Epidermal Growth Factor-stimulated Cells Research Articles

Novel tyrosine phosphorylated and cardiolipin-binding protein CLPABP functions as mitochondrial RNA granule

We identified a new protein containing the pleckstrin homology (PH) domain through tyrosine phosphoproteomics using epidermal growth factor-stimulated cells. The tandem PH domains of this protein can bind to mitochondria-specific phospholipid, cardiolipin or its dehydro product, phosphatidic acid; therefore, we have designated this protein as cardiolipin and phosphatidic acid-binding protein (CLPABP). In this study, we show that CLPABP is localized on the tubulin network and the mitochondrial surface in the granular form along with other proteins and RNA. The affinity of CLPABP to mitochondria is elevated depending on the extent of tyrosine phosphorylation. The CLPABP complex contains various proteins related to cytoplasmic mRNA metabolism. The unique subcellular localization of CLPABP requires its PH domains and a multifunctional protein, SF2p32, as its binding protein. The CLPABP granule also contains the cytochrome c transcript, which may be mediated by the RNA-binding protein HuR. Immunofluorescence staining reveals that the CLPABP granule is colocalized with cytochrome c and various ribosomal proteins that are present in the CLPABP complex. Therefore, the CLPABP RNA–protein complex may play a role in transporting cytochrome c mRNA and its translated product to the mitochondria.

A Role for Akt in Epidermal Growth Factor-Stimulated Cell Cycle Progression in Cultured Hepatocytes: Generation of a Hyperproliferative Window after Adenoviral Expression of Constitutively Active Akt

Epidermal growth factor (EGF) stimulation of cell cycle progression in cultured primary hepatocytes has previously been reported to be dependent on the mammalian target of rapamycin (mTOR) elements of the phosphoinositide 3-kinase (PI3K) signaling cascade and not the Akt pathway. Here we have established conditions of combined treatment of rat hepatocytes with insulin and EGF that favor cell cycle progression. The resulting cell population expresses albumin and retains receptor regulation of the signaling pathways leading to glycogen phosphorylase activation. We then investigated the hypothesis that the Akt limb of the PI3K pathway plays a central role in this insulin/EGF enhancement of cell cycle progression. The phosphorylation of Akt, central to the PI3K pathway, was increased by both insulin (sustained) and EGF (transient). The stimulation of Akt phosphorylation was inhibited in a concentration-dependent manner by the PI3K inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). Cell cycle progression in these cultures was reduced, but not abolished, by this inhibitor. The mTOR inhibitor, rapamycin, also inhibited entry into S phase. The novel Akt inhibitor A-443654 [(S)-1-(1H-indol-3-ylmethyl)-2-[5-(3-methyl-1H-indazol-5-yl)-pyridin-3-yloxy]-ethylamine] blocked both EGF-stimulated cell cycle progression and phosphorylation of the Akt substrate glycogen synthase kinase-3. Infection of cells with an adenoviral vector expressing a constitutively active form of Akt but not a kinase-dead form increased hepatocyte proliferation probably through enhanced cell cycle progression and reduced apoptosis. These results show that the Akt element of the PI3K cascade is necessary for EGF-stimulated cell cycle progression and provide evidence that the sustained elevation of Akt alone generates a hyperproliferative window in hepatocyte cultures.

GIT1 Is a Scaffold for ERK1/2 Activation in Focal Adhesions

GIT1 (G protein-coupled receptor kinase-interacting protein 1) has been shown to regulate focal adhesion disassembly. We previously reported that GIT1 associates with MEK1 and acts as a scaffold to enhance ERK1/2 activation. Here, we show that GIT1 co-localizes with ERK1/2 in focal adhesions and regulates cell migration in vascular smooth muscle cells, HEK293 cells, and HeLa cells. Immunofluorescence showed that GIT1 co-localized with phospho-ERK1/2 in focal adhesions after epidermal growth factor stimulation. Because Src is required for both GIT1 tyrosine phosphorylation and focal adhesion disassembly, we studied the effects of Src on GIT1-ERK1/2 interactions. PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) inhibited association of GIT1 with ERK1/2, and their co-localization in focal adhesions was dramatically decreased in SYF-/- cells. GIT1 small interfering RNA significantly inhibited ERK1/2 recruitment to and activation in focal adhesions. GIT1 small interfering RNA and mutated GIT1 lacking the MEK1 binding domain significantly decreased epidermal growth factor-stimulated cell spreading and migration, suggesting that GIT1-mediated events such as ERK1/2 activation are required for spreading and migration. In summary, the present study further supports a key role for GIT1 (a MEK1-binding protein) as a scaffold for signal transduction in focal adhesions.

