Epidermal growth factor-induced activation and translocation of c-Src to the cytoskeleton depends on the actin binding domain of the EGF-receptor

In the epidermal growth factor (EGF)-receptor signal transduction cascade, the non-receptor tyrosine kinase c-Src has been demonstrated to become activated upon EGF stimulation. In this paper we show that c-Src associates with the cytoskeleton and co-isolates with actin filaments upon EGF treatment of NIH-3T3 cells transfected with the EGF receptor. Immunofluorescence studies using CLSM show colocalization of F-actin and endogenous c-Src predominantly around endosomes and not on stress fibers and cell–cell contacts. Stimulation of EGF receptor-transfected NIH-3T3 cells with EGF induces an activation and translocation of c-Src to the cytoskeleton. These processes depend upon the presence of the actin binding domain of the EGF-receptor since in cells that express EGF-receptors lacking this domain, EGF fails to induce an activation and translocation to the cytoskeleton of c-Src. These data suggest a role for the actin binding domain of the EGF-receptor in the translocation of c-Src.     

Ubiquitin binding and conjugation regulate the recruitment of Rabex-5 to early endosomes

Rab GTPases and ubiquitination are critical regulators of transmembrane cargo sorting in endocytic and lysosomal targeting pathways. The endosomal protein Rabex-5 intersects these two layers of regulation by being both a guanine nucleotide exchange factor (GEF) for Rab5 and a substrate for ubiquitin (Ub) binding and conjugation. The ability of trafficking machinery components to bind ubiquitinated proteins is known to have a function in cargo sorting. Here, we demonstrate that Ub binding is essential for the recruitment of Rabex-5 from the cytosol to endosomes, independently of its GEF activity and of Rab5. We also show that monoubiquitinated Rabex-5 is enriched in the cytosol. These observations are consistent with a model whereby a cycle of Ub binding and monoubiquitination regulates the association of Rabex-5 with endosomes.     

Role of actin cytoskeleton in LPS-induced NF-kappaB activation and nitric oxide production in murine macrophages

Lipopolysaccharide (LPS) is a major cell wall component of Gram-negative bacteria and is known to cause actin cytoskeleton reorganization in a variety of cells including macrophages. Actin cytoskeleton dynamics influence many cell signaling pathways including the NF-kappaB pathway. LPS is also known to induce the expression of many pro-inflammatory genes via the NF-kappaB pathway. Here, we have investigated the role of actin cytoskeleton in LPS-induced NF-kappaB activation and signaling leading to the expression of iNOS and nitric oxide production. Using murine macrophages, we show that disruption of actin cytoskeleton by either cytochalasin D (CytD) or latrunculin B (LanB) does not affect LPS-induced NF-kappaB activation and the expression of iNOS, a NF-kappaB target gene. However, disruption of actin cytoskeleton caused significant reduction in LPS-induced nitric oxide production indicating a role of actin cytoskeleton in the post-translational regulation of iNOS.     

Epidermal growth factor-mediated activation of an S6 kinase in Swiss mouse 3T3 cells

Extracts from epidermal growth factor (EGF)-stimulated Swiss mouse 3T3 cells are up to 10 times more potent in phosphorylating ribosomal protein S6 than extracts from quiescent cells. Preparation of extracts in the absence of phosphatase inhibitors leads to a time-dependent loss of kinase activity. In order of potency, the most efficient phosphatase inhibitors in protecting the S6 kinase activity are phosphotyrosine followed by p-nitrophenyl phosphate, beta-glycerol phosphate, and phosphoserine. The kinetics of kinase activation following EGF treatment are rapid and transient. The maximum increase is observed between 15 and 30 min with only 20-30% of the activity remaining after 2 h. Phosphorylation of S6 in the intact cell follows a similar pattern of activation, reaching a maximum between 30 and 60 min and then slowly returning to basal levels by approximately 3 h. The activation of protein synthesis is also rapid; however, in contrast to the transient activation of the S6 kinase and S6 phosphorylation, it remains persistently high for at least 6 h following EGF treatment. Comparison of these events with EGF binding shows that about 50% of the cell surface binding sites are lost within 10 min of exposure to EGF, and about 25% remain after 2 h. Finally, sodium orthovanadate, which is known to mimic the mitogenic effect of EGF, also leads to activation of the S6 kinase, however, with distinct kinetics and by an apparent EGF receptor-independent pathway.     

