Calphostin-C stimulates epidermal growth factor receptor phosphorylation and internalization via light-dependent mechanism

Calphostin-C with perylenequinone structure is known to bind the regulatory domain of protein kinase C (PKC) and to inhibit kinase activity in vitro in a light-dependent fashion. We have found that calphostin-C induces substantial serine and threonine phosphorylation of the epidermal growth factor (EGF) receptor in a light-dependent fashion in the EGF receptor-hyperproducing squamous carcinoma cell line NA. Tryptic phospho-peptide mapping and phospho-amino acid analysis revealed that calphostin-C–-enhanced phosphorylation was on threonine 669, serine 671, serine 1046/1047, and serine 1166. However, caiphostin-C did not inhibit phosphorylation of the 80 K protein, a cytosolic major substrate of PKC (MARCKS). Staurosporine, a potent PKC inhibitor with affinity for the catalytic domain of PKC, inhibited phosphorylation of the 80 K protein and 12-O-tetradecanoyl-13-phorbol acetate induction of EGF receptor phosphorylation but did not inhibit the calphostin-C induction of the EGF receptor phosphorylation. These results suggest that the target of calphostin-C in vivo is different from that of staurosporine and thus calphostin-C in vivo does not inhibit PKC. Furthermore, calphostin-C enhanced the internalization of phosphorylated EGF receptor. Thus, calphostin-C apparently activates a novel signal transduction pathway which involves phosphorylation and internalization of the EGF receptor via light-dependent mechanism. © 1994 Wiley-Liss, Inc.     

Epidermal growth factor receptor internalization and biosynthesis in the diabetic rat.

The number of surface EGF receptors as well as their internalization rate and biosynthesis were analyzed in hepatocytes freshly isolated from control, streptozotocin-diabetic, and insulin-treated diabetic rats. All three parameters were decreased in diabetic animals and values were corrected by insulin treatment. Moreover, the inhibition of synthesis was specific for the EGF receptor since the other biosynthetically labeled proteins were not affected. These data demonstrate that the reduced number of hepatocyte surface EGF receptors results from an inhibition of EGF-receptor synthesis which is not compensated by a reduced internalization rate.     

In vivo inhibition of epidermal growth factor receptor autophosphorylation prevents receptor internalization

The question whether epidermal growth factor (EGF)-induced receptor endocytosis requires the prior autophosphorylation via the EGF receptor (EGFR) kinase domain has been a matter of long-standing debate. In the airway epithelial cell line NCI-H292, the EGFR kinase domain inhibitor BIBW 2948 BS was found to inhibit both autophosphorylation and subsequent internalization of the endogenous EGFR with similar IC₅₀ values. Applying an ex vivo EGFR internalization assay in a clinical study, the in vivo effect of inhalatively administered BIBW 2948 BS was determined directly at the targeted receptor in airway tissues from COPD patients. In these experiments, the in vivo inhibition of the EGFR kinase domain prevented the EGF-induced internalization of EGFR.     

Epidermal growth factor-stimulated tyrosine phosphorylation of caveolin-1. Enhanced caveolin-1 tyrosine phosphorylation following aberrant epidermal growth factor receptor status

