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A Short Lived Protein Involved in the Heat Shock Sensing Mechanism Responsible for Stress-activated Protein Kinase 2 (SAPK2/p38) Activation

1999; Elsevier BV; Volume: 274; Issue: 53 Linguagem: Inglês

10.1074/jbc.274.53.37591

1083-351X

Sonia Dorion, Julie Bérubé, Jacques Huot, Jacques Landry,

Endoplasmic Reticulum Stress and Disease

The stress-activated protein kinase 2 (SAPK2/p38) is activated by various environmental stresses and also by a vast array of agonists including growth factors and cytokines. This implies the existence of multiple proximal signaling pathways converging to the SAPK2/p38 activation cascade. Here, we show that there is a sensing mechanism highly specific to heat shock for activation of SAPK2/p38. After mild heat shock, cells became refractory to reinduction of the SAPK2/p38 pathway by a second heat shock. This was not the result of a toxic effect because the cells remained fully responsive to reinduction by other stresses, cytokines, or growth factors. Neither the activity of SAPK2/p38 itself nor the accumulation of the heat shock proteins was essential in the desensitization process. The cells were not desensitized to heat shock by other treatments that activated SAPK2/p38. Moreover, inhibiting SAPK2/p38 activity during heat shock did not block desensitization. Also, overexpression of HSP70, HSP27, or HSP90 by gene transfection did not cause desensitization, and inhibiting their synthesis after heat shock did not prevent desensitization. Desensitization rather appeared to be linked closely to the turnover of a putative upstream activator of SAPK2/p38. Cycloheximide induced a progressive and eventually complete desensitization. The effect was specific to heat shock and minimally affected activation by other stress inducers. Inhibiting protein degradation with MG132 caused the constitutive activation of SAPK2/p38, which was blocked by a pretreatment with either cycloheximide or heat shock. The results thus indicate that there is a sensing pathway highly specific to heat shock upstream of SAPK2/p38 activation. The pathway appears to involve a short lived protein that is the target of rapid successive up- and down-regulation by heat shock. The stress-activated protein kinase 2 (SAPK2/p38) is activated by various environmental stresses and also by a vast array of agonists including growth factors and cytokines. This implies the existence of multiple proximal signaling pathways converging to the SAPK2/p38 activation cascade. Here, we show that there is a sensing mechanism highly specific to heat shock for activation of SAPK2/p38. After mild heat shock, cells became refractory to reinduction of the SAPK2/p38 pathway by a second heat shock. This was not the result of a toxic effect because the cells remained fully responsive to reinduction by other stresses, cytokines, or growth factors. Neither the activity of SAPK2/p38 itself nor the accumulation of the heat shock proteins was essential in the desensitization process. The cells were not desensitized to heat shock by other treatments that activated SAPK2/p38. Moreover, inhibiting SAPK2/p38 activity during heat shock did not block desensitization. Also, overexpression of HSP70, HSP27, or HSP90 by gene transfection did not cause desensitization, and inhibiting their synthesis after heat shock did not prevent desensitization. Desensitization rather appeared to be linked closely to the turnover of a putative upstream activator of SAPK2/p38. Cycloheximide induced a progressive and eventually complete desensitization. The effect was specific to heat shock and minimally affected activation by other stress inducers. Inhibiting protein degradation with MG132 caused the constitutive activation of SAPK2/p38, which was blocked by a pretreatment with either cycloheximide or heat shock. The results thus indicate that there is a sensing pathway highly specific to heat shock upstream of SAPK2/p38 activation. The pathway appears to involve a short lived protein that is the target of rapid successive up- and down-regulation by heat shock. heat shock protein(s) mitogen-activated protein kinase extracellular signal-regulated kinase stress-activated protein kinase Jun N-terminal kinase epidermal growth factor tumor necrosis factor glutathione S-transferase hemagglutinin 4-morpholinepropanesulfonic acid MAP-activated protein activating transcription factor-2 Exposure of mammalian cells to heat stress induces two major signaling events, the transcriptional activation of genes coding for the heat shock proteins (HSP(s))1 (1Wu C. Clos J. Giorgi G. Haroun R.I. Kim S.