Effect of EGF on nuclear-cytoplasmic distribution of proteasomes in A-431 cells

The intracellular distribution of proteasomes was studied using immunofluorescent method. In nonstimulated cells proteasomes were observed both in the cytoplasm and nuclei of A-431 cells. When 100 ng/ml EGF was added for 15 min, proteasomes were located mainly in the nuclei. Later (up to 1 h) proteasomes released from the nuclei and were observed mainly in the cytoplasm. Tyrphostin AG1478, an inhibitor of tyrosine kinase, and U73122, an inhibitor of phospholipase C, prevent, proteasome export from the nuclei after EGF treatment. In contrast, a proteasome inhibitor--lactacystin has no effect on this process. The EGF-dependent tyrosine phosphorylation of EGF receptor is blocked by tyrhostin AG1478 and U733122. Lactacystin did not alter the induction of EGF receptor tyrosine phosphorylation, triggered by EGF. It is concluded that intracellular distribution of proteasomes depends on tyrosine activity of EGF receptor.     

Phospholipase C negatively regulates tyrosine phosphorylation of EGF receptor in A-431 cells

It is known that EGF induces tyrosine phosphorylation and internalization of the EGF receptor in A-431 cells. U73122, an inhibitor of phospholipase C, induces tyrosine phosphorylation of the EGF receptor and its association with phospholipase C still in nonstimulated cells. In U73122 treated cells EGF exerted no effect on these processes. Receptor-mediated endocytosis was not observed in A-431 cells treated with U73122. The reorganization of actin cytoskeleton was detected in U73122 cells.     

Rapid uptake of tyrphostin into A431 human epidermoid cells is followed by delayed inhibition of epidermal growth factor (EGF)-stimulated EGF receptor tyrosine kinase activity.

Treatment of A431 human epidermoid cells with epidermal growth factor (EGF; 20 nM) results in decreased proliferation. This is associated with blockage of the cells in the S and/or G2 phases of the cell cycle. We found that tyrphostin, a putative tyrosine kinase inhibitor, in the range of 50 to 100 microM, partially reversed the growth-inhibitory and cell cycle changes induced by EGF. By using high-pressure liquid chromatography with electrochemical detection, we found that tyrphostin was readily incorporated into A431 cells, reaching maximal levels within 1 h. Although tyrphostin (50 to 100 microM) had no effect on high-affinity binding of EGF to its receptor in A431 cells for up to 24 h, the compound partially inhibited EGF-stimulated EGF receptor tyrosine kinase activity. However, this effect was evident only after prolonged treatment of the cells (4 to 24 h) with the drug. When the peak intracellular concentration of tyrphostin occurred (1 h), no inhibition of tyrosine kinase activity was observed. After both 1 and 24 h, tyrphostin was a less effective inhibitor of tyrosine kinase activity than the potent tumor promoter 12-O-tetradecanoyl phorbol-13-acetate, which almost completely blocked EGF receptor autophosphorylation. On the basis of our data, we hypothesize that tyrphostin is not a competitive inhibitor of the EGF receptor tyrosine kinase in intact cells and that it functions by an indirect mechanism.     

The release of Hsp70 from A431 carcinoma cells is mediated by secretory-like granules

In our earlier work we have demonstrated that the treatment of squamous carcinoma cell line A431 with a pharmacological inhibitor of phospholipase C activity, U73122, resulted in fast release of stress-inducible heat shock protein 70 (Hsp70) into the extracellular medium (Evdonin et al., Cancer Cell Int., 4, 2, 2004). The purpose of the present study was to identify cellular organelles involved in the release of Hsp70 from A431 cells. We determined that Hsp70 is present in granules located at the periphery of cells, which had been treated with U73122 or subjected to heat shock. An inhibitor of the classical protein export pathway, brefeldin A was found to prevent the U73122-induced appearance of Hsp70 in the extracellular medium and in the peripheral granules. These findings suggest that vesicular transport is involved in Hsp70 release. The Hsp70-containing granules did not carry markers specific for lipid bodies, endosomes, or lysosomes. However, they were positive for a marker of secretory granules, i.e. chromogranin A. The levels of extracellular Hsp70 and chromogranin A were found to increase simultaneously. The secretory-like granule-dependent transport of Hsp70 was also studied in minimally transformed human HaCaT keratinocytes. We found that after U73122 and heat stress treatment, HaCaT cells secreted Hsp70 in a manner similar to A431 cells. Collectively our results suggest that human keratinocyte-derived cells release Hsp70 in the extracellular medium through a pathway involving secretory-like granules.     

