Inactivation of dual-specificity phosphatases is involved in the regulation of extracellular signal-regulated kinases by heat shock and hsp72

Abstract

Extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) dramatically enhance survival of cells exposed to heat shock. Using Cos-7 cells and primary human fibroblasts (IMR90 cells), we demonstrated that heat shock activates ERKs via two distinct mechanisms: stimulation of the ERK-activating kinases, MEK1/2, and inhibition of ERK dephosphorylation. Under milder heat shock conditions, activation of ERKs proceeded mainly through stimulation of MEK1/2, whereas under more severe heat shock MEK1/2 could no longer be activated and the inhibition of ERK phosphatases became critical. In Cos-7 cells, nontoxic heat shock caused rapid inactivation of the major ERK phosphatase, MKP-3, by promoting its aggregation, so that in cells exposed to 45 degrees C for 20 min, 90% of MKP-3 became insoluble. MKP-3 aggregation was reversible and, 1 h after heat shock, MKP-3 partially resolubilized. The redistribution of MKP-3 correlated with an increased rate of ERK dephosphorylation. Similar heat-induced aggregation, followed by partial resolubilization, was found with a distinct dual-specificity phosphatase MKP-1 but not with MKP-2. Therefore, MKP-3 and MKP-1 appeared to be critical heat-labile phosphatases involved in the activation of ERKs by heat shock. Expression of the major heat shock protein Hsp72 inhibited activation of MEK1/2 and prevented inactivation of MKP-3 and MKP-1. Hsp72DeltaEEVD mutant lacking a chaperone activity was unable to protect MKP-3 from heat inactivation but interfered with MEK1/2 activation similar to normal Hsp72. Hence, Hsp72 suppressed ERK activation by both protecting dual-specificity phosphatases, which was dependent on the chaperone activity, and suppressing MEK1/2, which was independent of the chaperone activity.

FIG. 1.

Duration and extent of ERK activation in cells subjected to various stresses. (A and C) Duration of ERK signal in cells subjected to various HS conditions. Cos-7 cells (A) or IMR90 cells (C) were exposed to the indicated HS conditions or left untreated (Cont.). Cells were harvested at the indicated time points after HS, and ERK activation was assayed by immunoblotting of cell lysates with anti-phospho-ERK antibody or tubulin to control for equal loading. (B and D) Activation of ERKs in cells subjected to various stresses. Cos-7 cells (B) or IMR90 cells (D) were subjected to HS (Cos-7 cells, 43°C for 30 min; IMR90 cells, 45°C for 30 min) or treated with 1 mM menadione for 30 min or 1 μM PDB for 7 min. ERK activation was assayed as in panels A and C.

FIG. 2.

MEK1/2 activation in cells subjected to various stresses. (A and C) Activation of MEK1/2 in cells subjected to various HS conditions. Cos-7 cells (A) or IMR90 cells (C) were exposed to the indicated HS conditions or left untreated (lanes C). Cells were harvested, and MEK1/2 activation was assayed by immunoblotting of cell lysates with anti-phospho-MEK1/2 antibody. The same membranes were reblotted with anti-tubulin antibody to control for equal loading. (B and D) Activation of MEK1/2 in cells subjected to various stresses. Cos-7 cells (B) or IMR90 cells (D) were subjected to HS (Cos-7 cells, 43°C for 30 min; IMR90 cells, 45°C for 30 min) or treated with 1 mM menadione for 30 min or 1 μM PDB for 7 min. MEK1/2 activation was assayed as in panels A and C.

FIG. 3.

Dephosphorylation of ERKs and MEK in cell subjected to various stresses. (A) Dephosphorylation of ERKs in unstimulated cells and in cells heated at 43°C for 15 min. Cos-7 cells were either left untreated (Backg) or heated at 43°C for 15 min. Further phosphorylation was prevented, as described in Materials and Methods. The extent of ERK phosphorylation was assayed at the indicated time points after ATP depletion as in Fig. 1A and C. The same membrane was reblotted with antitubulin antibody to control for equal loading. (B and C). Quantification of the rates of ERK dephosphorylation in cells subjected to different stresses. Cos-7 cells (B) and IMR90 cells (C) were either left untreated (Background), exposed to indicated HS conditions, or treated with 1 mM menadione for 30 min or 1 μM PDB for 7 min. ERK dephosphorylation was assayed as in panel A. Each curve represents the average of at least three experiments. (D) Quantification of the rates of phMEK1/2 dephosphorylation in IMR90 cells subjected to different stresses. IMR90 cells were exposed to HS at 45°C for 15 min or treated with 1 mM menadione for 30 min or 1 μM PDB for 7 min. The extent of MEK1/2 phosphorylation was assayed at the indicated time points after ATP depletion. Each curve represents the average of at least three experiments.