-J. Rabindran S.K. Westwood J.T. Wisniewski J. Yim G. Morimoto R.I. Tissières A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994: 395-416Google Scholar, 2Morimoto R.I. Jurivich D.A. Kroeger P.E. Mathur S.K. Murphy S.P. Nakai A. Sarge K. Abravaya K. 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Chem. 1999; 274: 5061-5069Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). There is evidence for a role of each of the MAP kinase cascades in the cell response to stress and apoptosis signaling. SAPK1/JNK and SAPK2/P38 activities were in most cases associated with promotion of apoptosis, whereas ERK activity is in general associated with protection (26Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5045) Google Scholar). In some circumstances, however, SAPK1/JNK and SAPK2/p38 can have a protective function (27Zechner D. Craig R. Hanford D.S. McDonough P.M. Sabbadini R.A. Glembotski C.C. J. Biol. Chem. 1998; 273: 8232-8239Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 28Nemoto S. Xiang J. Huang S. Lin A. J. Biol. Chem. 1998; 273: 16415-16420Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 29Roulston A. Reinhard C. Amiri P. Williams L.T. J. Biol. Chem. 1998; 273: 10232-10239Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). Activation of SAPK2/p38 appears particularly significant in the case of heat shock because one of the heat shock proteins, HSP27, is phosphorylated by MAPKAP kinase-2, a physiological substrate of SAPK2/p38 (6Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1507) Google Scholar, 30Huot J. Lambert H. Lavoie J.N. Guimond A. Houle F. Landry J. Eur. J. Biochem. 1995; 227: 416-427Crossref PubMed Scopus (173) Google Scholar). SAPK2/p38-mediated phosphorylation has been shown to be essential for some activities of HSP27. Upon phosphorylation, high molecular weight HSP27 multimers dissociate into monomers and dimers providing a pool of active monomeric HSP27 which can modify the dynamics of actin filaments (31Lambert H. Charette S.J. Bernier A.F. Guimond A. Landry J. J. Biol. Chem. 1999; 274: 9378-9385Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 32Benndorf R. Hayess K. Ryazantsev S. Wieske M. Behlke J. Lutsch G. J. Biol. Chem. 1994; 269: 20780-20784Abstract Full Text PDF PubMed Google Scholar). This activity results in the stabilization of the microfilaments in response to oxidative stress (33Lavoie J.N. Lambert H. Hickey E. Weber L.A. Landry J. Mol. Cell. Biol. 1995; 15: 505-516Crossref PubMed Scopus (570) Google Scholar, 34Guay J. Lambert H. Gingras-Breton G. Lavoie J.N. Huot J. Landry J. J. Cell Sci. 1997; 110: 357-368Crossref PubMed Google Scholar, 35Landry J. Huot J. Biochem. Soc. Symp. 1999; 64: 79-89PubMed Google Scholar). However, strong activation of SAPK2/p38 and phosphorylation of HSP27 coupled with a high level of expression of HSP27 can also in some circumstances result in bleb formation and increased toxicity, illustrating the necessity of a tight control over SAPK2/p38 activation (36Huot J. Houle F. Rousseau S. Deschesnes R.G. Shah G.M. Landry J. J. Cell Biol. 1998; 143: 1361-1373Crossref PubMed Scopus (266) Google Scholar). HSP27 becomes phosphorylated within 20 min upon exposure of cells to elevated temperature (10Landry J. Chrétien P. Laszlo A. Lambert H. J. Cell. Physiol. 1991; 147: 93-101Crossref PubMed Scopus (125) Google Scholar). Phosphorylation is maximal immediately or soon after the triggering heat shock and returns to the control level after 5 h when cells are fully thermotolerant. Intriguingly, HSP27 is not phosphorylated when a second heat shock is applied during thermotolerance. Desensitization starts at 2 h after the priming treatment, peaks at 5 h, at which time no reinduction of HSP27 phosphorylation is observed, and vanishes by 15 h. In fact, a perfect temporal correlation has been found between the capacity of the cells to resist HSP27 phosphorylation and the thermotolerant state. This has led to the suggestion that some factors induced by the priming heat shock and generated along with the development of thermotolerance block the signaling pathway leading to HSP27 phosphorylation (10Landry J. Chrétien P. Laszlo A. Lambert H. J. Cell. Physiol. 