Induction of hsp 72/73 by herbimycin A, an inhibitor of transformation by tyrosine kinase oncogenes

Herbimycin A, which has been known to inactivate and degrade p60v-src tyrosine kinase, induced an elevated synthesis of a protein with a molecular size of 70 kDa in A431 human epidermoid carcinoma cells. This protein showed the same migration distance on SDS-polyacrylamide gel electrophoresis as that of the protein induced in the cells by heat shock treatment, and this 70-kDa protein was identified as a member of the heat shock protein 70 family (hsp70) through immunoprecipitation with anti-hsp72/73 antibody and partial digestion with V8 protease. The induced level of the 70-kDa protein was dependent on the length of period and the concentration of herbimycin A treatment. Cellular fractionation and indirect immunofluorescence analyses revealed that the 70-kDa protein induced by herbimycin A was localized in the cytoplasm, in contrast to the nuclear distribution of hsp70 induced by heat treatment. Induction of hsp70 by herbimycin A was also observed in several other cells, including HeLa S3 cells, chicken embryo fibroblasts, NIH3T3 cells, and Rous sarcoma virus-transformed NIH3T3 cells.     

A model of glucocorticoid receptor unfolding and stabilization by a heat shock protein complex

It has recently been reported that incubation of avian progesterone receptors, mouse glucocorticoid receptors, or the viral tyrosine kinase pp60^src with rabbit reticulocyte lysate reconstitutes their association with the 90 kDa heat shock protein, hsp90. The reassociation is thought to require unfolding of the steroid receptor or pp60^src before hsp90 can bind. The unfoldase activity may be provided by hsp70, which is also present in the reconstituted receptor heterocomplex. In this paper we review evidence that hsp70 and hsp90 are associated in cytosolic heterocomplexes that contain a limited number of other proteins. From an analysis of known receptor-hsp interactions and a predicted direct interaction between hsp90 and hsp70 we have developed an admittedly very speculative model of glucocorticoid receptor unfolding and stabilization. One important feature of the model is that the receptor becomes attached to a heat shock protein heterocomplex rather than undergoing independent unfolding and stabilization events. The model requires that hsp70 and hsp90 bind directly to the receptor at independent sites. Importantly, the model accomodates the stoichiometry of 2 hsp90 per 1 molecule of receptor that has been assayed in the untransformed GR heterocomplex in cytosols prepared from hormone-free cells.     

Subnuclear Trafficking of Glucocorticoid Receptors In Vitro: Chromatin Recycling and Nuclear Export

We have used digitonin-permeabilized cells to examine in vitro nuclear export of glucocorticoid receptors (GRs). In situ biochemical extractions in this system revealed a distinct subnuclear compartment, which collects GRs that have been released from chromatin and serves as a nuclear export staging area. Unliganded nuclear GRs within this compartment are not restricted in their subnuclear trafficking as they have the capacity to recycle to chromatin upon rebinding hormone. Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality. In addition, chromatin-released receptors export from nuclei of permeabilized cells in an ATP- and cytosol-independent process that is stimulated by sodium molybdate, other group VI-A transition metal oxyanions, and some tyrosine phosphatase inhibitors. The stimulation of in vitro nuclear export by these compounds is not unique to GR, but is restricted to other proteins such as the 70- and 90-kD heat shock proteins, hsp70 and hsp90, respectively, and heterogeneous nuclear RNP (hnRNP) A1. Under analogous conditions, the 56-kD heat shock protein, hsp56, and hnRNP C do not export from nuclei of permeabilized cells. If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited. Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.     