Par-4-mediated recruitment of Amida to the actin cytoskeleton leads to the induction of apoptosis

Par-4 (prostate apoptosis response-4) sensitizes cells to apoptotic stimuli, but the exact mechanisms are still poorly understood. Using Par-4 as bait in a yeast two-hybrid screen, we identified Amida as a novel interaction partner, a ubiquitously expressed protein which has been suggested to be involved in apoptotic processes. Complex formation of Par-4 and Amida occurs in vitro and in vivo and is mediated via the C-termini of both proteins, involving the leucine zipper of Par-4. Amida resides mainly in the nucleus but displays nucleo-cytoplasmic shuttling in heterokaryons. Upon coexpression with Par-4 in REF52.2 cells, Amida translocates to the cytoplasm and is recruited to actin filaments by Par-4, resulting in enhanced induction of apoptosis. The synergistic effect of Amida/Par-4 complexes on the induction of apoptosis is abrogated when either Amida/Par-4 complex formation or association of these complexes with the actin cytoskeleton is impaired, indicating that the Par-4-mediated relocation of Amida to the actin cytoskeleton is crucial for the pro-apoptotic function of Par-4/Amida complexes in REF52.2 cells. The latter results in enhanced phosphorylation of the regulatory light chain of myosin II (MLC) as has previously been shown for Par-4-mediated recruitment of DAP-like kinase (Dlk), suggesting that the recruitment of nuclear proteins involved in the regulation of apoptotic processes to the actin filament system by Par-4 represents a potent mechanism how Par-4 can trigger apoptosis.     

The actin cytoskeleton is required for the trafficking of the B cell antigen receptor to the late endosomes

The B cell antigen receptor (BCR) plays two central roles in B cell activation: to internalize antigens for processing and presentation, and to initiate signal transduction cascades that both promote B cells to enter the cell cycle and facilitate antigen processing by accelerating antigen transport. An early event in B cell activation is the association of BCR with the actin cytoskeleton, and an increase in cellular F-actin. Current evidence indicates that the organization of actin filaments changes in response to BCR-signaling, making actin filaments good candidates for regulation of BCR-antigen targeting. Here, we have analyzed the role of actin filaments in BCR-mediated antigen transport, using actin filament-disrupting reagents, cytochalasin D and latrunculin B, and an actin filament-stabilizing reagent, jasplakinolide. Perturbing actin filaments, either by disrupting or stabilizing them, blocked the movement of BCR from the plasma membrane to late endosomes/lysosomes. Cytochalasin D-treatment dramatically reduced the rate of internalization of BCR, and blocked the movement of the BCR from early endosomes to late endosomes/lysosomes, without affecting BCR-signaling. Thus, BCR-trafficking requires functional actin filaments for both internalization and movement to late endosomes/lysosomes, defining critical control points in BCR-antigen targeting.     

Annexin V relocates to the platelet cytoskeleton upon activation and binds to a specific isoform of actin.

We have previously reported that stimulation of platelets causes a relocation of annexin V to the cytoplasmic side of the plasma membrane where it associates with actin. This study examined the association of annexin V with the platelet cytoskeleton and its binding to actin, following both physiological activation with thrombin and Ca2+ -ionophore activation. The time-dependence of annexin V incorporation into the detergent-extracted cytoskeleton following activation with thrombin was also measured. Although calcium from the intracellular stores was enough to relocate intracellular annexin V to the cytoskeleton, this relocation was further enhanced by influx of extracellular calcium. The association of annexin V with the cytoskeleton was found to be unaffected by the action of cytochalasin E, however, annexin V was solubilized when DNase I was used to depolymerize the membrane cytoskeleton, and spontaneously re-associated with the actin filaments when re-polymerization was induced in vitro. Using a bifunctional crosslinking reagent we have identified an 85-kDa complex in both membrane and cytoskeleton fractions containing annexin V and actin. Direct binding to actin filaments was only observed in high [Ca2+], however, inclusion of an extract from thrombin-stimulated platelets lowered the [Ca2+] requirement for the binding of annexin V to F-actin to physiological levels. We also show that GST-annexin V mimics the physiological binding of annexin V to membranes, and that this GST-annexin V binds directly to a specific isoform of actin. Immunoprecipitation using antibodies against annexin V copurify annexin V and gamma- but not beta-actin from activated platelets. This is the first report of a possible preferential binding of annexin V to a specific isoform of actin, namely gamma-actin. The results of this study suggest a model in which annexin V that relocates to the plasma membrane and binds to gamma-actin in an activation-dependent manner forms a strong association with the platelet cytoskeleton.     