1991; 147: 93-101Crossref PubMed Scopus (125) Google Scholar). In the present study, we investigated the mechanisms responsible for inhibiting HSP27 phosphorylation in heat-induced thermotolerant cells. The results showed that the desensitization process occurs at a proximal step in the signaling pathway upstream of SAPK2/p38 phosphorylation and activation. Desensitization was strictly homologous affecting exclusively heat shock induction of the SAPK2/p38 pathway. We infer that there is a sensing mechanism highly specific to heat shock in the activation pathway of SAPK2/p38, and we showed that it involves a short lived protein whose activity depends on a tight regulation between synthesis and degradation. [γ32-P]ATP (3,000 Ci/mmol) was purchased from NEN Life Sciences Products. H2O2, MG132 (N-carbobenzoxyl-Leu-Leu-norleucinal), cycloheximide, emetine, puromycin, and EGF were from Sigma Chemical Co. Thrombin was from Life Technologies, Inc. TNF-α and SB203580 were from Calbiochem. Recombinant HSP27 and ATF2-GST were purified from Escherichia coli transformed with appropriate plasmids (37Landry J. Lambert H. Zhou M. Lavoie J.N. Hickey E. Weber L.A. Anderson C.W. J. Biol. Chem. 1992; 267: 794-803Abstract Full Text PDF PubMed Google Scholar, 38Dérijard B. Raingeaud J. Barrett T. Wu I.-H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-684Crossref PubMed Scopus (1415) Google Scholar). Chemicals for electrophoresis were obtained from Bio-Rad and Fisher Scientific. Anti-HA is a mouse monoclonal antibody recognizing the YPYDVPDYA peptide sequence from human influenza hemagglutinin protein (Roche Molecular Biochemicals Corporation). All other antibodies used are polyclonal antibodies raised in rabbit. Anti-GST-MAPKAP kinase-2 recognizes the p45 and p54 isoforms of MAPKAP kinase-2 (30Huot J. Lambert H. Lavoie J.N. Guimond A. Houle F. Landry J. Eur. J. Biochem. 1995; 227: 416-427Crossref PubMed Scopus (173) Google Scholar); anti-p38, SAPK2/p38 (34Guay J. Lambert H. Gingras-Breton G. Lavoie J.N. Huot J. Landry J. J. Cell Sci. 1997; 110: 357-368Crossref PubMed Google Scholar); L2R3, the Chinese hamster HSP27 (33Lavoie J.N. Lambert H. Hickey E. Weber L.A. Landry J. Mol. Cell. Biol. 1995; 15: 505-516Crossref PubMed Scopus (570) Google Scholar); anti-HSP70 (no. 799), the inducible form of HSP70 (39Tanguay R.M. Wu Y. Khandjian E.W. Dev. Genet. 1993; 14: 112-118Crossref PubMed Scopus (141) Google Scholar); anti-HSP90αβ (no. 214), the α and β forms of HSP90 2T. C. Wu and R. M. Tanguay, unpublished data. ; and phospho-p38MAPK, the phosphorylated and activated form of SAPK2/p38 (New England Biolabs). Chinese hamster CCL39 and human HeLa cells were cultivated in Dulbecco's modified Eagle's medium containing 2.2 g/liter NaHCO3 and 4.5 g/liter glucose, and supplemented with 5% or 10% fetal bovine serum, respectively. Cultures were maintained at 37 °C in a 5% CO2humidified atmosphere. Exponentially growing cells (106cells/60 × 15-mm culture dish plated the day before the experiment) were used for all treatments except for mitogenic stimulation where serum-starved cells, deprived for 24 h, were used. For heat shock treatment, the dishes were sealed with Parafilm and immersed into a circulating water bath thermoregulated at 44 ± 0.05 °C for the indicated period of time. All other inducers used were added directly into culture medium, and cells were maintained at 37 °C for the duration of treatments. The plasmids pSVHa27WT (31Lambert H. Charette S.J. Bernier A.F. Guimond A. Landry J. J. Biol. Chem. 1999; 274: 9378-9385Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), βAPrhsp70 (40Mosser D.D. Duchaine J. Massie B. Mol. Cell. Biol. 1993; 13: 5427-5438Crossref PubMed Scopus (190) Google Scholar), and pcDNA3-HA-p38 (41Berra E. Municio M.M. Sanz L. Frutos S. Diaz-Meco M.T. Moscat J. Mol. Cell. Biol. 1997; 17: 4346-4354Crossref PubMed Scopus (161) Google Scholar) were used for expression of Chinese hamster HSP27, inducible human HSP70, and HA-tagged SAPK2/p38, respectively. pAM-HSP90β contains the human HSP90β cDNA (42Hickey E. Brandon S.E. Smale G. Lloyd D. Weber L.A. Mol. Cell. Biol. 