Chemokine receptors CXCR-1/2 activate mitogen-activated protein kinase via the epidermal growth factor receptor in ovarian cancer cells

Ovarian cancer typically disseminates widely in the abdomen, a characteristic that limits curative therapy. The mechanisms that promote ovarian cancer cell migration are incompletely understood. We studied model SK-OV-3 ovarian cancer cells and observed robust expression of the alpha chemokine receptors CXCR-1 and CXCR-2. Interleukin-8 (IL-8) treatment caused shape changes in the cells, with membrane ruffling and formation/retraction of thin actin-like projections, as detected by time-lapse microscopy. Stimulation of the CXCR-1/2 receptors by human interleukin 8 (IL-8) rapidly activated the p44/42 mitogen-activated protein (extracellular signal-regulated kinase (Erk1/2)) kinase pathway. Treatment of SK-OV-3 cells with the inhibitors genestein and herbimycin A indicated that tyrosine kinases were involved in the IL-8 activation of Erk1 and Erk2. Of note, IL-8 induced transient phosphorylation of the epidermal growth factor (EGF) receptor and its association with the adaptor molecules Shc and Grb2. This transactivation of the EGF receptor was dependent on intracellular Ca(2+) mobilization. Furthermore AG1478, a specific inhibitor of the EGF receptor kinase, blocked Erk1 and Erk2 activation. c-Src kinase was not involved in the IL-8-mediated phosphorylation of the EGF receptor, but was critical for Shc phosphorylation and downstream Erk1/2 kinase activation. These results suggest important "cross-talk" between chemokine and growth factor pathways that may link signals of cell migration and proliferation in ovarian cancer.     

Protein kinase C regulates integrin-induced activation of the extracellular regulated kinase pathway upstream of Shc

Adhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, we monitored the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells using pharmacological and genetic approaches. We found that inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduced extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCalpha, PKCdelta, or PKCepsilon was sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms were activated following adhesion. PKC was required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc.     

Muscarinic M3 and epidermal growth factor receptors activate mutually inhibitory signaling cascades in human neuroblastoma SH-SY5Y cells

Regulatory interactions among individual receptor-coupled signal transduction systems are critically important for establishing cellular responses in the face of multiple stimuli. In this study, potential regulatory interactions between signal transduction systems activated by growth factor receptors and by G-protein-coupled receptors were examined using human neuroblastoma SH-SY5Y cells which express endogenous epidermal growth factor (EGF) and muscarinic M3 receptors. Activation of muscarinic receptors with carbachol was found to inhibit EGF-induced signaling, including tyrosine phosphorylation of the adaptor protein Cbl and of the EGF receptor, and complex formation between Shc proteins and the EGF receptor and Grb2. Protein kinase C, which is activated by muscarinic M3 receptors, mediated this inhibitory cross-talk. Activation of EGF receptors was found to inhibit muscarinic receptor-induced tyrosine phosphorylation of focal adhesion kinase and paxillin. Reactive oxygen species, which are formed as components of the EGF signaling cascade, mediated this inhibitory cross-talk. These mutual inhibitory interactions demonstrate novel mechanisms for neuronal integration of multiple signals generated by activation of receptors by neurotransmitters and growth factors.     

Plasma membrane phospholipid scramblase 1 promotes EGF-dependent activation of c-Src through the epidermal growth factor receptor

Phospholipid scramblase (PLSCR1) is a multiply palmitoylated, calcium-binding endofacial membrane protein proposed to mediate transbilayer movement of plasma membrane phospholipids. PLSCR1 is a component of membrane lipid rafts and has been shown to both physically and functionally interact with activated epidermal growth factor (EGF) receptors and other raft-associated cell surface receptors. Cell stimulation by EGF results in Tyr phosphorylation of PLSCR1, its association with both Shc and EGF receptors, and rapid cycling of PLSCR1 between plasma membrane and endosomal compartments. We now report evidence that upon EGF stimulation, PLSCR1 is phosphorylated by c-Src, within the tandem repeat sequence 68VYNQPVYNQP77. The in vivo interaction between PLSCR1 and Shc requires the Src-mediated phosphorylation on tyrosines 69 and 74. In in vitro pull down studies, phosphorylated PLSCR1 was found to bind directly to Shc through the phosphotyrosine binding domain. Consistent with the potential role of PLSCR1 in growth factor signaling pathways, granulocyte precursors derived from mice deficient in PLSCR1 show impaired proliferation and maturation under cytokine stimulation. Using PLSCR1-/- embryonic fibroblasts and kidney epithelial cells, we now demonstrate that deletion of PLSCR1 from the plasma membrane reduces the activation of c-Src by EGF, implying that PLSCR1 normally facilitates receptor-dependent activation of this kinase. We propose that PLSCR1, through its interaction with Shc, promotes Src kinase activation through the EGF receptor.     