FIG. 4.

MKP-3 shifts from soluble to insoluble fraction upon HS of Cos-7 cells. (A) Effect of MKP-3(C/S) on dephosphorylation of ERK in control and UV-irradiated cells. Cos-7 cells were transfected with the plasmid expressing MKP-3(C/S) or vector alone. Transfected cells were either left untreated (Cont.) or UV irradiated at 400 J/m² and left for 15 min. The extent of ERK phosphorylation was assayed at the indicated time points after ATP depletion as in Fig. 1A and C. The same membrane was reblotted with anti-MKP-3 antibody to control for MKP-3(C/S) expression or with anti-tubulin antibody to control for equal loading. (B) Redistribution of MKP-3 from soluble to insoluble fraction in cells heated at 43°C for 15 min. Cos-7 cells were transfected with either the plasmid expressing myc-tagged MKP-3 or empty vector (Mock). At 48 h after transfection, untreated cells or cells heated at 43°C for 15 min were harvested and lysed. Lysates were fractionated into soluble and insoluble fractions as described in Materials and Methods and then assayed for the presence of MKP-3 by immunoblotting with anti-myc antibody. The same samples were also immunoblotted with anti-ERK1, anti-PP5, and anti-MEK1/2 antibodies. S, supernatant; P, pellet. (C and D) Redistribution of MKP-3 from soluble to insoluble fractions in cells exposed to various stresses. Cos-7 cells transfected same as for panel B were subjected to the indicated HS conditions or were treated with 1 mM menadione for 30 min. Either right after the treatments or 1 h later the cells were harvested, and cell lysates were assayed as for panel B. Each bar on the graph in panel C represents average of at least three experiments. In panel D, the data represent a typical experiment. Sup., supernatant; Pel., pellet. (E) Redistribution of MKP-1 and MKP-2 from soluble to insoluble fractions in IMR90 cells heated at 45°C for 15 min. IMR90 cells were subjected to the indicated HS. Lysates were fractionated into soluble and insoluble fractions as described in Materials and Methods and assayed for the presence of MKP-1 and MKP-2 by immunoblotting with the corresponding antibody. S, supernatant; P, pellet.

FIG. 5.

Effect of MKP-3 resolubilization and de novo synthesis of MKP-1 on the rate of ERK dephosphorylation during recovery from HS. (A and B) Dephosphorylation of ERKs in cells heated at 43°C for 15 min (A) or 30 min (B). Cos-7 cells were heated at 43°C for 15 min. The rate of ERK dephosphorylation (measured as described for Fig. 3A) was assay either immediately after HS (□) or after 1 h of recovery at 37°C with (○) or without (⋄) the protein synthesis inhibitor emetine. Each curve represents the average of at least three experiments. (C) Induction of MKP-1 in cells heated at 43°C for 15 min. Cos-7 cells were heated at 43°C for 15 min, and the cells were either immediately harvested or incubated with or without emetine. The induction of MKP-1 was assayed in cell lysates by immunoblotting with anti-MKP-1 antibody.

FIG. 6.

Activation of ERKs and MEK1/2 by various stresses in cells expressing MKP-3. (A) Activation of ERKs by various stresses in cells transfected (tranf.) with either MKP-3-expressing plasmid or vector alone (Mock tranf.). Cos-7 cells transfected as described for Fig. 4A were treated with menadione (Men; 1 mM, 30 min) or PDB (1 μM, 7 min), UV irradiated (400 J/m²), or subjected to HS at 43°C for 30 min. Immediately after these treatments, cells were harvested and ERK activation was assayed. (B) Activation of MEK1/2 by various stresses in cells transfected with either MKP-3-expressing plasmid or vector alone. This experiment was carried out as described for panel A, but the samples were reblotted with anti-phospho-MEK1/2 antibody. The band marked with an asterisk is a cross-reacting protein that provides an internal control for equal loading. Lanes C, untreated controls.