1989; 9: 2615-2626Crossref PubMed Scopus (228) Google Scholar) inserted at theSalI site of the expression vector pALTER-MAX (Promega). CCL39 cells were plated 24 h before transfection at a concentration of 0.75 × 106cells/75 cm2culture flask. Transfection by calcium phosphate precipitation was done as described before using 21 μg of plasmid (7 μg of pcDNA3-HA-p38 and 14 μg of pSVHa27WT, βAPrhsp70, PAM-HSP90, or carrier DNA) per flask (11Landry J. Chrétien P. Lambert H. Hickey E. Weber L.A. J. Cell Biol. 1989; 109: 7-15Crossref PubMed Scopus (582) Google Scholar). The cells were replated 24 h later and used 48 h after transfection. After treatments, cells were scraped and extracted in lysis buffer containing 20 mm MOPS, pH 7.0, 10% glycerol, 80 mm β-glycerophosphate, 5 mm EGTA, 0.5 mm EDTA, 1 mmNa3VO4, 5 mmNa4P2O7, 50 mm NaF, 1% Triton X-100, 1 mm benzamidine, 1 mmdithiothreitol, and 1 mm phenylmethylsulfonyl fluoride. The extracts were vortexed and centrifuged at 17,000 × gfor 12 min at 4 °C. The clarified supernatants were either used immediately for immunoprecipitation or stored at −80 °C. The further steps were carried out at 4 °C. The supernatant was diluted four times in buffer I (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1 mm EDTA, 1 mm EGTA, 1 mm MgCl2, 1 mmNa3VO4, 1% Triton X-100, and 1 mmphenylmethylsulfonyl fluoride). Antibodies were added in limiting concentrations, and the mixtures were incubated for 1 h. 10–15 μl of protein A-Sepharose (Amersham Pharmacia Biotech) 50% v/v in buffer I were added, and the mixtures were incubated for 30 min. Samples were centrifuged for 15 s and washed three times with 300 μl of buffer I. Immunoprecipitates were used directly for the kinase assays. Kinase activities were assayed in immune complexes. MAPKAP kinase-2 activity was measured using 1 μg of recombinant HSP27 as substrate (30Huot J. Lambert H. Lavoie J.N. Guimond A. Houle F. Landry J. Eur. J. Biochem. 1995; 227: 416-427Crossref PubMed Scopus (173) Google Scholar). The assays were carried out in 25 μl of kinase buffer K (100 μm ATP, 3 μCi of [γ32-P]ATP, 40 mm p-nitrophenyl phosphate, 20 mm MOPS, pH 7.0, 10% glycerol, 15 mm MgCl2, 0.05% Triton X-100, 1 mmdithiothreitol, 1 mm leupeptin, 0.1 mmphenylmethylsulfonyl fluoride, and 0.3 μg of protein kinase A inhibitor). The kinase activity was assayed for 30 min at 30 °C and was stopped by the addition of 10 μl of SDS-polyacrylamide gel electrophoresis loading buffer. Immunoprecipitated SAPK2/p38 was assayed analogously using ATF2-GST as substrate (34Guay J. Lambert H. Gingras-Breton G. Lavoie J.N. Huot J. Landry J. J. Cell Sci. 1997; 110: 357-368Crossref PubMed Google Scholar). The kinase assay buffer for SAPK2/p38 contained 50 mm HEPES, pH 7.4, 50 mm β-glycerophosphate, 50 mmMgCl2, 0.2 mm Na3VO4, 2 μg ATF2-GST, and 4 μCi of [γ32-P]ATP. Kinase activities were evaluated by measuring incorporation of the radioactivity into the specific substrates after resolution by SDS-polyacrylamide gel electrophoresis and quantification using PhosphorImager (Molecular Dynamics). To ensure equal loading of the kinases on different lanes, immune complexes were analyzed by Western blotting using specific antibodies. Proteins were separated through 10% SDS/polyacrylamide gels and transferred onto nitrocellulose as described previously (33Lavoie J.N. Lambert H. Hickey E. Weber L.A. Landry J. Mol. Cell. Biol. 1995; 15: 505-516Crossref PubMed Scopus (570) Google Scholar). After reacting the membranes with the specific antibodies, proteins were detected using an ECL detection kit (Amersham Pharmacia Biotech) or by iodinated secondary antibodies and quantified using PhosphorImager analysis. Cells that had become thermotolerant after a mild heat shock are refractory to heat-induced phosphorylation of HSP27 (10Landry J. Chrétien P. Laszlo A. Lambert H. J. Cell. Physiol. 1991; 147: 93-101Crossref PubMed Scopus (125) Google Scholar). We investigated whether desensitization could be correlated with the loss of responsiveness of MAPKAP kinase-2 and SAPK2/p38, the two upstream activators of HSP27 phosphorylation. In CCL39 cells, MAPKAP kinase-2 and SAPK2/p38 are activated maximally right after a 20-min heat shock (Fig. 1, A andB ). The activities of both kinases rapidly returned to basal level within the next 3–5 h. This is in perfect agreement with the previously published kinetics of HSP27 phosphorylation, which is maximal within 20 min and is back to normal basal levels within 3–5 h after the priming treatment (10Landry J. Chrétien P. Laszlo A. Lambert H. J. Cell. Physiol. 