Induction of connective tissue growth factor by activation of heptahelical receptors. Modulation by Rho proteins and the actin cytoskeleton

Expression of connective tissue growth factor (CTGF) was induced in renal mesangial cells by activation of heptahelical receptors by serotonin (5-HT) and lysophosphatidic acid (LPA). Induction of CTGF mRNA was transient with maximal expression after 1 to 2 h, whereas induction of CTGF by transforming growth factor beta (TGF-beta) increased over time. In contrast to the induction of other early response genes (Egr-1 and cyclooxygenase-2), LPA-mediated induction of CTGF was pertussis toxin-insensitive and independent of p42/44 MAP kinase activation. 5-HT-mediated CTGF induction was due to activation of 5-HT(2A) receptors and likewise independent of p42/44 MAP kinase activation. Upon stimulation, enhanced levels of CTGF protein were detected in cellular homogenates, whereas no protein was detectable in cell culture supernatants. Inhibition of proteins of the Rho family by toxin B abrogated basal as well as CTGF expression stimulated by LPA, 5-HT, and TGF-beta. Inhibition of the downstream mediator of RhoA, the Rho kinase by Y-27632 partially reduced induction of CTGF by LPA and TGF-beta. Toxin B not only affected gene expression, but disrupted the actin cytoskeleton similarly as observed after treatment with cytochalasin D. Disassembly of actin stress fibers by cytochalasin D partially reduced basal and stimulated CTGF expression. These data indicate that an intact actin cytoskeleton is critical for the expression of CTGF. Elimination of the input of Rho proteins by toxin B, however, was significantly more effective and their effect on CTGF expression thus goes beyond disruption of the cytoskeleton. These findings thus establish activation of heptahelical receptors coupled to pertussis toxin-insensitive G proteins as a novel signaling pathway to induce CTGF. Proteins of the Rho family and an intact cytoskeleton were identified as critical determinants of CTGF expression induced by LPA and 5-HT, and also by TGF-beta.     

DAP12 couples c-Fms activation to the osteoclast cytoskeleton by recruitment of Syk

We examined the mechanism by which M-CSF regulates the cytoskeleton and function of the osteoclast, the exclusive bone resorptive cell. We show that binding of M-CSF to its receptor c-Fms generates a signaling complex comprising phosphorylated DAP12, an adaptor containing an immunoreceptor tyrosine-based activation motif (ITAM) and the nonreceptor tyrosine kinase Syk. c-Fms tyrosine 559, the exclusive binding site of c-Src, is necessary for regulation of DAP12/Syk signaling. Deletion of either of these molecules yields osteoclasts that fail to reorganize their cytoskeleton. Retroviral transduction of null precursors with wild-type or mutant DAP12 or Syk reveals that the SH2 domain of Syk and the ITAM tyrosine residues and transmembrane domain of DAP12 mediate M-CSF signaling. Our data provide genetic and biochemical evidence that uncovers an epistatic signaling pathway linking the receptor tyrosine kinase c-Fms to the immune adaptor DAP12 and the cytoskeleton.     

F-BAR-containing adaptor CIP4 localizes to early endosomes and regulates Epidermal Growth Factor Receptor trafficking and downregulation

Cdc42-Interacting Protein-4 (CIP4) family adaptors have been implicated in promoting Epidermal Growth Factor Receptor (EGFR) internalization, however, their unique or overlapping functions remain unclear. Here, we show that although CIP4 was not required for early events in clathrin-mediated endocytosis of EGFR, CIP4 localizes to vesicles containing EGFR and Rab5. Furthermore, expression of constitutively active Rab5 led to accumulation of CIP4 and the related adaptor Toca-1 in giant endosomes. Using a mutagenesis approach, we show that localization of CIP4 to endosomes is mediated in part via the curved phosphoinositide-binding face of the CIP4 F-BAR domain. Downregulation of CIP4 in A431 epidermoid carcinoma cells by RNA interference led to elevated EGFR levels, compared to control cells. Although surface expression of EGFR was not affected by CIP4 silencing, EGF-induced transit of EGFR from EEA1-positive endosomes to lysosomes was reduced compared to control cells. This correlated with more robust activation of ERK kinase and entry to S phase in CIP4-depleted A431 cells, compared to control cells. The combined silencing of CIP4 and Toca-1 was more effective in driving cells into S phase, suggesting a partial redundancy in their functions. Overall, our results implicate CIP4 and Toca-1 in regulating late events in EGFR trafficking from endosomes that serves to limit sustained ERK activation within the endosomal compartment.     