P90 ribosomal S6 kinase 2 is associated with and dephosphorylated by protein phosphatase 2Cdelta.

RSK2 (p90 ribosomal S6 kinase 2) is activated via the ERK (extracellular-signal-regulated kinase) pathway by phosphorylation on four sites: Ser227 in the activation loop of the N-terminal kinase domain, Ser369 in the linker, Ser386 in the hydrophobic motif and Thr577 in the C-terminal kinase domain of RSK2. In the present study, we demonstrate that RSK2 is associated in vivo with PP2Cdelta (protein phosphatase 2Cdelta). In epidermal growth factorstimulated cells, RSK2 is partially dephosphorylated on all four sites in an Mn2+-dependent manner, leading to reduced protein kinase activity. Furthermore, PP2Cd is phosphorylated by ERK on Thr315 and Thr333 in the catalytic domain. Mutation of Thr315 and Thr333 to alanine in a catalytically inactive mutant PP2Cdelta (H154D) (His154-->Asp) increases the association with RSK2 significantly, whereas mutation to glutamate, mimicking phosphorylation, reduces the binding of RSK2. The domains of interaction are mapped to the N-terminal extension comprising residues 1-71 of PP2Cd and the N-terminal kinase domain of RSK2. The interaction is specific, since PP2Cd associates with RSK1-RSK4, MSK1 (mitogen- and stress-activated kinase 1) and MSK2, but not with p70 S6 kinase or phosphoinositide-dependent kinase 1. We conclude that RSK2 is associated with PP2Cd in vivo and is partially dephosphorylated by it, leading to reduced kinase activity.

Interaction Protein for Cytohesin Exchange Factors 1 (IPCEF1) Binds Cytohesin 2 and Modifies Its Activity

The ADP-ribosylation factor 6 (ARF6) small GTPase functions as a GDP/GTP-regulated switch in the pathways that stimulate actin reorganization and membrane ruffling. The formation of active ARF6GTP is stimulated by guanine nucleotide exchange factors (GEFs) such as cytohesins, which translocate to the plasma membrane in agonist-stimulated cells by binding the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate through the pleckstrin homology domain with subsequent ARF6 activation. Using cytohesin 2 as bait in yeast two-hybrid screening, we have isolated a cDNA encoding a protein termed interaction protein for cytohesin exchange factors 1 (IPCEF1). Using yeast two-hybrid and glutathione S-transferase pull-down assays coupled with deletion mutational analysis, the specific domains required for the cytohesin 2-IPCEF1 interaction were mapped to the coiled-coil domain of cytohesin 2 and the C-terminal 121 amino acids of IPCEF1. IPCEF1 also interacts with the other members of the cytohesin family of ARF GEFs, suggesting that the interaction with IPCEF1 is highly conserved among the cytohesin family of ARF GEFs. The interaction of cytohesin 2 and IPCEF1 in mammalian cells was demonstrated by immunoprecipitation. Immunofluorescence analysis revealed that IPCEF1 co-localizes with cytohesin 2 to the cytosol in unstimulated cells and translocates to the plasma membrane via binding to cytohesin 2 in epidermal growth factor-stimulated cells. However, a deletion mutant of IPCEF1 that lacks the cytohesin 2 binding site failed to co-migrate with cytohesin 2 to the membrane in stimulated cells. The functional significance of the IPCEF1-cytohesin 2 interaction is demonstrated by showing that IPCEF1 increases the in vitro and in vivo stimulation of ARFGTP formation by cytohesin 2.