Caveolin-1 is the major coat protein of caveolae and has been reported to interact with various intracellular signaling molecules including the epidermal growth factor (EGF) receptor. To investigate the involvement of caveolin-1 in EGF receptor action, we used mouse B82L fibroblasts transfected with (a) wild type EGF receptor, (b) a C-terminally truncated EGF receptor at residue 1022, (c) a C-terminally truncated EGF receptor at residue 973, or (d) a kinase-inactive EGF receptor (K721M). Following EGF treatment, there was a distinct electrophoretic mobility shift of the caveolin-1 present in cells expressing the truncated forms of the EGF receptor, but this shift was not detectable in cells bearing either normal levels of the wild type EGF receptor or a kinase-inactive receptor. This mobility shift was also not observed following the addition of other cell stimuli, such as platelet-derived growth factor, insulin, basic fibroblast growth factor, or phorbol 12-myristate 13-acetate. Analysis of caveolin-1 immunoprecipitates from EGF-stimulated or nonstimulated cells demonstrated that the EGF-induced mobility shift of caveolin-1 was associated with its tyrosine phosphorylation in cells expressing truncated EGF receptors. Maximal caveolin-1 phosphorylation was achieved within 5 min after exposure to 10 nM EGF and remained elevated for at least 2 h. Additionally, several distinct phosphotyrosine-containing proteins (60, 45, 29, 24, and 20 kDa) were co-immunoprecipitated with caveolin-1 in an EGF-dependent manner. Furthermore, the Src family kinase inhibitor, PP1, does not affect autophosphorylation of the receptor, but it does inhibit the EGF-induced mobility shift and phosphorylation of caveolin-1. Conversely, the MEK inhibitors PD98059 and UO126 could attenuate EGF-induced mitogen-activated protein kinase activation, they do not affect the EGF-induced mobility shift of caveolin-1. Because truncation and overexpression of the EGF receptor have been linked to cell transformation, these results provide the first evidence that the tyrosine phosphorylation of caveolin-1 occurs via an EGF-sensitive signaling pathway that can be potentiated by an aberrant activity or expression of various forms of the EGF receptor.     

Epidermal growth factor (EGF) stimulates EGF receptor synthesis

Epidermal growth factor (EGF) binds to the extracellular domain of a specific 170,000-dalton transmembrane glycoprotein; this results in rapid removal of both ligand and receptor from the cell surface. In WB cells, a rat hepatic epithelial cell line, ligand-directed receptor internalization leads to receptor degradation. We tested whether the EGF receptor was replenished at a constitutive or enhanced rate following EGF binding by immunoprecipitating biosynthetically labeled EGF receptor from cells cultured with [35S]methionine. EGF stimulated receptor synthesis within 2 h in a dose-dependent manner; this was particularly evident when examining the nascent form of the receptor. To determine the site of EGF action, total WB cell RNA was transferred to nitrocellulose paper after electrophoresis and was hybridized to cDNA probes from both the external and cytoplasmic coding regions of the human EGF receptor. EGF increased receptor mRNA by 3-5-fold. Therefore, at least in some cells, the surface action of EGF that leads to EGF receptor degradation is counterbalanced by a positive effect on receptor synthesis.     

Epidermal growth factor or okadaic acid stimulates phosphorylation of eukaryotic initiation factor 4F

Eukaryotic initiation factor 4F, a multi-protein mRNA cap binding complex, was isolated by m7GTP-Sepharose affinity chromatography from human mammary epithelial cells (184A1N4) incubated with [32P] orthophosphate. Treatment of cells with epidermal growth factor resulted in enhanced phosphorylation of both p28 (eIF-4E) and p220 subunits. The identities of the p28 and p220 subunits were confirmed by immunoprecipitation. The phosphorylation was both rapid and sustained in duration; p28 attained maximal levels (2-3-fold) within 30 min of treatment and remained elevated for at least 2 h, while p220 reached one-half maximal levels by 30 min, and maximal levels (3-4-fold) by 2 h of treatment. Two phosphorylated isoforms of p28 and multiple phosphorylated forms of p220 were detected by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphoamino acid analysis of 6 N HCl hydrolyzates of p28 and p220 isolated from epidermal growth factor-treated and control cells indicated that serine is the predominant phosphorylated amino acid in both instances. In no case was phosphotyrosine observed. Pretreatment of cells with 1 microM okadaic acid resulted in the hyperphosphorylation of both p28 and p220 subunits. These results suggest that mitogenic growth factors and cellular serine/threonine phosphatases (pp1 and/or pp2A) serve essential roles in regulating phosphorylation levels of eukaryotic initiation factor 4F and support the concept that translational control is a component of the signal transduction mechanisms involved in growth regulation.     

Transient epidermal growth factor (EGF)-dependent suppression of EGF receptor autophosphorylation during internalization.