1991; 147: 93-101Crossref PubMed Scopus (125) Google Scholar). We examined the reactivation of these two kinases by heat shock at various times after a priming heat shock treatment. Neither kinase was reactivated when the cells were restimulated at any time between 5 and 10 h postpriming although both were present at normal levels as determined by immunoblot using anti MAPKAP kinase-2 and SAPK2/p38 antibodies (Fig. 1 C). At later times (24 h), both kinases were activated normally as shown previously for HSP27 phosphorylation (10Landry J. Chrétien P. Laszlo A. Lambert H. J. Cell. Physiol. 1991; 147: 93-101Crossref PubMed Scopus (125) Google Scholar). Similar kinetics of desensitization was observed in HeLa cells (data not shown). In the case of SAPK2/p38, the results were confirmed using a phospho-specific antibody, which indicated that the failure to activate SAPK2/p38 correlated with lack of phosphorylation by the upstream kinases (data not shown). The desensitization appeared to be total and not just a mere displacement in the dose-response curve for heat shock. Whereas a 20-min heat shock induced a maximal MAPKAP kinase-2 activation in naive cells, treatment for up to 1 h did not induce MAPKAP kinase-2 activity in fully desensitized cells (Fig. 1 D). The Western blot analysis shown in Fig. 1 C indicated that heat-induced desensitization of MAPKAP kinase-2 and SAPK2/p38 activation were not caused by a down-regulated expression of MAPKAP kinase-2 or SAPK2/p38 in thermotolerant cells. To determine whether these enzymes were physically available and functional, we evaluated if the desensitization process also affected MAPKAP kinase-2 activation by other known activators of the pathway. Cells were first exposed to heat shock and then restimulated with various growth factors, cytokines, or stress agents. In control cells, thrombin and TNF-α elicited a strong activation of MAPKAP kinase-2 (Fig. 2,A and B). The ability of thrombin and TNF-α to activate MAPKAP kinase-2 was unaffected in heat-desensitized cells. Similarly, heat-desensitized cells remained fully responsive to restimulation with all other agonists tested at 5 h after heat shock, namely EGF, platelet-derived growth factor, readdition of serum to serum-deprived cells, sphingomyelinase, and phorbol ester (data not shown). The ability of physical or chemical stresses to activate the SAPK2/p38 pathway in heat-desensitized cells was also tested by looking either directly at the activity of SAPK2/p38 or at the activity of MAPKAP kinase-2. Both H2O2 and sodium arsenite induced a normal activation of the pathway at all times after the desensitizing heat shock (Fig. 2, C and D). Similarly, sorbitol induction of the pathway was about 75% of the control response at 5 h after the priming heat shock (data not shown). Hence heat shock-induced desensitization of the SAPK2/p38 pathway was not caused by a general toxic response and affected specifically the heat shock-sensitive pathway. Thus, the failure to induce phosphorylation of HSP27 in heat-induced thermotolerant cells was the result of a total inhibition of some proximal elements of the heat shock signaling pathway preventing phosphorylation and activation of SAPK2/p38 and activation of the HSP27 kinase MAPKAP kinase-2. Having shown clear homologous desensitization of the SAPK2/p38 pathway activation by heat shock, we next tested if other agonists were also capable of eliciting a desensitization to heat shock. Cells pretreated with EGF showed virtually total desensitization of MAPKAP kinase-2 activation to restimulation with EGF (Fig. 3 A). MAPKAP kinase-2 activation was not induced above the residual level remaining after the first stimulation. In contrast, heat shock induction was unaffected in EGF-desensitized cells. Similarly, thrombin treatment induced homologous desensitization of MAPKAP kinase-2 activation but did not desensitize to heat shock (Fig. 3 B). One conclusion of these results is that activation of SAPK2/p38 is not a sufficient condition for heat desensitization. The possibility that it could