Epidermal growth factor induces acute matrix contraction and subsequent calpain-modulated relaxation.

During wound healing, dermal fibroblasts switch from a migratory, repopulating phenotype to a contractile, matrix-reassembling phenotype. The mechanisms controlling this switch are unknown. A possible explanation is suggested by the finding that chemokines that appear late in wound repair prevent growth factor-induced cell-substratum de-adhesion by blocking calpain activation. In this study, we tested the specific hypothesis that fibroblast contraction of the matrix is promoted by a pro-repair growth factor, epidermal growth factor, and is modulated by calpain-mediated release of adhesions. We employed an isometric force transduction system designed to measure the contraction of a collagen matrix under tension by a population of NR6 fibroblasts transfected with the human epidermal growth factor receptor. By maintaining a fixed level of strain, we could monitor both the initial contraction and subsequent relaxation of the matrix. Epidermal growth factor stimulated a transient, dose-dependent increase in matrix contraction that peaked within 60 minutes and then decayed over the ensuing 3 to 6 hours. Calpain inhibitor I (ALLN) prevented epidermal growth factor-stimulated cell de-adhesion and resulted in a significantly slower decay of matrix contraction, with only a slight decrease of the peak magnitude of contraction. The mitogen-activated protein kinase kinase-1-selective inhibitor PD 98059 that blocks signaling through the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway, required for epidermal growth factor receptor-mediated activation of calpain and de-adhesion, does not significantly affect the magnitude of matrix contraction within minutes of epidermal growth factor addition, but slows the decay similarly to calpain inhibition. Epidermal growth factor receptor signaling thus stimulates the complementary mechanisms of intracellular contractile force generation and calpain-mediated de-adhesion, which are known to coordinately facilitate cell migration. These findings suggest that calpain can act as a functional switch for transmission of intracellular contractile force to the surrounding matrix, with calpain-mediated de-adhesion reducing this transmission and corresponding matrix contraction. Countervailing processes that down-regulate calpain activation can, accordingly, direct the transition of cell function from locomotion to matrix contraction.

Palmitoylation of Caveolin-1 at a Single Site (Cys-156) Controls Its Coupling to the c-Src Tyrosine Kinase: TARGETING OF DUALLY ACYLATED MOLECULES (GPI-LINKED, TRANSMEMBRANE, OR CYTOPLASMIC) TO CAVEOLAE EFFECTIVELY UNCOUPLES c-Src AND CAVEOLIN-1 (TYR-14)

Caveolin-1 was initially identified as a phosphoprotein in Rous sarcoma virus-transformed cells. Previous studies have shown that caveolin-1 is phosphorylated on tyrosine 14 by c-Src and that lipid modification of c-Src is required for this phosphorylation event to occur in vivo. Phosphocaveolin-1 (Tyr(P)-14) localizes within caveolae near focal adhesions and, through its interaction with Grb7, augments anchorage-independent growth and epidermal growth factor-stimulated cell migration. However, the cellular factors that govern the coupling of caveolin-1 to the c-Src tyrosine kinase remain largely unknown. Here, we show that palmitoylation of caveolin-1 at a single site (Cys-156) is required for coupling caveolin-1 to the c-Src tyrosine kinase. Furthermore, upon evaluating a battery of nonreceptor and receptor tyrosine kinases, we demonstrate that the tyrosine phosphorylation of caveolin-1 by c-Src is a highly selective event. We show that Src-induced tyrosine phosphorylation of caveolin-1 can be inhibited or uncoupled by targeting dually acylated proteins (namely carcinoembryonic antigen (CEA), CD36, and the NH(2)-terminal domain of Galpha(i1)) to the exoplasmic, transmembrane, and cytoplasmic regions of the caveolae membrane, respectively. Conversely, when these proteins are not properly targeted or lipid-modified, the ability of c-Src to phosphorylate caveolin-1 remains unaffected. In addition, when purified caveolae preparations are preincubated with a myristoylated peptide derived from the extreme N terminus of c-Src, the tyrosine phosphorylation of caveolin-1 is abrogated; the same peptide lacking myristoylation has no inhibitory activity. However, an analogous myristoylated peptide derived from c-Yes also has no inhibitory activity. Thus, the inhibitory effects of the myristoylated c-Src peptide are both myristoylation-dependent and sequence-specific. Finally, we investigated whether phosphocaveolin-1 (Tyr(P)-14) interacts with the Src homology 2 and/or phosphotyrosine binding domains of Grb7, the only characterized downstream mediator of its function. Taken together, our data identify a series of novel lipid-lipid-based interactions as important regulatory factors for coupling caveolin-1 to the c-Src tyrosine kinase in vivo.