To study the activity of the epidermal growth factor (EGF) receptor during EGF-directed internalization, liver epithelial cells were exposed to EGF at 37 degrees C for various periods of time, washed, and homogenized at 0 degrees C. EGF receptor autophosphorylation was assessed in homogenates using [gamma-32P]ATP. Autophosphorylation was stimulated 3- to 6-fold in homogenates of cells incubated with EGF (100 ng/ml) for 15 min but was at or below basal levels in homogenates of cells treated with EGF for 2.5-5 min. This was surprising because immunoblotting revealed that EGF receptor phosphotyrosine (P-Tyr) content in intact cells was near maximal from 30 s to 5 min after EGF treatment. Excess EGF (1 microgram/ml), added after homogenization but prior to the assay, increased autophosphorylation in homogenates of cells that had not been treated with EGF, but failed to increase activity in homogenates of cells treated with EGF in culture for 2.5-5 min. Suppression of tyrosine phosphorylation of an exogenous kinase substrate was also observed at times paralleling the suppression of EGF receptor autophosphorylation. The transient suppression of receptor autophosphorylation in the cell-free assay was not explained by persistent occupation of autophosphorylation sites by phosphate added in the intact cells. The sites were greater than 80% dephosphorylated during the homogenization. Additionally phosphatase inhibition that prevented the normal loss of EGF receptor P-Tyr in intact cells at 15 min did not affect the pattern of early (2.5-5 min) suppression and later (15 min) stimulation of autophosphorylation measured in the cell-free assay. The suppression was not explained by activation of protein kinase C in that depletion of greater than 95% of cellular protein kinase C activity by an 18-h incubation of cells with 10 microM 12-O-tetradecanoylphorbol 13-acetate (TPA) did not affect the early suppression of autophosphorylation in EGF-treated cells. Moreover, under the conditions tested, activation of protein kinase C by short-term treatment (0.5-10 min) with TPA or angiotensin II did not appreciably alter subsequent autophosphorylation in the cell-free assay. In contrast, a 30 degrees C preincubation of homogenates from cells with suppressed EGF receptor autophosphorylation led to the recovery of the ability of EGF to stimulate EGF receptor autophosphorylation. These results suggest that a rapid reversible protein kinase C-independent process prevents detection of EGF receptor kinase activity during an early phase of EGF-dependent receptor internalization.     

Epidermal growth factor stimulates the phosphorylation of pyruvate kinase in freshly isolated rat hepatocytes.

Epidermal growth factor (EGF) has previously been shown to stimulate gluconeogenesis in rat liver by decreasing the activity of pyruvate kinase [(1988) Biochem. J. 255, 361-364]. Here we investigate the mechanism underlying the inactivation of the enzyme. EGF was found to increase the incorporation of phosphate into pyruvate kinase, with maximal phosphorylation achieved only after 10 min in the presence of the growth factor. The increase in phosphorylation was not additive with that caused by cyclic AMP. Phosphoamino acid analysis of pyruvate kinase isolated from cells treated with EGF indicated that EGF increases phosphorylation solely on serine residues. The exact site of EGF-mediated phosphorylation has yet to be identified.     

Epidermal growth factor stimulates tyrosine phosphorylation of specific proteins in permeabilized human fibroblasts

We have investigated the epidermal growth factor (EGF)-stimulated tyrosine-specific protein kinase activity in quiescent cultures of diploid human fibroblasts that have a well characterized mitogenic response to EGF. We developed a method of permeabilizing cells with digitonin or other agents that permitted the rapid labeling of cellular proteins with exogenously added [gamma-32P]ATP while allowing only about 25% of marker cytosolic enzymes to escape from the cells. When phosphatases were inhibited with zinc and vanadate, EGF induced up to 8-fold stimulation of the incorporation of radioactivity from [gamma-32P]ATP into a 35-kDa band on sodium dodecyl sulfate gels. Alkali treatment of gels showed that EGF stimulated the phosphorylation of bands with apparent molecular masses of 170, 45, 35, 26, 22, and 21 kDa. Phosphoamino acid analysis was performed on the 170- and 35-kDa bands and revealed that the EGF-stimulated phosphorylation was on tyrosyl residues. The 35-kDa band was resolved into four spots by two-dimensional gel electrophoresis. The most acidic form was the most prominent and it was precipitated by an antiserum against a 35-kDa protein from A-431 cells; heretofore, this protein has only been reported to be phosphorylated in an EGF-dependent manner by A-431 membranes in vitro (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). This antiserum also precipitated a 35-kDa phospho-protein from extracts of intact [32P]orthophosphate-labeled fibroblasts which was phosphorylated on tyrosine in an EGF-dependent manner. None of the forms of the 35-kDa phosphoproteins labeled in permeabilized cells were immunologically related to the 34-kDa protein that is a substrate for the tyrosyl kinase encoded by Rous sarcoma virus. Other mitogens (serum, insulin, platelet-derived growth factor, and thrombin) did not detectably stimulate phosphorylation in permeabilized cells.     

