[Oxidized glutathione induces activation of the epidermal growth factor receptor and MAP kinases ERK 1,2].

Ligand-independent activation ("transactivation") of the epidermal growth factor receptor (EGFR) was demonstrated upon cell stimulation with cytokines, activators of G-protein-coupled receptors and various stressors. Recently, we showed transactivation of EGFR and activation of transcription factor STAT3, rather than STAT1, induced by glutathione disulfide (GSSG) and glutoxim in epidermoid carcinoma A431 cells (Burova et al., Dokl. Akad. Nauk., 2005, 404: 1-3). Glutoxim (PHARMA-VAM, Moscow) is a pharmacological synthetic analogue of GSSG, whose therapeutic use as an immunomodulator has been permitted. In this study, we investigated dynamics of EGFR activation upon A431 cell stimulation with GSSG and glutoxim. The time course of activation has a sinuous pattern. It has been shown that the intrinsic EGFR tyrosine kinase is responsible for the receptor phosphorylation induced by GSSG and glutoxim. Here, we also demonstrated the activation of ERK 1,2 upon treatment of A431 cells and HER14 cells (HIN 3T3 fibroblasts transfected with full-length EGFR) with these drugs. ERK 1,2 activation was abolished by AG1478, a pharmacological inhibitor of EGFR tyrosine kinase, implicating intrinsic EGFR tyrosine kinase in this process.

[EGF-dependent signaling pathways are activated under heat stress in A431 carcinoma cells].

EGF receptor transactivation and activation of EGF-dependent signaling pathways under heat shock conditions were studied. Heating A431 cells at 42 degrees C induced both EGF receptor tyrosine phosphorylation and appearance of phosphorylated forms of key components of its downstream signaling pathways - phospholipase Cgamma1 (PLCgamma1), transcription factor STAT3, and EPK1/2. It is suggested that EGF receptor is transactivated under heat shock in A431 cells. Pretreatment of heat-shocked cells with a specific inhibitor of EGF receptor tyrosine kinase tyrphostin AG1478 does not prevent EGF receptor and EPK 1/2 tyrosine phosphorylation. In contrast, tyrphostin AG1478 abrogates tyrosine phosphorylation of PLCgamma1 and STAT3. This suggested that the intrinsic EGF receptor tyrosine kinase is not involved in EGF receptor transactivation, but is sufficient for PLCgamma1 and STAT3 activation in stress conditions. The effect of a conditioned medium of heated cells was investigated to check whether autocrine mechanism is involved in EGF receptor transactivation. The conditioned medium of heated cells induced both tyrosine phosphorylation of EFG receptor and ERK 1/2. Simultaneously, neither PLCgamma1, not STAT3 phosphorylation were detected. Here, for the first time, we demonstrated the involvement of autocrine mechanism in EGF receptor transactivation under heat stress in A431 carcinoma cells, but additional intracellular events are essential for activation of EGF receptor downstream signaling pathways.

[Effect of nocodazole on the activation of transcription factors STAT1 and STAT3 in A431 cells]. / Vliianie nokodazola na aktivatsiiu transkriptsionnykh faktorov STAT1 i STAT3 v kletkakh A431.

The STAT transcription factors (signal transducers and activators of transcription), STAT1 and STAT3, are involved in signal transduction from growth factors and different cytokine receptors. STAT1 and STAT3 activation mechanisms are not sufficiently investigated, but they are known to depend upon both cell type and stimulus for either of them. Recently, we have shown that nocodazole blocked EGF-induced STAT1 transport to the nucleus. Here, we have compared STAT1 and STAT3 activation in response to IFNgamma, IFNalpha and epidermal growth factor (EGF) in A431 cells. We have shown the STAT1 activation by all these agents; unlike, STAT3 was activated by EGF only. STAT1 and STAT3 activation upon EGF is blocked by both nocodazole and Src-kinase family inhibitor. STAT1 activation upon IFNgamma influence is blocked by nocodazole, but does not depend on the activity of Src-family kinases. The increased STAT3 phosphorylation results from a combined action of Src-kinase inhibitor and IFNgamma. IFNalpha-induced activation of STAT1 was not inhibited by either nocodazole or Src-kinase inhibitor. Taken together, the data obtained suggest that the activation of both STAT1 and STAT3 in A431 cells is accomplished by different mechanisms.

[Dynamics of the interaction of Stat1 transcription factor with the internalized EGF receptor and karyopherins alpha in response to stimulation of cells with epidermal growth factor]. / Dinamika vzaimodeistviia transkriptsionnogo faktor STAT1 s internalizovannym retseptorom EFR i karioferinami alpha v otvet na stimuliatsiiu kletok épidermal'nym faktorom rosta.

The present paper deals with two aspects of EGF-induced signal transduction via transcriptional factor STAT1. Utilizing vesicle fractionation in Percoll density gradient followed by co-immunoprecipitation, we observed the association of STAT1 with EGF receptor internalized in early endosomes. Co-immunoprecipitation studies with antikaryopherin alpha antibody showed the binding of activated STAT1 to nuclear import factors--karyopherins alpha. Both complexes demonstrate similar dynamics upon EGF treatment: they are formed at the early times, cannot be detected within 15 min, and reappear in 20 min or later. The complex of STAT1 and karyopherin alpha is localized in the cytoskeletal fraction.