Point Mutants of c-Raf-1 RBD with Elevated Binding to v-Ha-Ras

A mutational analysis of the Ras-binding domain (RBD) of c-Raf-1 identified three amino acid positions (Asn(64), Ala(85), and Val(88)) where amino acid substitution with basic residues increases the binding of RBD to recombinant v-Ha-Ras. The greatest increase in binding (6-9-fold) was observed with the A85K-RBD mutant. The elevated binding for the A85K-RBD and V88R-RBD mutants was also detected with Ras expressed in cultured mammalian cells, namely NIH-3T3 and BAF cells. None of the wild type residues in RBD positions Asn(64), Ala(85), and Val(88) have been previously implicated in the interaction with Ras (Block, C., Janknecht, R., Herrmann, C., Nassar, N., and Wittinghofer, A. (1996) Nat. Struct. Biol. 3, 244-251; Nassar, N., Horn, G., Herrmann, C., Scherer, A., McCormick, F., and Wittinghofer, A. (1995) Nature 375, 554-560). The discovery of elevated binding among the mutants in these positions implies that additional RBD residues can be used to generate the Ras. RBD complex. These findings are of particular significance in the design of Ras antagonists based on the RBD prototype. The A85K-RBD mutant can be used to develop an assay for measuring the level of activated Ras in cultured cells; Sepharose-linked A85K-RBD.GST fusion protein served as an activation-specific probe to precipitate Ras.GTP but not Ras.GDP from epidermal growth factor-stimulated cells. A85K-RBD precipitates up to 5-fold more Ras.GTP from mammalian cells than wild type RBD.

C-Raf-mediated Inhibition of Epidermal Growth Factor-stimulated Cell Migration

Epidermal growth factor stimulates migration of a number of cell types, yet the signaling pathways that regulate epidermal growth factor-stimulated migration are poorly defined. In this report, we employ a transient transfection migration assay to assess the role of components of the Ras-mitogen-activated protein (MAP) kinase signaling pathway in epidermal growth factor-stimulated chemotaxis of rat embryo fibroblasts. Expression of dominant negative Ras blocks epidermal growth factor-mediated chemotaxis, while constitutively active Ras has no effect on chemokinesis or chemotaxis. PD98059 and U0126, inhibitors of MAP kinase kinase (MEK) activity, decreased epidermal growth factor-stimulated migration, while kinase-defective MEK1, an inhibitor of MAP kinase activation, enhanced migration. To understand the paradoxical effects of these molecules on epidermal growth factor-induced migration, we examined the role of c-Raf on migration. Expression of either wild type c-Raf or the catalytic domain of c-Raf effectively inhibited epidermal growth factor-stimulated cell migration. We suggest that, whereas Ras activity is necessary to promote epidermal growth factor-stimulated migration, sustained activation of c-Raf may be important in down-regulating migratory signaling pathways triggered by epidermal growth factor receptor activation. Further, activation of c-Raf upon inhibition of the MEK-MAP kinase pathway may contribute to the inhibition of cell migration observed with pharmacological MEK inhibitors.

Wiskott-Aldrich syndrome protein is associated with the adapter protein Grb2 and the epidermal growth factor receptor in living cells.

