Inhibitors of tyrosine and ser/thr phosphatases modulate the heat shock response

Abstract

Following heat shock the expression of heat shock genes is regulated by the heat shock transcription factor, HSF, known to bind to arrays of the heat shock element, NGAAN, upstream of the heat shock genes. Phosphorylation of HSF is necessary for its activation. We report that the treatment of Chinese hamster HA-1 cells with 250 nM of okadaic acid (OA), a ser/thr phosphatase inhibitor, leads to an increase in activated HSF after heat shock. This is followed by the activation of the transcription of heat shock genes as assayed by the increase in the synthesis of beta-galactosidase in an HA-1 cell line containing the heat shock promoter ligated to the beta-galactosidase gene. To investigate the specificity of OA, we used other phosphatase inhibitors. We found that treatment of HA-1 cells with 500 microM of sodium vanadate, an inhibitor of tyr/phosphatases, resulted in a three to fivefold reduction in HSF activation and binding to the heat shock element following heat shock. Such reduction in HSF activation virtually abolished beta-galactosidase induction. Reduced HSP synthesis was further confirmed by SDS-PAGE and Western blot analysis using anti-HSP-70 and 28 antibodies. Sodium vanadate treatment of heat shocked cells greatly reduced levels of thermotolerance. These results show that ser/thr and specifically tyr/phosphatase inhibitors modulate the signal transduction pathway of HSF activation.

Glycogen Synthase Kinase 3β and Extracellular Signal-Regulated Kinase Inactivate Heat Shock Transcription Factor 1 by Facilitating the Disappearance of Transcriptionally Active Granules after Heat Shock

“…In most human cell lines, HSF-1 appears as punctate granules throughout the nucleus upon heat shock (11,29,41,55). The appearance and distribution of HSF-1 foci are similar when different fixatives (paraformaldehyde, methanol, or glutaraldehyde), or different antibodies to HSF-1 (11,29,41,55) are used, indicating that HSF-1 granule appearance after heat shock is not an artifact of sample preparation.…”

“…Electrophoretic mobility shift analysis with whole-cell extracts has been described in detail previously (41,43,74). Briefly, after each treatment, cells were rinsed with PBS and lysed in 100 l of extraction buffer (10 mM HEPES [pH 7.9], 0.4 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, 5% glycerol, 0.5 mM PMSF).…”

TNFα mediates susceptibility to heat-induced apoptosis by protein phosphatase-mediated inhibition of the HSF1/hsp70 stress response

“…Thus, PPs appear as key linker molecules between the cellular effects of TNFa, the activation of HSF1/hsp70 stress responses and cellular susceptibility to apoptosis. Several points of evidence support this concept: (i) PPs PP1/PP2a and PP2b can be activated by TNFa, 29,39 (ii) activation of these PPs increases susceptibility to apoptotic cell death, 28,29 (iii) activation of PP1/PP2a and PP2b (calcineurin) as well as elevated intracellular calcium levels inhibit phosphorylation of HSF1 and thus activation of the HSF1/hsp70 stress response, [40][41][42] (iv) hsp70, which acts in a negative-feedback loop on HSF1 activation, 18 induces PP1/PP2a as well as PP2b and thus blocks continuing activation of HSF1 43,44 and (v) ERK and NF-kB signaling, which maintained a certain responsiveness of HSF1/hsp70 and also protected cells from apoptotic death, is a concurring mechanism to PPs, since their mutual inhibition has been repeatedly demonstrated. 45,46 In summary, the data presented provide insights into the interplay of signals essential for cell survival, stress resistance and apoptosis.…”

Activation of heat shock protein (hsp)70 and proto-oncogene expression by alpha1 adrenergic agonist in rat aorta with age.

“…Upon stress, HSF1 is converted to a trimer which binds to multiple copies of a consensus nucleotide sequence in the promoter region of heat shock genes termed the heat shock element (16,17). Increased expression of hsp70 is associated with phosphorylation of HSF1 and is more pronounced in the presence of okadaic acid but diminished in the presence of a tyrosine phosphatase inhibitor (18)(19)(20). Thiol reducing agents have been shown to inhibit heat-induced synthesis of heat shock proteins, abundance of hsp70 mRNA, hsp70 gene promoter activity, and HSF DNA binding activity, suggesting involvement of a redox mechanism in the heat shock signal transduction pathway (21,22).…”