Protein kinase Calpha activates c-Src and induces podosome formation via AFAP-110

We report that the actin filament-associated protein AFAP-110 is required to mediate protein kinase Calpha (PKCalpha) activation of the nonreceptor tyrosine kinase c-Src and the subsequent formation of podosomes. Immunofluorescence analysis demonstrated that activation of PKCalpha by phorbol 12-myristate 13-acetate (PMA), or ectopic expression of constitutively activated PKCalpha, directs AFAP-110 to colocalize with and bind to the c-Src SH3 domain, resulting in activation of the tyrosine kinase. Activation of c-Src then directs the formation of podosomes, which contain cortactin, AFAP-110, actin, and c-Src. In a cell line (CaOV3) that has very little or no detectable AFAP-110, PMA treatment was unable to activate c-Src or effect podosome formation. Ectopic expression of AFAP-110 in CaOV3 cells rescued PKCalpha-mediated activation of c-Src and elevated tyrosine phosphorylation levels and subsequent formation of podosomes. Neither expression of activated PKCalpha nor treatment with PMA was able to induce these changes in CAOV3 cells expressing mutant forms of AFAP-110 that are unable to bind to, or colocalize with, c-Src. We hypothesize that one major function of AFAP-110 is to relay signals from PKCalpha that direct the activation of c-Src and the formation of podosomes.     

EphB1 recruits c-Src and p52Shc to activate MAPK/ERK and promote chemotaxis

Eph receptors and their ligands (ephrins) play an important role in axonal guidance, topographic mapping, and angiogenesis. The signaling pathways mediating these activities are starting to emerge and are highly cell- and receptor-type specific. Here we demonstrate that activated EphB1 recruits the adaptor proteins Grb2 and p52Shc and promotes p52Shc and c-Src tyrosine phosphorylation as well as MAPK/extracellular signal-regulated kinase (ERK) activation. EphB1-mediated increase of cell migration was abrogated by the MEK inhibitor PD98059 and Src inhibitor PP2. In contrast, cell adhesion, which we previously showed to be c-jun NH2-terminal kinase (JNK) dependent, was unaffected by ERK1/2 and Src inhibition. Expression of dominant-negative c-Src significantly reduced EphB1-dependent ERK1/2 activation and chemotaxis. Site-directed mutagenesis experiments demonstrate that tyrosines 600 and 778 of EphB1 are required for its interaction with c-Src and p52Shc. Furthermore, phosphorylation of p52Shc by c-Src is essential for its recruitment to EphB1 signaling complexes through its phosphotyrosine binding domain. Together these findings highlight a new aspect of EphB1 signaling, whereby the concerted action of c-Src and p52Shc activates MAPK/ERK and regulates events involved in cell motility.     

Inducible phosphorylation of cortactin is not necessary for cortactin-mediated actin polymerisation

Cortactin is an SH3 domain-containing protein that contributes to the formation of dynamic cortical actin-associated structures, such as lamellipodia and membrane ruffles. It was originally identified as a substrate for the protein kinase Src; however, the role of tyrosine phosphorylation in the translocation of cortactin to the cell periphery and in the subsequent actin polymerisation is still unclear. Recently, two serine/threonine kinases, Pak1 and Erk, have been implicated in the regulation of cortactin. Therefore, we systematically investigated whether phosphorylation on either tyrosine or serine/threonine residues is necessary for cortactin function. In COS7 cells over-expressing Vav2 or treated with EGF, we could not detect tyrosine phosphorylation, although cortactin was translocated to cell periphery and induced membrane ruffle formation. In addition, the selective MEK inhibitor, PD98059, did not influence in vivo the ability of cortactin to bind to and induce membrane ruffles upon Vav2 over-expression or short-term EGF treatment. Finally, using a constitutively active Pak1 mutant, Pak1 T423E, we showed that Pak1 is not capable of phosphorylating cortactin either in vitro or in COS7 cells. These results suggest that cortactin-mediated actin polymerisation at cell periphery requires only Rac activation but neither tyrosine nor serine/threonine phosphorylation.     