Src homology domains [i.e., Src homology domain 2 (SH2) and Src homology domain 3 (SH3)] play a critical role in linking receptor tyrosine kinases to downstream signaling networks. A well-defined function of the SH3-SH2-SH3 adapter Grb2 is to link receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), to the p21ras-signaling pathway. Grb2 has also been implicated to play a role in growth factor-regulated actin assembly and receptor endocytosis, although the underlying mechanisms remain unclear. In this study, we show that Grb2 interacts through its SH3 domains with the human Wiskott-Aldrich syndrome protein (WASp), which plays a role in regulation of the actin cytoskeleton. We find that WASp is expressed in a variety of cell types and is exclusively cytoplasmic. Although the N-terminal SH3 domain of Grb2 binds significantly stronger than the C-terminal SH3 domain to WASp, full-length Grb2 shows the strongest binding. Both phosphorylation of WASp and its interaction with Grb2, as well as with another adapter protein Nck, remain constitutive in serum-starved or epidermal growth factor-stimulated cells. WASp coimmunoprecipitates with the activated EGFR after epidermal growth factor stimulation. Purified glutathione S-transferase-full-length-Grb2 fusion protein, but not the individual domains of Grb2, enhances the association of WASp with the EGFR, suggesting that Grb2 mediates the association of WASp with EGFR. This study suggests that Grb2 translocates WASp from the cytoplasm to the plasma membrane and the Grb2-WASp complex may play a role in linking receptor tyrosine kinases to the actin cytoskeleton.

Transformation by pp60src or stimulation of cells with epidermal growth factor induces the stable association of tyrosine-phosphorylated cellular proteins with GTPase-activating protein.

GTPase-activating protein (GAP) is a cytosolic protein that stimulates the rate of hydrolysis of GTP (GTP to GDP) bound to normal p21ras, but does not catalyze the hydrolysis of GTP bound to oncogenic, activated forms of the ras protein. Transformation of cells with v-src or activated transforming variants of c-src or stimulation of cells with epidermal growth factor resulted in the stable association of GAP with two tyrosine-phosphorylated cellular proteins of 64 kDa (p64) and 190 kDa (p190). Analysis of GAP immune complexes isolated from extracts of metabolically labeled src-transformed cells and epidermal growth factor-stimulated cells indicated that tyrosine phosphorylation of p64 and p190 appeared to be coincident with the stable association of these proteins with GAP. Quantitation of the amount of p64 associated with GAP in v-src-transformed cells, however, indicated that only 15 to 25% of tyrosine-phosphorylated p64 was found in complex with GAP. Mutations within the SH2 region of pp60src that render activated pp60src defective for transformation inhibited the efficient formation of complexes between GAP and the tyrosine-phosphorylated forms of p64 and p190. From these data, we suggest that tyrosine phosphorylation and stable association of p64 with GAP is an important step in mediating cellular signaling through the p21ras-GAP pathway.

Transformation by pp60srcor Stimulation of Cells with Epidermal Growth Factor Induces the Stable Association of Tyrosine-Phosphorylated Cellular Proteins with GTPase-Activating Protein

GTPase-activating protein (GAP) is a cytosolic protein that stimulates the rate of hydrolysis of GTP (GTP to GDP) bound to normal p21ras, but does not catalyze the hydrolysis of GTP bound to oncogenic, activated forms of the ras protein. Transformation of cells with v-src or activated transforming variants of c-src or stimulation of cells with epidermal growth factor resulted in the stable association of GAP with two tyrosine-phosphorylated cellular proteins of 64 kDa (p64) and 190 kDa (p190). Analysis of GAP immune complexes isolated from extracts of metabolically labeled src-transformed cells and epidermal growth factor-stimulated cells indicated that tyrosine phosphorylation of p64 and p190 appeared to be coincident with the stable association of these proteins with GAP. Quantitation of the amount of p64 associated with GAP in v-src-transformed cells, however, indicated that only 15 to 25% of tyrosine-phosphorylated p64 was found in complex with GAP. Mutations within the SH2 region of pp60src that render activated pp60src defective for transformation inhibited the efficient formation of complexes between GAP and the tyrosine-phosphorylated forms of p64 and p190. From these data, we suggest that tyrosine phosphorylation and stable association of p64 with GAP is an important step in mediating cellular signaling through the p21ras-GAP pathway.