Epidermal growth factor (EGF) stimulates association and kinase activity of Raf-1 with the EGF receptor.

Raf-1 serine- and threonine-specific protein kinase is transiently activated in cells expressing the epidermal growth factor (EGF) receptor upon treatment with EGF. The stimulated EGF receptor coimmunoprecipitates with Raf-1 kinase and mediates protein kinase C-independent phosphorylation of Raf-1 on serine residues. Hyperphosphorylated Raf-1 has lower mobility on sodium dodecyl sulfate gels and has sixfold-increased activity in immunocomplex kinase assay with histone H1 or Raf-1 sequence-derived peptide as a substrate. Raf-1 activation requires kinase-active EGF receptor; a point mutant lacking tyrosine kinase activity in inactive in Raf-1 coupling and association. It is noteworthy that tyrosine phosphorylation of c-Raf-1 induced by EGF was not detected in these cells. These observations suggest that Raf-1 kinase may act as an important downstream effector of EGF signal transduction.     

Insulin-like growth factor type-I receptor internalization and recycling mediate the sustained phosphorylation of Akt

Previously we demonstrated that insulin-like growth factor-I mediates the sustained phosphorylation of Akt, which is essential for long term survival and protection of glial progenitors from glutamate toxicity. These prosurvival effects correlated with prolonged activation and stability of the insulin-like growth factor type-I receptor. In the present study, we investigated the mechanisms whereby insulin-like growth factor-I signaling, through the insulin-like growth factor type-I receptor, mediates the sustained phosphorylation of Akt. We showed that insulin-like growth factor-I stimulation induced loss of receptors from the cell surface but that surface receptors recovered over time. Blocking receptor internalization inhibited Akt phosphorylation, whereas inhibition of receptor trafficking blocked receptor recovery at the cell surface and the sustained phosphorylation of Akt. Moreover the insulin-like growth factor type-I receptor localized with the transferrin receptor and Rab11-positive endosomes in a ligand-dependent manner, further supporting the conclusion that this receptor follows a recycling pathway. Our results provide evidence that ligand stimulation leads to internalization of the insulin-like growth factor type-I receptor, which mediates Akt phosphorylation, and that receptor recycling sustains Akt phosphorylation in glial progenitors. Mathematical modeling of receptor trafficking further supports these results and predicts an additional kinetic state of the receptor consistent with sustained Akt phosphorylation.     

Epidermal growth factor: structure, location, phosphorylation and regulation of its receptor

Epidermal growth factor (EGF) is a Mr 6045 polypeptide first characterized for its ability to stimulate mitogenesis in epidermal and epithelial cells. The first step in the action of the growth factor is its binding to specific, high affinity membrane receptors. These receptors have been studied in a number of tissues and cell culture lines. The level of EGF receptors is modulated by many agents. EGF down-regulates its receptor. In addition, the number of EGF receptors is decreased by other growth factors (platelet-derived growth factor; transforming growth factor), by many tumor promoters and by viral transformation. Several hormones also can regulate EGF binding in its target tissues.     