Regional myocardial ischemia-induced activation of MAPKs is associated with subcellular redistribution of caveolin and cholesterol

Ischemia-reperfusion activates ERK and p38 MAPK in cardiac membranes, but the role of caveolae in MAPK signaling during this stress has not been studied. The purpose of this study was to determine the effect of in vivo myocardial ischemia-reperfusion on the level and distribution of caveolin-1 and -3 and cholesterol as well as MAPK activation in caveolin-enriched fractions. Adult male rats were subjected to in vivo regional myocardial ischemia induced by 25 min of coronary artery occlusion and 10 min (n = 5) or 2 h (n = 4) of reperfusion. Another group of rats served as appropriate nonischemic time controls (n = 4). A discontinuous sucrose density gradient was used to isolate caveolae/lipid rafts from ischemic and nonischemic heart tissue. Caveolin-1 and -3, as well as cholesterol, were enriched in the light fractions. A redistribution of caveolin-3 and a reduction in caveolin-1 and cholesterol levels in the light fractions occurred after 10 min of reperfusion. The ERKs were activated in ischemic zone light and heavy fractions by 10 min of reperfusion. p44 ERK was activated after 2 h of reperfusion only in the light fractions, whereas p42 ERK phosphorylation was increased in the light and heavy fractions. Although no p38 MAPK activation occurred after 10 min of reperfusion, 2 h of reperfusion caused significant activation of p38 MAPK in nonischemic zone light and heavy fractions. These results show the importance of caveolar membrane/lipid rafts in MAPK signaling and suggest that subcellular compartmentation of p44/p42 ERKs and p38 MAPK may play distinct roles in the response to myocardial ischemia-reperfusion.     

Signaling from E-cadherins to the MAPK pathway by the recruitment and activation of epidermal growth factor receptors upon cell-cell contact formation

E-cadherins are well characterized cell surface molecules expressed in epithelial cells, which play a major role in cell adhesion through the establishment of calcium-dependent homophilic interactions at sites of cell-cell contacts. They are also integral components of morphogenetic programs controlling the maintenance of the structural and functional integrity of epithelia. Accumulated evidence indicates that the E-cadherin-mediated cell adhesion system is highly regulated from inside the cells by a number of intracellular signaling pathways. Recently available information suggests that E-cadherins may also play a role in the transduction of signals from the outside of the cell to the cytoplasm. However, the nature of the biochemical routes regulated by E-cadherins is still largely unknown. In this study, we set out to explore the possibility that E-cadherins may regulate the activity of MAPK, a key signaling pathway involved in cell fate decisions, upon the formation of cell-cell contacts among neighboring cells. By using an immortalized non-tumorigenic keratinocyte cell line, HaCat, as a model system, we provide evidence that the assembly of calcium-dependent adherens junctions leads to a rapid and remarkable increase in the state of activation of MAPK and that this event is mediated by E-cadherins. Furthermore, we found that E-cadherins stimulate the MAPK pathway through the ligand-independent activation of epidermal growth factor receptors and the consequent activation of a biochemical route leading to the stimulation of MAPKs. These findings suggest that E-cadherins can initiate outside-in signal transducing pathways through the engagement of tyrosine kinase receptors for epidermal growth factor, thus providing a novel molecular mechanism whereby these cell adhesion molecules may ultimately control the fate of normal and transformed epithelial cells.     

Distinct pathways for the early recruitment of myosin II and actin to the cytokinetic furrow

Equatorial organization of myosin II and actin has been recognized as a universal event in cytokinesis of animal cells. Current models for the formation of equatorial cortex favor either directional cortical transport toward the equator or localized de novo assembly. However, this process has never been analyzed directly in dividing mammalian cells at a high resolution. Here we applied total internal reflection fluorescence microscope (TIRF-M), coupled with spatial temporal image correlation spectroscopy (STICS) and a new analytical approach termed temporal differential microscopy (TDM), to image the dynamics of myosin II and actin during the assembly of equatorial cortex. Our results indicated distinct and at least partially independent mechanisms for the early equatorial recruitment of myosin and actin filaments. Cortical myosin showed no detectable directional flow during early cytokinesis. In addition to equatorial assembly, we showed that localized inhibition of disassembly contributed to the formation of the equatorial myosin band. In contrast to myosin, actin filaments underwent a striking flux toward the equator. Myosin motor activity was required for the actin flux, but not for actin concentration in the furrow, suggesting that there was a flux-independent, de novo mechanism for actin recruitment along the equator. Our results indicate that cytokinesis involves signals that regulate both assembly and disassembly activities and argue against mechanisms that are coupled to global cortical movements.     