Sequence-specific peptide aptamers,interacting with the intracellular domain of the epidermal growth factor receptor,interfere with Stat3 activation and inhibit the growth of tumor cells

Receptor tyrosine kinases of the epidermal growth factor (EGF) receptor family regulate essential cellular functions such as proliferation, survival, migration, and differentiation but also play central roles in the etiology and progression of tumors. We have identified short peptide sequences from a random peptide library integrated into the thioredoxin scaffold protein, which specifically bind to the intracellular domain of the EGF receptor (EGFR). These molecules have the potential to selectively inhibit specific aspects of EGF receptor signaling and might become valuable as anticancer agents. Intracellular expression of the aptamer encoding gene construct KDI1 or introduction of bacterially expressed KDI1 via a protein transduction domain into EGFR-expressing cells results in KDI1.EGF receptor complex formation, a slower proliferation, and reduced soft agar colony formation. Aptamer KDI1 did not summarily block the EGF receptor tyrosine kinase activity but selectively interfered with the EGF-induced phosphorylation of the tyrosine residues 845, 1068, and 1148 as well as the phosphorylation of tyrosine 317 of p46 Shc. EGF-induced phosphorylation of Stat3 at tyrosine 705 and Stat3-dependent transactivation were also impaired. Transduction of a short synthetic peptide aptamer sequence not embedded into the scaffold protein resulted in the same impairment of EGF-induced Stat3 activation.     

Insulin receptor substrates 1 and 2 but not Shc can activate the insulin receptor independent of insulin and induce proliferation in CHO-IR cells

Ligand-activated insulin receptor (IR) attracts and phosphorylates various substrates such as insulin receptor substrates 1-4 (IRS) and Shc. To investigate how binding affinity for substrate affects signalling we generated chimeric receptors with the beta-chain of the insulin receptor containing NPXY motives with different affinities for receptor substrates. We found that the extent of receptor tyrosine phosphorylation positively correlates with binding affinity towards IRS1/2 but not towards Shc. Moreover, overexpression of IRS1 or IRS2 but not of Shc increased IR tyrosine phosphorylation in a dose-dependent manner, also independent of insulin. Molecular truncations of IRS1 revealed that neither the isolated PH and PTB domains nor the C-terminus with the tyrosine phosphorylation sites alone are sufficient for substrate-dependent receptor activation. Overexpression of IRS1 and IRS2 impaired insulin-induced internalization of the IR in a dose-dependent manner suggesting that IRS proteins prevent endosome-associated receptor dephosphorylation/inactivation. IRS1 and IRS2 could therefore target the activated IR to different cellular compartments. Overexpression of IRS1 and IRS2 inhibited insulin-stimulated activation of the MAP kinases Erk1/2 while it increased/induced activation of Akt/PKB. Finally, overexpression of IRS1 and IRS2 but not of Shc induced DNA synthesis in starved CHO-IR cells independent of exogenous growth factors. Our results demonstrate that variations in cellular IRS1 and IRS2 concentration affect insulin signalling both upstream and downstream and that IRS proteins could play instructive rather than just permissive roles in signal transmission.     

Cholecystokinin stimulates extracellular signal-regulated kinase through activation of the epidermal growth factor receptor,Yes, and protein kinase C. Signal amplification at the level of Raf by activation of protein kinase Cepsilon