Transactivation of hsp70-1/2 in geldanamycin-treated human non-small cell lung cancer H460 cells: involvement of intracellular calcium and protein kinase C

Geldanamycin is an antitumor drug that binds HSP90 and induces a wide range of heat shock proteins, including HSP70s. In this study we report that the induction of HSP70s is dose-dependent in geldanamycin-treated human non-small cell lung cancer H460 cells. Analysis of the induction of HSP70s specific isoform using LC-ESI-MS/MS analysis and Northern blotting showed that HSP70-1/2 are the major inducible forms under geldanamycin treatment. Transactivation of hsp70-1/2 was determined by electrophoretic mobility-shift assay using heat shock element (HSE) as a probe. The signaling pathway mediators involved in hsp70-1/2 transactivation were screened by the kinase inhibitor scanning technique. Pretreatment with serine/threonine protein kinase inhibitors H7 or H8 blocked geldanamycin-induced HSP70-1/2, whereas protein kinase A inhibitor HA1004, protein kinase G inhibitor KT5823, and myosin light chain kinase inhibitor ML-7 had no effect. Furthermore, the protein kinase C (PKC)-specific inhibitor Ro-31-8425 and the Ca2+-dependent PKC inhibitor G?-6976 diminished geldanamycin-induced HSP70-1/2, suggesting an involvement of the PKC in the process. In addition, geldanamycin treatment causes a transient increase of intracellular Ca2+. Chelating intracellular Ca2+ with BAPTA-AM or depletion of intracellular Ca2+ store with A23187 or thapsigargin significantly decreased geldanamycin-transactivated HSP70-1/2 expression. Taken together, our results demonstrate that geldanamycin-induced specific HSP70-1/2 isoforms expression in H460 cells through signaling pathway mediated by Ca2+ and PKC.     

Epidermal growth factor receptor metabolism and protein kinase activity in human A431 cells infected with Snyder-Theilen feline sarcoma virus or harvey or Kirsten murine sarcoma virus.

When human A431 cells, which carry high numbers of epidermal growth factor (EGF) receptors, are exposed to EGF, the total content of phosphotyrosine in cell protein is increased, the EGF receptor becomes phosphorylated at tyrosine, and new phosphotyrosine-containing 36,000- and 81,000-dalton proteins are detected. We examined the properties of A431 cells infected with Snyder-Theilen feline sarcoma virus, whose transforming protein has associated tyrosine protein kinase activity, and Harvey and Kirsten sarcoma viruses, whose transforming proteins do not. In all cases, the infected cells were more rounded and more capable of anchorage-independent growth than the uninfected cells. EGF receptors were assayed functionally by measuring EGF binding and structurally by metabolic labeling and immunoprecipitation. In no case did infection appear to alter the rate of EGF receptor synthesis, but infection reduced EGF receptor stability by about 50% for cloned Harvey sarcoma virus-infected cells and by 80% for cloned feline sarcoma virus-infected cells. The corresponding reductions in EGF binding were 70 and 90%, respectively. The proteins of feline sarcoma virus-infected A431 cells contained an increased amount of phosphotyrosine, and the 36,000- and 81,000-dalton phosphoproteins were detected. The EGF receptor was not detectably phosphorylated at tyrosine, however, unless the cells were exposed to EGF. The Harvey and Kirsten sarcoma virus-infected cells did not exhibit elevated levels of phosphotyrosine either in the total cell proteins or in the EGF receptor, nor were the 36,000- and 81,000-dalton proteins detectable. However, these phosphoproteins were found in the infected cells after EGF treatment. Thus, all of the infected A431 cells exhibited reduced EGF binding and increased degradation of EGF receptors, yet their patterns of protein phosphorylation were distinct from those of EGF-treated A431 cells.     