Sequential processing of epidermal growth factor in early and late endosomes of rat liver.

We have used isolated perfused rat livers to examine the intracellular processing of 125I-epidermal growth factor (EGF) and to determine where in the endocytic pathway the hydrolases which degrade EGF are acting. Following uptake of 125I-EGF at 37 or 16 degrees C, subcellular fractions enriched in endosomes and lysosomes were isolated, and their 125I-EGF content was examined by reverse-phase high performance liquid chromatography. Three forms of EGF processed at their carboxyl termini are generated in endosomes. At 37 degrees C, EGF is first processed in early endosomes by a carboxypeptidase B-like protease and is further processed in late endosomes by a trypsin-like protease and then a carboxypeptidase B-like protease. At 16 degrees C, entry of EGF into late endosomes is slowed, and only the first processed form is generated over 60 min. Longer perfusions (180 min) at 16 degrees C result in some processing (7%) by proteases found in late endosomes. EGF-horseradish peroxidase cytochemistry confirmed that the additional processing detected at 180 min correlated with movement of EGF from tubulovesicular to multivesicular endosomes. These results, combined with in vitro incubations of EGF in isolated endosomal and lysosomal fractions, suggest that different proteases are active at selective points in the endocytic pathway and that the full complement of proteases needed for complete degradation of EGF is active only in lysosomes.     

Selective membrane recruitment of EEA1 suggests a role in directional transport of clathrin-coated vesicles to early endosomes

The molecular mechanisms ensuring directionality of endocytic membrane trafficking between transport vesicles and target organelles still remain poorly characterized. We have been investigating the function of the small GTPase Rab5 in early endocytic transport. In vitro studies have demonstrated a role of Rab5 in two membrane fusion events: the heterotypic fusion between plasma membrane-derived clathrin-coated vesicles (CCVs) and early endosomes and in the homotypic fusion between early endosomes. Several Rab5 effectors are required in homotypic endosome fusion, including EEA1, which mediates endosome membrane docking, as well as Rabaptin-5 x Rabex-5 complex and phosphatidylinositol 3-kinase hVPS34. In this study we have examined the localization and function of Rab5 and its effectors in heterotypic fusion in vitro. We report that the presence of active Rab5 is necessary on both CCVs and early endosomes for a heterotypic fusion event to occur. This process requires EEA1 in addition to the Rabaptin-5 complex. However, whereas Rab5 and Rabaptin-5 are symmetrically distributed between CCVs and early endosomes, EEA1 is recruited selectively onto the membrane of early endosomes. Our results suggest that EEA1 is a tethering molecule that provides directionality to vesicular transport from the plasma membrane to the early endosomes.     

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.     

Epidermal growth factor stimulation of trophoblast differentiation requires MAPK11/14 (p38 MAP kinase) activation

Cultured human term villous cytotrophoblasts (CT) have been reported to be nonproliferating but differentiate when exposed to epidermal growth factor (EGF). Here we show that CT differentiate into chorionic gonadotropin (beta-hCG/CGB)-expressing cells when cultured with medium alone. The addition of EGF decreases CGB secretion and prolongs production for up to 13 days. EGF stimulates the phosphorylation (activation) of the signaling intermediate p38 (MAPK11/14), and blocking phosphorylation pharmacologically with either SB203580 or SB202190 strongly inhibited spontaneous and EGF-stimulated secretion of CGB. In addition, EGF-stimulated fusion of cytotrophoblasts into syncytial units was strongly inhibited by SB203580. EGF upregulated trophoblast proliferation (measured by bromodeoxyuridine uptake) and SB203580 increased this proliferation after 5 days. In agreement with these observations, EGF and SB203580 increased expression of the G1-phase-specific gene cyclin-D1 (CCND1) and SB203580 downmodulated its inhibitor p21 (CDKN1A). When added to villous explant cultures, EGF did nothing to the pattern of CGB secretion, but addition of SB203580 prevented the normal surge in secretion during syncytial regeneration over Days 3-7. These data support the hypothesis that EGF-stimulated cytotrophoblast differentiation to syncytium requires MAPK11/14 activation, and that cytotrophoblast proliferation can be stimulated in culture by EGF and enhanced by MAPK11/14 inhibition with a consequent reduction of differentiation.