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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.

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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.

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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.

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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.

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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.

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The ability of 26S proteasomes from the human proerythroleukaemic cell line K562 to degrade high-molecular-weight cytoplasmic RNAs, particularly specific messenger RNA, has been detected. The addition of hemin to K562 cells in the culture media leads to redistribution of proteasomes and their migration mainly to the cytoplasm. The human wild type p53 gene mRNA was shown to be specifically nucleolized by proteasomes. These particles displayed endoribonuclease activity towards mRNA for Renilla sp. luciferase. Proteasomes also specifically degraded Alu-containing mRNAs. A supposition is made about the involvement of proteasomes in stability control of specific RNA groups.

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Proteosomes from human proerythroleukaemic cell line K562 are found to degrade high molecular weight cytoplasmic RNAs, particularly ribosomal and specific messenger RNA. This activity was observed to be endoribonucleotylic. The induction of differentiation by erythroid pathway in K562 cells invokes augmentation of endonuclease activity in proteasomes. The number of characteristics of this enzymatic activity was investigated. Specificity of endonuclease of these RNPs is shown to be Ca- and Mg-dependent. Dephosphorylation of protein subunits suppresses RNase activity of proteasomes. Endonuclease of proteasomes is thermolabile. The examined activity depends on the secondary structure of substrate RNA. Protein subunits are responsible for ribonuclease activity of proteasomes rather than for low molecular weight RNAs associated with the complex.

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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.

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A comparative study was made of reactive oxygen species (ROS) in rat embryo fibroblasts and their transformants. Primary rat embryo fibroblasts (REF), REF transformed by the complementing oncogenes E1A plus cHa-ras (cell line E1A + Ras), and REF transformed by E1A plus E1B-19 kDa (cell line E1A + E1B) were studied. ROS generation was measured with microfluorometric assay using fluorescent probe 2',7'-dichlorofluorescin diacetate. It has been shown that the block of REF and E1A + 1B cells in the G1/S under serum-starved conditions (0.5% serum) for 24-48 h was paralleled by a decrease in ROS generation. Activation of serum-starved REF and E1A + 1B cells with 10% serum resulted in reactivation of cell cycle and gradual increase in ROS generation. The maximum intracellular level of ROS correlated in time with the phase of DNA synthesis. Serum-starved E1A + Ras cells were not stopped in the G1/S and ROS production of these cells was not dependent on serum growth factors. The prolonged cultivation of E1A + Ras cells in the medium with low serum content (0.5%) caused a sharp increase in ROS generation, which was accompanied by apoptotic death.

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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.

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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.

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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.

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The review is devoted to the participation of phospholipase C gamma 1 in cell signaling. A great attention is paid to mechanisms of activation of phospholipase C gamma and its interaction with other signaling molecules. The role of phospholipase C gamma binding to cytoskeletal elements in cell signaling is discussed.

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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.

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