Intracellular distribution of proteasomes in A-431 cells

The intracellular proteasome distribution in A-431 cells was shown using methods of cell fractionation and immunofluorescence. In growing cells the distribution of proteasomes was EGF-dependent. In unstimulated cells and within 30 min of EGF treatment, proteasomes were localized in the cytoplasm and nuclei, but not on the plasma membrane. After 30 min of EGF treatment they were observed on the plasma membrane as well. In A-431 cells cultivated for 24 h in the medium with a lowered serum concentration, proteasomes were detected on the plasma membrane already in unstimulated cells. It is suggested that dephosphorylation of the EGF receptor and signalling proteins in unstimulated cells may depend on the proteolytic activity of proteasomes.     

Enhancement of epidermal growth factor (EGF) and insulin-stimulated tyrosine phosphorylation of endogenous substrates by sodium selenate.

Sodium selenate stimulated tyrosine phosphorylation of the epidermal growth factor (EGF) receptor in A431 cells and enhanced the tyrosine phosphorylation of endogenous proteins in response to EGF in A431 cells and insulin in NIH 3T3 HIR3.5 cells. These effects occurred without changes in ligand binding, were not abolished by mercaptoethanol in the case of the EGF receptor, and appeared distinct from the effects of vanadate. These results support a role for selenium or selenoproteins in regulating EGF and insulin receptor tyrosine kinase activity and suggest a mechanism whereby selenium-containing compounds contribute to cell growth.     

Autophosphorylation of EGF receptors in the A431 cell line with an increased intracellular concentration of Ca(2+) ions

The increase in the intracellular concentration of Ca2+ in A431 cells induced by the calcium ionophore A23187 leads to phosphorylation of epidermal growth factor (EGF) receptors at serine and/or threonine residues. This process is accompanied by the decrease in the level of EGF receptor autophosphorylation at tyrosine residues. Preincubation of cells in a A23187-containing medium in the presence of phorbol-12-myristoyl-13-acetate leads to a further decrease of the phosphotyrosine content in EGF receptors. At increased intracellular concentrations of Ca2+ preincubation of A431 cells with the protein kinase C inhibitor H-7 has no effect on the degree of EGF receptor autophosphorylation. Down-regulation of cellular protein kinase C does not change the A23187-induced effect either. The data obtained suggest that the decreased autophosphorylation of EGF receptors induced by Ca2+ is not due to the activation of cellular protein kinase C.     

Cytoskeletal and Morphological Changes Associated with the Specific Suppression of the Epidermal Growth Factor Receptor Tyrosine Kinase Activity in A431 Human Epidermoid Carcinoma

The epidermal growth factor (EGF) receptor is well known as a mediator of mitogenic signaling and its tyrosine kinase activity has been suggested as a viable target in cancer chemotherapy. To explore the consequences of abolishing the kinase activity of this receptor, we have utilized a potent and specific inhibitor of the enzyme, PD 153035, to sustain a long-term suppression of its activity. This compound inhibits EGF receptor autophosphorylation in cells with an IC₅₀in the low nanomolar range and does not block PDGF or FGF receptor kinase until concentrations are greater than 10 μM.[1] Human epidermoid carcinoma A431 cells were grown in the presence of PD 153035 and were passed weekly until cells grew in the presence of 1 μMinhibitor. These cells, referred to as A431R, showed a remarkable change in morphology, becoming flattened and spread out. A comparison of the sensitivity of EGF receptor autophosphorylation to PD 153035 between A431 and A431R showed a similar dose response, indicating that the cells had not developed any defect in the kinase which might make it resistant to the inhibitor. Likewise, EGF receptor autophosphorylation in response to exogenously added EGF, as well as receptor internalization, was similar between the two cell lines. Furthermore, analysis of A431R cells by flow cytometry showed no significant change in DNA content or percentage of cells in any one phase of the cell cycle compared to the parent line.¹²⁵I-labeled EGF/receptor binding studies showed that receptor number in the A431R cells was equivalent to that of the parent line; however, the Scatchard plot was linear, in contrast to the typical biphasic plot obtained with the parent cells, implying a loss of high-affinity receptors. Cytoskeletal preparations from both cell lines indicated that the A431R had fourfold less EGF receptor associated with the cytoskeleton than A431. This was accompanied by a remarkable increase in polymerized actin stress fibers throughout the A431R cells, which most likely accounts for their flattened morphology. The A431R cells also exhibited a twofold increase in the expression of focal adhesion kinase, which is consistent with a greater contact area for their cell surface and increase in focal adhesions. Finally, although the A431R cells have a doubling time of 24 h, similar to that of the parent line, these cells stop growing as the monolayer approaches confluence, reminiscent of the contact inhibition seen in nontransformed cells. These data indicate that long-term suppression of the EGF receptor tyrosine kinase activity in A431 human epidermoid carcinoma results in certain cellular properties which are more consistent with a differentiated and nontransformed phenotype.     