Epidermal growth factor receptor signal transactivation

Cross-communication between heterologous signaling systems and the epidermal growth factor receptor (EGFR) has been shown to be critical for a variety of biological responses: EGFR transactivation when G-protein-coupled receptors (GPCRs) are stimulated represents the paradigm of an interreceptor network that is dependent on G-proteins, kinases, metalloproteases, and growth factor precursors. Investigating the mechanism of this process will help expand our knowledge of physiological regulatory mechanisms and diverse pathophysiological disorders.     

Colony stimulating factor-1 (CSF-1) stimulates temperature dependent phosphorylation and activation of the RAF-1 proto-oncogene product.

The serine/threonine kinase RAF-1 is phosphorylated in intact macrophages in response to CSF-1 at 37 degrees C The augmented phosphorylation of RAF-1 and a concomitant increase in RAF-1 associated serine/threonine kinase activity are kinetically later events than CSF-1 induced protein tyrosine phosphorylation. Furthermore, phosphoamino acid analysis of RAF-1 reveals the presence of phosphoserine, trace amounts of phosphothreonine but no phosphotyrosine and the phosphorylated RAF-1 does not react with anti-phosphotyrosine antibodies. In contrast to CSF-1 induced protein tyrosine phosphorylation, RAF-1 phosphorylation and activation are temperature dependent and do not occur at 4 degrees C. Furthermore, coprecipitation experiments failed to reveal any noncovalent association of RAF-1 with the CSF-1 receptor. Thus, while RAF-1 is not a direct substrate for the CSF-1 receptor tyrosine kinase in vivo, its temperature dependent phosphorylation and activation represent an intriguing aspect of the CSF-1 response.     

Epidermal growth factor stimulates the phosphorylation of the calcium-dependent 35,000-dalton substrate in intact A-431 cells

We have previously reported the isolation of a 35-kDa protein from A-431 cells that, in the presence of Ca2+, is an excellent in vitro substrate for the epidermal growth factor (EGF) receptor/kinase present in membrane preparations (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). In this communication we demonstrate that the phosphorylation of the 35-kDa protein is markedly enhanced in intact, 32P-labeled, A-431 cells following exposure of the cells to EGF. The 35-kDa protein immunoprecipitated from cells treated with EGF is phosphorylated to a 20-120-fold greater extent than comparable preparations from control cells. Both phosphotyrosine and phosphoserine residues are detected in the protein after treatment of the cells with EGF. EGF-dependent phosphorylation of the 35-kDa protein is barely detected unless the intact cells are exposed to EGF for periods greater than 5 min. We suggest that endosomes containing internalized EGF X receptor/kinase complexes are primarily responsible for the observed phosphorylation of the 35-kDa protein in intact cells.     

Heparin-binding growth factor 1 stimulates tyrosine phosphorylation in NIH 3T3 cells.

Tyrosine phosphorylation of cellular proteins induced by heparin-binding growth factor 1 (HBGF-1) was studied by using the murine fibroblast cell line NIH 3T3 (clone 2.2). HBGF-1 specifically induced the rapid tyrosine phosphorylation of polypeptides of Mr 150,000, 130,000, and 90,000 that were detected with polyclonal and monoclonal antiphosphotyrosine (anti-P-Tyr) antibodies. The concentration of HBGF-1 required for half-maximal induction of tyrosine phosphorylation of the Mr-150,000 Mr-130,000, and Mr-90,000 proteins was approximately 0.2 to 0.5 ng/ml, which was consistent with the half-maximal concentration required for stimulation of DNA synthesis in NIH 3T3 cells. HBGF-1-induced tyrosine phosphorylation of the Mr-150,000 and Mr-130,000 proteins was detected within 30 s, whereas phosphorylation of the Mr-90,000 protein was not detected until 3 min after HBGF-1 stimulation. All three proteins were phosphorylated maximally after 15 to 30 min. Phosphoamino acid analysis of the Mr-150,000 and Mr-90,000 proteins confirmed the phosphorylation of these proteins on tyrosine residues. Phosphorylation of the Mr-150,000 and Mr-90,000 proteins occurred when cells were exposed to HBGF-1 at 37 degrees C but not at 4 degrees C. Exposure of cells to sodium orthovanadate, a potent P-Tyr phosphatase inhibitor, before stimulation with HBGF-1 resulted in enhanced detection of the Mr-150,000, Mr-130,000, and Mr-90,000 proteins by anti-P-Tyr antibodies. Anti-P-Tyr affinity-based chromatography was used to adsorb the HBGF-1 receptor affinity labeled with 125I-HBGF-1. The cross-linked HBGF-1 receptor-ligand complex was eluded with phenyl phosphate as two components: Mr 170,000 and 150,000. P-Tyr, but not phosphoserine or phosphothreonine, inhibited adsorption of the (125)I-HBGF-1-receptor complex to the anti-P-Tyr antibody matrix. Treatment of cells with sodium orthovanadate also enhanced recognition of the cross-linked (125)I-HBGF-1-receptor complex by the anti-P-Tyr matrix. These data suggest that (i) the (125)I-HBGF-1-receptor complex is phosphorylated on tyrosine residues and (ii) HBGF-1-induced signal transduction involves, in part, the tyrosine phosphorylation of at least three polypeptides.     

