influencing cell mechanics via alterations of receptor function remains unexplored. It has been shown that the addition of sialic acid to terminal glycans impacts growth, development, and cancer progression. In addition, the sialyltransferase ST6Gal-I promotes epidermal growth factor receptor (EGFR) activity, and we have shown EGFR is an ‘allosteric mechano-organizer’ of integrin tension. Here, we investigated the impact of ST6Gal-I on cell mechanics. Using DNA-based tension gauge tether probes of variable thresholds, we found that high ST6Gal-I activity promotes increased integrin forces and spreading in Cos-7 and OVCAR3, OVCAR5, and OV4 cancer cells. Further, employing inhibitors and function-blocking antibodies against β1, β3, and β5 integrins and ST6Gal-I targets EGFR, tumor necrosis factor receptor, and Fas cell surface death receptor, we validated that the observed phenotypes are EGFR-specific. We found that while tension, contractility, and adhesion are extracellular-signal-regulated kinase pathway-dependent, spreading, proliferation, and invasion are phosphoinositide 3-kinase-Akt serine/threonine kinase dependent. Using total internal reflection fluorescence microscopy and flow cytometry, we also show that high ST6Gal-I activity leads to sustained EGFR membrane retention, making it a key regulator of cell mechanics. Our findings suggest a novel sialylation-dependent mechanism orchestrating cellular mechanics and enhancing cell motility via EGFR signaling.     

Threonine 680 Phosphorylation of FLJ00018/PLEKHG2, a Rho Family-specific Guanine Nucleotide Exchange Factor,by Epidermal Growth Factor Receptor Signaling Regulates Cell Morphology of Neuro-2a Cells

FLJ00018/PLEKHG2 is a guanine nucleotide exchange factor for the small GTPases Rac and Cdc42 and has been shown to mediate the signaling pathways leading to actin cytoskeleton reorganization. The function of FLJ00018 is regulated by the interaction of heterotrimeric GTP-binding protein Gβγ subunits or cytosolic actin. However, the details underlying the molecular mechanisms of FLJ00018 activation have yet to be elucidated. In the present study we show that FLJ00018 is phosphorylated and activated by β₁-adrenergic receptor stimulation-induced EGF receptor (EGFR) transactivation in addition to Gβγ signaling. FLJ00018 is also phosphorylated and activated by direct EGFR stimulation. The phosphorylation of FLJ00018 by EGFR stimulation is mediated by the Ras/mitogen-activated protein kinase (MAPK) pathway. Through deletion and site-directed mutagenesis studies, we have identified Thr-680 as the major site of phosphorylation by EGFR stimulation. FLJ00018 T680A, in which the phosphorylation site is replaced by alanine, showed a limited response of the Neuro-2a cell morphology to EGF stimulation. Our results provide evidence that stimulation of the Ras/MAPK pathway by EGFR results in FLJ00018 phosphorylation at Thr-680, which in turn controls changes in cell shape.     

EGFR kinase activity is required for TLR4 signaling and the septic shock response

Mammalian Toll-like receptors (TLR) recognize microbial products and elicit transient immune responses that protect the infected host from disease. TLR4—which signals from both plasma and endosomal membranes—is activated by bacterial lipopolysaccharides (LPS) and induces many cytokine genes, the prolonged expression of which causes septic shock in mice. We report here that the expression of some TLR4-induced genes in myeloid cells requires the protein kinase activity of the epidermal growth factor receptor (EGFR). EGFR inhibition affects TLR4-induced responses differently depending on the target gene. The induction of interferon-β (IFN-β) and IFN-inducible genes is strongly inhibited, whereas TNF-α induction is enhanced. Inhibition is specific to the IFN-regulatory factor (IRF)-driven genes because EGFR is required for IRF activation downstream of TLR—as is IRF co-activator β-catenin—through the PI3 kinase/AKT pathway. Administration of an EGFR inhibitor to mice protects them from LPS-induced septic shock and death by selectively blocking the IFN branch of TLR4 signaling. These results demonstrate a selective regulation of TLR4 signaling by EGFR and highlight the potential use of EGFR inhibitors to treat septic shock syndrome.