pp60cSrc Is a Caspase-3 Substrate and Is Essential for the Transformed Phenotype of A431 Cells

The protein tyrosine kinase c-Src is a major signal transduction element in many growth factor receptor signals for proliferation and transformation. We showed recently that c-Src is a mediator of antiapoptotic signals through regulation of the antiapoptotic gene Bcl-X_L. A431 cells overexpress the EGF receptor (EGFR) and possess high Src activity. In A431 cells, Src is activated by the EGFR, and inhibition of the EGF receptor results in c-Src inhibition. In this study we show that (i) inhibition of the EGFR kinase or Src kinase by specific inhibitors results in growth inhibition and inhibition of colony formation in soft agar. The relative efficacies of the EGFR kinase inhibitor and of the Src kinase inhibitor are similar suggesting the major role src plays in the oncogenic signaling of EGFR in A431 cells. (ii) The Src kinase inhibitor PP1 sensitizes A431 cells to CDDP-induced apoptosis. (iii) CDDP induces caspase-3-dependent cleavage of the c-Src C-terminal portion and a concomitant reduction in Bcl-X_L levels. We conclude that c-Src is an important antiapoptotic signaling molecule downstream of the EGF receptor that contributes to the transformed phenotype of A431 cells.     

Involvement of ATP in the nuclear and nucleolar functions of the 70 kd heat shock protein.

The major heat shock protein, hsp70, is an ATP-binding protein which is synthesized in very large amounts in response to stress. In unstressed, or recovered, mammalian cells it is found in both nucleus and cytoplasm. Under these conditions, its interaction with nuclei is weak, and it is readily released from them upon lysis of cells in isotonic buffer. After heat shock, hsp70 binds tightly first to some nuclear component(s) and then to nucleoli. It can be released from these binding sites rapidly and specifically in vitro by as little as 1 microM ATP, but not by non-hydrolysable ATP analogues. Studies of hsp70 deletion mutations show that the ability of mutants to be released by ATP correlates with their ability to migrate to heat-shocked nucleoli and aid their repair in vivo. We propose a model in which ATP-driven cycles of binding and release of hsp70 help to solubilize aggregates of proteins or RNPs that form after heat shock. Cells also contain proteins related to hsp70 that are synthesized in the absence of stress. The most abundant of these shows the same behaviour as hsp70 after heat shock, and thus may perform a related function in both normal and stressed cells.     

Prolonged induction of p21Cip1/WAF1/CDK2/PCNA complex by epidermal growth factor receptor activation mediates ligand-induced A431 cell growth inhibition

