[The extracellular heat shock protein 70 and its functions].

Various stress factors induce the accumulation of intracellular heat shock proteins, in particular heat shock protein 70 (Hsp70), which is considered as the main component of cellular response to stress. Though initially Hsp70 was thought to be a typical intracellular protein, the strong evidence now exists demonstrating that the Hsp70 exits mammalian cells not only following necrotic cell death but also by a process involving its active release. This review discusses the mechanisms of Hsp70 release and immunomodulatory and signaling functions of extracellular Hsp70.

[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 EGF on ubiquitination and proteasome-dependent degradation of phospholipase C gamma1 in A431 cells]. / Vliianye EFR na ubikvitinilirovanie i proteasomozavisimuiu degradatsiiu fosfolipazy C gamma1 v kletkakh A431.

Phospholipase C gamma 1 (PLC gamma 1), an enzyme participating in phosphoinositide turnover, is one of the key elements in cell signaling. Here it is shown that treatment of A431 carcinoma cells with proteasome inhibitors Mg132 and lactacystin results in increasing the PLC gamma 1 intracellular level. Simultaneously, several additional bands with lower electrophoretic mobilities were detected on immunoblots, using anti-PLC gamma 1 antibodies. PLC gamma 1 ubiquitinilation was shown using immunoprecepitation. In control A 431 cells, PLC gamma 1 is ubiquitinilated, but the addition of EGF greatly induces the ubiquitinilation of the protein. Association of PLC gamma 1 with ubiquitin-ligase c-Cb1 was shown. Dynamics of ubiquitinilation under EGF treatment is in a close agreement with that of association of PLC gamma 1 and c-Cb1. It is concluded that PLC gamma 1 is ubiquitinilated and degraded by proteasomes. PLC gamma 1 ubiquitinilation is an EGF-dependent process.

[Effect of EGF on the intracellular distribution of heat-shock proteins in A431 cells]. / Vliianie IeFR na vnutrikletochnoe raspredelenie belkov teplovogo shoka v kletkakh A431.

The intracellular distribution of hsp70 and hdj1 was studied using immunofluorescent method. In nonstimulated cells hsp70 and hdj1 were observed in the cytoplasm of A431 cells. When 100 ng/ml EGF was added for 15 min, both hsp70 and hdj1 were accumulated in the nuclei. Later on (up to 1 h) hsp70 was exported from the nuclei to be observed mainly in the cytoplasm, whereas hdj1 remained in the nuclei. In cells exposed to tyrphostin AG1478, this inhibitor of tyrosine kinase activity of EGF receptor prevented EGF-dependent accumulation of hsp70 and hdj1 in the nuclei. U73122, an inhibitor of phospholipase C activity, induced tyrosine phosphorylation of EGF receptor without EGF stimulation. In cells treated with U73122, both hsp70 and hdj1 were detected in the nuclei of non-stimulated cells. It is concluded that the intracellular distribution of heat shock proteins in A431 cells depends on tyrosine kinase activity of EGF receptor. Here we report for the first time the influence of EGF on the intracellular redistribution of heat shock proteins.

[EGF receptor degrades via the proteasome-dependent pathway in A-431 cells]. / Retseptor EFR degradiruet po proteasom-zavisimomu puti v kletkakh A-431.

It is known that a lot of cell receptors degrade by ubiquitine-proteasome pathway. Here we show that degradation of the epidermal growth factor (EGF) receptor is proteasome-dependent. Treatment of A-431 cells with lactacystine, an inhibitor of proteolytic activities of 26S proteasomes, induces accumulation of the receptor in cells. Incubation of cell lysates with isolated 26S proteasomes leads to diminishing EGF receptor in these cells. Active (tyrosine phosphorylated) EGF receptor is a target of proteolysis by proteasomes.

[Phospholipase C negatively regulates tyrosine phosphorylation of EGF receptor in A-431 cells]. / Fosfolipaza C negativno reguliruet fosforilirovanie po tirozinu retseptora EFR v kletkakh A-431.

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.

[Effect of EGF on nuclear-cytoplasmic distribution of proteasomes in A-431 cells]. / Vliianie EFR na iaderno-tsitoplazmaticheskoe raspredelenie proteasom v kletkakh A-431.

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.

[Tissue specificity of subunit composition and EGF-dependent changes in the subunit pattern of 20S-proteasomes]. / Tkanespetsifichnost' sub''edinichnogo sostava i EFR-zavisimye izmeneniia nabora sub''edinits 20S-proteasom.