FIG. 7.

Hsp72 and Hsp72ΔEEVD suppress HS mediated activation of ERKs and MEK1/2. (A and B) Quantification of the rates of ERK dephosphorylation in cells overexpressing the normal or mutant form of Hsp72 or preheated at 45°C for 30 min. Cos-7 cells (A) or IMR90 cells (B) were infected with adenovirus expressing either GFP (□), Hsp72 (⋄), or Hsp72ΔEEVD (○). At 36 h postinfection, cells were heated for 30 min at 43°C (A) or 45°C (B), and the rate of ERK dephosphorylation was assayed. The percentage of ERK activity was normalized to GFP-expressing cells. Each curve represents the average of at least three experiments. Alternatively, in panel B IMR90 cells were subjected to HS at 45°C for 30 min, recovered at 37°C for 18 h, and subjected to a second HS at 45°C for 30 min (preheated). (C and D) Hsp72 and Hsp72ΔEEVD suppress background and HS-induced ERKs and MEK1/2 activity. Cos-7 cells (C) and IMR90 cells (D) were infected as described for panels A and B and either left untreated (Cont.) or heated at 43°C (A) or 45°C (B). The extent of ERK and MEK1/2 activation was assayed by immunoblotting cell lysates with anti-phospho-ERK or anti-phosph-MEK1/2 antibody. (E) Expression of Hsp72 or Hsp72ΔEEVD in IMR90 cells infected with the corresponding adenovirus. IMR90 cells were infected with GFP, Hsp72, or Hsp72ΔEEVD as described above. Cell lysates were immunoblotted with SPA820 antibody, which recognizes both normal and mutant Hsp72 proteins.

FIG. 8.

Hsp72 but not Hsp72ΔEEVD prevents aggregation of MKP-3 in cells exposed to HS. (A and B) Cos-7 cells were transfected with the plasmid expressing myc-tagged MKP-3, and 12 h later infected with adenovirus expressing GFP, Hsp72, or Hsp72ΔEEVD. At 36 h postinfection, the cells were heated at 43°C for 30 (A) or 15 min (B), and MKP-3 redistribution from soluble to insoluble fraction was assayed as described for Fig. 4A. Each bar on the graph represents the average of at least three experiments. (C) IMR90 cells were infected with adenovirus expressing GFP, Hsp72, or Hsp72ΔEEVD. At 36 h postinfection, cells were heated at 45° for 15 min, and MKP-1 redistribution from soluble to insoluble fraction was assayed as described for Fig. 4A. Alternatively, IMR90 cells were subjected to HS at 45°C for 30 min, recovered at 37°C for 18 h, and subjected to a second HS at 45°C for 15 min (preheated). Each bar on the graph represents the average of at least three experiments. Bars: , supernatant; ▪, pellet.

FIG. 8.

Hsp72 but not Hsp72ΔEEVD prevents aggregation of MKP-3 in cells exposed to HS. (A and B) Cos-7 cells were transfected with the plasmid expressing myc-tagged MKP-3, and 12 h later infected with adenovirus expressing GFP, Hsp72, or Hsp72ΔEEVD. At 36 h postinfection, the cells were heated at 43°C for 30 (A) or 15 min (B), and MKP-3 redistribution from soluble to insoluble fraction was assayed as described for Fig. 4A. Each bar on the graph represents the average of at least three experiments. (C) IMR90 cells were infected with adenovirus expressing GFP, Hsp72, or Hsp72ΔEEVD. At 36 h postinfection, cells were heated at 45° for 15 min, and MKP-1 redistribution from soluble to insoluble fraction was assayed as described for Fig. 4A. Alternatively, IMR90 cells were subjected to HS at 45°C for 30 min, recovered at 37°C for 18 h, and subjected to a second HS at 45°C for 15 min (preheated). Each bar on the graph represents the average of at least three experiments. Bars: , supernatant; ▪, pellet.

FIG. 9.

Effects of HS and Hsp72 on activation of ERKs.