Proliferation of some cultured human tumor cell lines bearing high numbers of epidermal growth factor (EGF) receptors is paradoxically inhibited by EGF in nanomolar concentrations. In the present study, we have investigated the biochemical mechanism of growth inhibition in A431 human squamous carcinoma cells exposed to exogenous EGF. In parallel, we studied a selected subpopulation, A431-F, which is resistant to EGF-mediated growth inhibition. We observed a marked reduction in cyclin-dependent kinase-2 (CDK2) activity when A431 and A431-F cells were cultured with 20 nM EGF for 4 h. After further continuous exposure of A431 cells to EGF, the CDK2 activity remained at a low level and was accompanied by persistent G1 arrest. In contrast, the early reduced CDK2 activity and G1 accumulation in A431-F cells was only transient. We found that, at early time points (4-8 h), EGF induces p21Cip1/WAF1 mRNA and protein expression in both EGF-sensitive A431 cells and EGF-resistant A431-F cells. But only in A431 cells, was p21Cip1/WAF1 expression sustained at a significantly increased level for up to 5 d after addition of EGF. Induction of p21Cip1/WAF1 by EGF could be inhibited by a specific EGF receptor tyrosine kinase inhibitor, tyrphostin AG1478, suggesting that p21Cip1/WAF1 induction was a consequence of receptor tyrosine kinase activation by EGF. We also demonstrated that the increased p21Cip1/WAF1 was associated with both CDK2 and proliferating cell nuclear antigen (PCNA). Taken together, our results demonstrate that p21Cip1/WAF1 is an important mediator of EGF-induced G1 arrest and growth inhibition in A431 cells.     

Effects of protein kinase C activation after epidermal growth factor binding on epidermal growth factor receptor phosphorylation

The possible role of epidermal growth factor (EGF) receptor phosphorylation at threonine 654 in modulating the protein-tyrosine kinase activity of EGF-treated A431 cells has been studied. It has been suggested that EGF could indirectly activate a protein-serine/threonine kinase, protein kinase C, that can phosphorylate the EGF receptor at threonine 654. Protein kinase C is known to be activated, and threonine 654 is phosphorylated, when A431 cells are exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). The protein-tyrosine kinase activity of EGF receptors is normally evidenced in EGF-treated cells by phosphorylation of the receptor at tyrosine. This is inhibited when TPA-treated cells are exposed to EGF. We now show that receptor phosphorylation at threonine 654 can also be detected in EGF-treated A431 cells, presumably due to indirect stimulation of protein kinase C or a similar kinase. Some receptor molecules are phosphorylated both at threonine 654 and at tyrosine. Since prior phosphorylation at threonine 654 inhibits autophosphorylation, we propose that protein kinase C can phosphorylate the threonine 654 of autophosphorylated receptors. This provides evidence for models in which protein kinase C activation, consequent upon EGF binding, could reduce the protein-tyrosine kinase activity of the EGF receptor. Indeed, we find that 12-O-tetradecanoylphorbol-13-acetate, added 10 min after EGF, further increases threonine 654 phosphorylation and induces the loss of tyrosine phosphate from A431 cell EGF receptors.     

Functional studies on the EGF receptor with an antibody that recognizes the intracellular portion of the receptor

An antibody against the human epidermal growth factor receptor (EGF), capable of activating its tyrosine kinase has been produced. Antibody 2913 recognizes only the cytoplasmic portion of the EGF receptor in A431 carcinoma cells, in normal human fibroblasts, and in a variety of other human tumor cell lines (Xu, Y.-A., Richert, N., Ito, S., Merlino, G. T., and Pastan, I. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7308-7313). Indirect immunofluorescence and electron microscopy show that the antibody binds to intact cells only after membrane permeabilization. Moreover the antibody immunoprecipitates the v-erb-B gene product in avian myeloblastosis virus-infected cells but does not recognize the secreted form (105 kDa) of the A431 cell EGF receptor which lacks the cytoplasmic domain. Antibody 2913 activates the EGF receptor kinase in solubilized A431 membranes causing autophosphorylation on tyrosine residues only. Tryptic peptide maps suggest that antibody 2913 and EGF stimulate phosphorylation of the same amino acid residues. By electron microscopy, the cytoplasmic portion of the receptor was followed throughout its endocytotic pathway. The results show that the kinase domain is rapidly degraded in lysosomes with no accumulation in the cytoplasm or in the nucleus.