Cholecystokinin (CCK) and related peptides are potent growth factors in the gastrointestinal tract and may be important for human cancer. CCK exerts its growth modulatory effects through G(q)-coupled receptors (CCK(A) and CCK(B)) and activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). In the present study, we investigated the different mechanisms participating in CCK-induced activation of ERK1/2 in pancreatic AR42J cells expressing both CCK(A) and CCK(B). CCK activated ERK1/2 and Raf-1 to a similar extent as epidermal growth factor (EGF). Inhibition of EGF receptor (EGFR) tyrosine kinase or expression of dominant-negative Ras reduced CCK-induced ERK1/2 activation, indicating participation of the EGFR and Ras in CCK-induced ERK1/2 activation. However, compared with EGF, CCK caused only small increases in tyrosine phosphorylation of the EGFR and Shc, Shc-Grb2 complex formation, and Ras activation. Signal amplification between Ras and Raf in a CCK-induced ERK cascade appears to be mediated by activation of protein kinase Cepsilon (PKCepsilon), because 1) down-modulation of phorbol ester-sensitive PKCs inhibited CCK-induced activation of Ras, Raf, and ERK1/2 without influencing Shc-Grb2 complex formation; 2) PKCepsilon, but not PKCalpha or PKCdelta, was detectable in Raf-1 immunoprecipitates, although CCK activated all three PKC isoenzymes. In addition, the present study provides evidence that the Src family tyrosine kinase Yes is activated by CCK and mediates CCK-induced tyrosine phosphorylation of Shc. Furthermore, we show that CCK-induced activation of the EGFR and Yes is achieved through the CCK(B) receptor. Together, our data show that different signals emanating from the CCK receptors mediate ERK1/2 activation; activation of Yes and the EGFR mediate Shc-Grb2 recruitment, and activation of PKC, most likely PKCepsilon, augments CCK-stimulated ERK1/2 activation at the Ras/Raf level.     

Rap2B-dependent stimulation of phospholipase C-epsilon by epidermal growth factor receptor mediated by c-Src phosphorylation of RasGRP3

Receptor tyrosine kinase regulation of phospholipase C-epsilon (PLC-epsilon), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors. PLC and Ca(2+) signaling by the EGF receptor, which activated both PLC-gamma1 and PLC-epsilon, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B. EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-epsilon, and Rap2B-dependent translocation of PLC-epsilon to the plasma membrane. GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca(2+) and expression of lipase-inactive PLC-gamma1 but not of PLC-epsilon. Expression of RasGRP3, a Ca(2+)/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca(2+) signaling by the EGF receptor. EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation. Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-gamma1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-epsilon.     

Multiple Grb2-Mediated Integrin-Stimulated Signaling Pathways to ERK2/Mitogen-Activated Protein Kinase: Summation of Both c-Src- and Focal Adhesion Kinase-Initiated Tyrosine Phosphorylation Events

Fibronectin receptor integrin-mediated cell adhesion triggers intracellular signaling events such as the activation of the Ras/mitogen-activated protein (MAP) kinase cascade. In this study, we show that the nonreceptor protein-tyrosine kinases (PTKs) c-Src and focal adhesion kinase (FAK) can be independently activated after fibronectin (FN) stimulation and that their combined activity promotes signaling to extracellular signal-regulated kinase 2 (ERK2)/MAP kinase through multiple pathways upstream of Ras. FN stimulation of NIH 3T3 fibroblasts promotes c-Src and FAK association in the Triton-insoluble cell fraction, and the time course of FN-stimulated ERK2 activation paralleled that of Grb2 binding to FAK at Tyr-925 and Grb2 binding to Shc. Cytochalasin D treatment of fibroblasts inhibited FN-induced FAK in vitro kinase activity and signaling to ERK2, but it only partially inhibited c-Src activation. Treatment of fibroblasts with protein kinase C inhibitors or with the PTK inhibitor herbimycin A or PP1 resulted in reduced Src PTK activity, no Grb2 binding to FAK, and lowered levels of ERK2 activation. FN-stimulated FAK PTK activity was not significantly affected by herbimycin A treatment and, under these conditions, FAK autophosphorylation promoted Shc binding to FAK. In vitro, FAK directly phosphorylated Shc Tyr-317 to promote Grb2 binding, and in vivo Grb2 binding to Shc was observed in herbimycin A-treated fibroblasts after FN stimulation. Interestingly, c-Src in vitro phosphorylation of Shc promoted Grb2 binding to both wild-type and Phe-317 Shc. In vivo, Phe-317 Shc was tyrosine phosphorylated after FN stimulation of human 293T cells and its expression did not inhibit signaling to ERK2. Surprisingly, expression of Phe-925 FAK with Phe-317 Shc also did not block signaling to ERK2, whereas FN-stimulated signaling to ERK2 was inhibited by coexpression of an SH3 domain-inactivated mutant of Grb2. Our studies show that FN receptor integrin signaling upstream of Ras and ERK2 does not follow a linear pathway but that, instead, multiple Grb2-mediated interactions with Shc, FAK, and perhaps other yet-to-be-determined phosphorylated targets represent parallel signaling pathways that cooperate to promote maximal ERK2 activation.