Epidermal growth factor stimulates cAMP accumulation in cultured rat cardiac myocytes.

We have previously shown that epidermal growth factor (EGF) augments cAMP accumulation in the heart and stimulates cardiac adenylyl cyclase via a G protein mediated mechanism (Nair et al., 1989). More recently, employing an antibody against the carboxy-terminus decapeptide of Gs alpha, we have demonstrated that Gs alpha mediates the effects of EGF on cardiac adenylyl cyclase (Nair et al., 1990). Since the heart comprises of a variety of cell types, the purpose of the studies presented here was to determine whether or not the effects of EGF on adenylyl cyclase were mediated in cardiac myocytes or noncardiomyocytes. Therefore, cultures of ventricular cardiomyocytes and noncardiomyocytes from neonatal rat hearts were established and characterized. Apart from the differences in cellular morphology, cardiomyocytes but not the noncardiomyocytes employed in our studies expressed the alpha- and beta-myosin heavy chain (MHC) mRNA and the beta-MHC protein. Additionally, as described previously, treatment of cardiomyocytes with thyroid hormone increased alpha-MHC mRNA and decreased the expression of beta-MHC mRNA, indicating that the cardiomyocytes employed in our studies were responding in a physiologically relevant manner. EGF in a time-dependent manner increased cAMP accumulation in the cardiomyocytes but not in noncardiomyocytes. Maximum and half-maximum effects were observed at 100 nM and 2 nM concentrations of EGF, respectively. As determined by the presence of immunoreactive EGF receptors and tyrosine phosphorylation of the 170 kDa protein in membranes of cardiomyocytes and noncardiomyocytes, both the cell populations contained functional EGF receptors. Therefore, the differential effects of EGF on cAMP accumulation in the two cell populations appear to be due to differential coupling of the EGF receptors to the adenylyl cyclase system rather than the absence of EGF receptors in noncardiomyocytes. Consistent with our previous findings in isolated membranes and perfused rat hearts, EGF-elicited increase in cAMP accumulation in cardiomyocytes did not involve activation of beta-adrenoreceptors and was abolished by prior treatment of cells with cholera toxin. Overall, our findings demonstrate that EGF-elicited increase in cAMP accumulation in the heart is the reflection of changes in cAMP content of cardiomyocytes and not noncardiomyocytes.     

表皮生长因子受体及其变异性质

EGFR主要由三个结构区域组成,即EGF结合区域、跨膜区域和依赖EGF蛋白激酶区域。EGFR被激活后,不仅能使许多底物蛋白磷酸化,自身也发生磷酸化,这些磷酸化作用在信息传导中有重要作用。EGFR与EGF结合后,其复合体以胞饮方式内容形成受体小体,除部分被降解外,大部分在高尔基GERL区加工后到达细胞核调节基因表达。本文通过对受体激活模型和用体外遗传变异方法对变异EGFR的研究阐述了EGFR的结构与功能的关系。