be required during heat-induced homologous desensitization was tested by determining the effect on heat-induced desensitization of SB203580, a pyridinil imidazol derivative that efficiently inhibits SAPK2/p38 activity (43Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1981) Google Scholar). As reported previously, SB203580 did not prevent SAPK2/p38 activation but led to a total inhibition of MAPKAP kinase-2 activation (Fig. 4) and HSP27 phosphorylation. Inhibition of SAPK2/p38 activity during the priming heat shock treatment had no effect on induction of desensitization, indicating that events occurring downstream of SAPK2/p38 were not required for inducing homologous heat desensitization.FIG. 4Heat desensitization does not require SAPK2/p38 activity. Panel A, exponentially growing CCL39 cells were pretreated (+SB) or not (−SB) for 1 h with the SAPK2/p38 inhibitor SB203580 (5 μm) and then exposed (HS) or not (Ctl) to a 20-min heat shock at 44 °C before being extracted. Panel B, cells were pretreated (+SB) or not (−SB) for 1 h with SB203580 (5 μm), exposed to a 20-min heat shock at 44 °C, and returned at 37 °C. 5 h later, the cells were exposed (HS) or not (Ctl) to a second identical heat shock before being extracted. Extracts were assayed for MAPKAP kinase-2 (black bars) or SAPK2/p38 (white bars) activities using recombinant HSP27 or ATF2-GST as substrates, respectively. Results are expressed as the ratio of the kinase activities of stimulated cells over the activity of unstimulated cells.View Large Image Figure ViewerDownload (PPT) Another possibility for explaining the heat-induced desensitization is that one of the heat shock proteins that accumulate after the first heat shock acts as a repressor of one of the elements in the heat-specific sensing pathway that triggers SAPK2/p38 activation. This appeared unlikely because the kinetics of accumulation of the HSP after heat shock does not match the kinetics of desensitization. For example, HSP27 and HSP70 concentrations peak at 10 and 14 h after heat shock, whereas desensitization is maximal at 5 h (10Landry J. Chrétien P. Laszlo A. Lambert H. J. Cell. Physiol. 1991; 147: 93-101Crossref PubMed Scopus (125) Google Scholar). Nevertheless, we tested directly the effect of overexpressing HSP70, HSP90, or HSP27 on heat activation of SAPK2/p38. HSP70, HSP90, or HSP27 was cotransfected with HA-tagged SAPK2/p38 in CCL39 cells. Under the conditions of transfection used, we calculated, after correcting for the transfection efficiency (about 20%), that individual transfected cells expressed amounts of HSP equivalent (HSP70 and HSP90) or 2-fold higher (HSP27) than what control cells express 5 h after heat shock. Epitope-tagged SAPK2/p38 was immunoprecipitated from the transfected cells at various times after shifting the temperature to 44 °C, and p38 activity was determined using ATF2-GST as substrate. As shown in Fig. 5 A overexpression of HSP90, HSP70, or HSP27 had no effect on heat activation of SAPK2/p38 compared with cells transfected with an empty vector. Furthermore, these cells were desensitized to heat shock to the same extent as control cells (Fig. 5 B). These results strongly suggested that HSP70, HSP90, or HSP27 generated during development of thermotolerance was not implicated in heat-induced homologous desensitization of SAPK2/p38 activation. This was supported further by the finding that the addition of cycloheximide during and after heat shock to inhibit HSP and total protein synthesis did not block desensitization of the SAPK2/p38 pathway (see below). Cycloheximide desensitized MAPKAP kinase-2 to activation by heat shock (Fig. 6 A). The capacity to activate MAPKAP kinase-2 with heat shock decreased progressively upon exposure to cycloheximide and was totally inhibited after 5 h, suggesting the existence of an essential element with a half-life in the order of 2–3 h in the SAPK2/p38 activation pathway. To determine whether this element was specific to heat shock, cells were exposed to cycloheximide for 5 h and then treated with H2O2, sodium arsenite, or sorbitol. In contrast to heat shock, activation of MAPKAP kinase-2 by these agents was not or only slightly affected by the cycloheximide pretreatment(Fig. 6 B ). Similar results were obtained using two other structurally and mechanistically unre