[The role of SRC kinase in activation of transcription factor STAT1]. / Rol' tirozinkinazy SRC v aktivatsii traskriptsionnogo faktora STAT1.

Transcription factor STAT1 (Signal Transducers and Activators of Transcription) takes part in signal transduction from receptors of growth factors and many cytokines, including interferons. In this paper, the role of tyrosinkinases Src and JAK2 was estimated in activation of STAT1 by epidermal growth factor (EGF) and hyperosmotic shock. Using a pharmacological inhibitor of Src kinases CGP77675 and cells with knockout c-src, it was shown that Src activated STAT1 upon stimulation by both epidermal growth factor and hypersomatic shock. In contrast, JAK2 activity exerted no influence on these processes.

[EGF-dependent association of 20S-proteasome and alpha-RNP particles with the epidermal growth factor receptor in A-431 cells]. / EFR-vyzvannaia assotsitsiia 20S-proteasom i alpha-RNP-chastits s retseptorom EFR v kletkakh A-431.

Here we demonstrate that the epidermal growth factor (EGF) induces association of prosomes (20S-proteasomes) with its receptor in A-431 cells. Additionally, ligand-dependent association of ribonucleoprotein particles (alpha-RNP), containing small ALU-like RNA, with the EGF receptor was demonstrated. A suggestion has been put forward on the involvement of prosomes and alpha-RNP in the EGF signal transmission to different stages of gene expression.

[Intact microtubule network is necessary for the EGF-induced transport of transcription factor STAT1 in the nucleus of A-431 cells]. / Intaktnaia set' mikrotrubochek neobkhodima dlia EFR-indutsirovannogo transporta transkriptsionnogo faktora STAT1 v iadro kletok A-431.

The mechanism by which transcription factor STAT1 is translocated from the cytoplasm to the cell nucleus is not clear. We put forward a hypothesis suggesting an important role of the cytoskeleton in signal transduction. The results of the present work show that the treatment of cells with nocodazole, a microtubule-disrupting drug, inhibits completely STAT1 import to the nucleus. However, the treatment of cells with cytochalasin B, which is known to depolymerize microfilaments, exerted no detectable effect on the transport of STAT1. The sensitivity to nocodazole treatment suggests that STAT1 may utilize a transport pathway that involves the tubulin cytoskeleton. These data throw light on some mechanism of a rapid and effective nonvesicular transport of STAT1.

[Dynamics of EGF-induced nuclear-cytoplasmic transport of transcription factor STAT1 in A431 cells]. / Dinamika EFR-zavisimogo iaderno-tsitoplazmaticheskogo pereraspredeleniia transkriptsionnogo faktora STAT1 v kletkakh A431.

Dynamics of nuclear translocation of transcription factor Stat1 in human epidermoid carcinoma A431 cells in response to the epidermal growth factor (EGF) was examined by immunofluorescent microscopy and in cytoplasmic and nuclear extracts by Western blot. In has been shown that a prolonged presence of EGF induces a rapid tyrosine phosphorylation and nuclear translocation of Stat1 (within 5 min). The maximum amount of this protein in the nucleus was reached 30 min after the cell treatment to be maintained at the same level for 5 h. To study the dynamics of the export of Stat1 from the nucleus, a gentle treatment of cells with acetate buffer, pH 4.5, was used for extracting the surface-bound EGF. In this case, a complete dephosphorylation of Stat1 in the cytoplasm was observed in 30 min and the export of the Stat1 from the nucleus lasted for 1-6 h. These studies suggest the existence of an EGF-dependent dynamic equilibrium between the import and export of Stat1 in A431 cells.

[The tyrosine kinase activity of the internalized epidermal growth factor receptor in A-431 cells]. / Tirozinkinaznaia aktivnost' internalizovannogo retseptora épidermal'nogo faktora rosta v kletkakh A-431.

The interaction between epidermal growth factor (EGF) and specific membrane receptors in A-431 cells results in stimulation of its tyrosine kinase (TK) activity. The intrinsic tyrosine kinase phosphorylates both the receptor itself and different intracellular substrates. Upon EGF binding, clustering and internalization of EGF-receptor (EGF-R) complexes are observed. In the present study by incubating A-431 cells with EGF at 4 or at 37 degrees C, eluting surface-bound EGF we were able to compare the TK-activity of internalized EGF receptors to those localized on the plasma membrane. By the method of the phosphorylation of exogenic substrate poly (Glu/Tyr) 4:1 in vitro it was shown that the total TK-activity was equal to 3 microM PO4- incorporated/mg protein/min. Under conditions, when 40% EGF-R complexes were internalized their activity consisted of 46% of the total after inactivation 60% surface-bound receptors. We draw a conclusion that the TK-activity of EGF-receptors is remained during their internalization.