The subunit pattern of 20S proteasomes from rat kidney, rat liver, human A-431 cells, human K-562 cells and mouse NIH 3T3 cells were studied. Proteasomes in cells of a common tissue origin appeared to be similar, independently of the intensity of cell proliferation. Unlike, proteasomes in cells of various types of tissue specificity differed from each other. Besides, EGF was shown to induce changes in the subunit pattern of proteasomes in A-431 cells.

[Intracellular distribution of proteasomes in A-431 cells]. / Vnutrikletochnoe raspredelenie proteasom v kletkakh A-431.

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.

[Participation of phosphoinositide-specific phospholipase Cgamma1 and STAT1 protein in the formation of latent complexes of signal proteins]. / Uchastie fosfoinozitid-spetsificheskoi fosfolipazy Cgamma1 i belka STAT1 v obrazovanii latentnykh kompleksov signal'nykh belkov.

It is known that the growth factor activates appropriate membrane receptors which become starting points of cascades of protein-protein interactions leading to cellular response. Recent data suggest that different signalling pathways may cross-talk during the cellular response. Here we show that phosphoinositide-specific phospholipase C gamma 1, one of the key elements in phosphoinositide pathway of signal transduction, is physically associated with members of the STAT pathway. The precipitation of phospholipase C gamma 1, using polyclonal antibody in A-431 cells, leads to co-immunoprecipitation of STAT1 alpha and STAT1 beta, as well as STAT3. The formation of such complexes was observed in both unstimulated and EGF stimulated cells. The participation of SH3-domains in the formation of such complexes is discussed.

[Association of phosphoinositide-specific phospholipase Cgamma1 with elements of cytoskeleton in A-431 cells]. / Sviaz' fosfoinozitid-spetsificheskoi fosfolipazy Cgamma1 s élementami tsitoekelta v kletkah A-431.

It is shown that phosphoinositide-specific phospholipase C gamma 1 (PLC gamma 1), a substrate of growth factor receptors, is associated with cytoskeleton in A-431 cells. PLC gamma 1 is co-localized only with the cortical actin but not with actin stress fibers. Since EGF receptor is also co-localized with the cortical actin it is concluded that PLC gamma 1 co-localized with actin is mediated by the EGF receptor. After the treatment with cytochalasin B PLC gamma 1 is co-localized with actin aggregates and cytoskeleton elements other than actin. Using double immunofluorescence PLC gamma 1 is shown to be associated with cytokeratin intermediate filaments. The cross-talking of different cytoskeleton elements and their participation in cell signaling is discussed.

[Proteins of the Stat family: role in cell signalling]. / Belki semeistva Stat: rol' v provedenii kletochnogo signala.

This review is devoted to the participation of STATs in cell signalling. A great attention is paid to the mechanisms of STATs activation and cross-talking of STAT-pathway of signal transduction with others. The role of STATs in cell proliferation, differentiation, apoptosis and transformation is discussed.

[The transcription factor STAT3 is associated with elements of the cytoskeleton in A-431 cells]. / Transkriptsionnyi faktor STAT3 sviazan s élementami tsitoskeleta v kletkakh A-431.

The transcription factor STAT3 (signal transducers and activators of transcription) takes part in cell signaling. Here we show that STAT3 is associated with the cytoskeleton in A-431 cells. The protein is determined in cytoskeletal (detergent insoluble) fraction of control cells and of cells after RGF treatment. STAT3 is diffusely distributed in the cytoplasm of control cells to be seen relocalized to the nuclei after EGF treatment. Besides, the protein is observed in cell membrane protrusions. Using double immunofluorescence, co-localization of action and STAT3 was shown in these structures.

[Transcription factors STAT1 and STAT3 are localized in different compartments of rat embryo fibroblasts transformed by E1A and Ha-Ras oncogenes]. / Transkriptsionnye faktory STAT1 i STAT3 lokalizovany v raznykh kompartmentakh émbrional'nykh fibroblastov krysy, transformirovannykh onkogenami E1A + Ha-Ras.

Intracellular distribution of STAT1 and STAT3 transcription factors in normal fibroblasts (REF) and in E1A + Ha-Ras transformed cells has been studied by means of indirect immunofluorescence. The obtained data evidence that in REF cells, in response to the growth factor addition, STAT1 and STAT3 proteins are redistributed from the cytoplasm to the nucleus. In transformants E1A + Ha-Ras, however, significantly different pictures can be seen: while STAT1 is found to be constitutively localized in the cell nuclei, STAT3 is predominantly revealed in the cytoplasm. The data obtained from fractionation of subcellular structures confirm in general the immunofluorescence results on the cytoplasmic localization of STAT3 protein in E1A + Ha-Ras transformants. Thus, transformation of REF cells with E1A + Ha-Ras oncogenes causes a constitutive activation of STAT1 and STAT3 transcription factors, the proteins, however, being distributed in different cell compartments.