Heat-shock-induced activation of stress MAP kinase is regulated by threonine- and tyrosine-specific phosphatases

Abstract

In eukaryotic species from yeast to human, stress-activated protein kinases (SAPKs), members of a MAP kinase (MAPK) subfamily, regulate the transcriptional response to various environmental stress. It is poorly understood how diverse forms of stress are sensed and transmitted to SAPKs. Here, we report the heat shock regulation of the fission yeast Spc1 SAPK, a homolog of human p38 and budding yeast Hog1p. Although osmostress and oxidative stress induce strong activation of the Wis1 MAPK kinase (MEK), which activates Spc1 through Thr-171/Tyr-173 phosphorylation, activation of Wis1 upon heat shock is relatively weak and transient. However, in heat-shocked cells, Pyp1, the major tyrosine phosphatase that dephosphorylates and inactivates Spc1, is inhibited for its interaction with Spc1, which leads to strong activation of Spc1. Subsequently, Spc1 activity is rapidly attenuated by Thr-171 dephosphorylation, whereas Tyr-173 remains phosphorylated. Thr-171 dephosphorylation is compromised in a strain lacking functional type 2C serine/threonine phosphatases (PP2C), Ptc1 and Ptc3. Moreover, Ptc1 and Ptc3 can dephosphorylate Thr-171 of Spc1 both in vivo and in vitro. These observations strongly suggest that PP2C enzymes play an important role in the attenuation of Spc1 activity in heat-shocked cells. Thus, transient activation of Spc1 upon heat shock is ensured by differential regulation of threonine and tyrosine phosphorylation.

Figure 1

Activity of the wild-type and constitutively active mutant of the Wis1 MEK under stress conditions. Strains carrying the wis1:myc (KS1878) or wis1DD:myc (KS2125) alleles were grown to mid-log phase at 30°C in YES medium and exposed to various forms of stress. Wis1myc and Wis1DDmyc, which have 12 copies of the myc tag at the carboxy-terminus, were purified by anti-myc immunoprecipitation, and their catalytic activity was measured with GST–Spc1 as substrate. Phosphorylation of GST–Spc1 was detected by immunoblotting with anti-phosphotyrosine (α-pTyr) antibodies. Anti-myc (α-myc) immunoblotting shows the amount of immunoprecipitated Wis1myc and Wis1DDmyc in the assay. (A) Activity of Wis1myc and Wis1DDmyc purified from unstressed cells (U) and cells exposed to osmotic (0.6 m KCl for 10 min; Os) or oxidative (0.3 mm H₂O₂ for 10 min; Ox) stress. (B) Activity of Wis1myc and Wis1DDmyc after the culture temperature was shifted from 30°C to 48°C at time 0. No significant change in Wis1DD activity was detected after the stress treatments in repeated experiments.

Figure 2

Dephosphorylation of Spc1 Tyr-173 is inhibited in heat-shocked cells. (A) Wild-type strain (KS2096) carrying chromosomal spc1⁺ tagged with the HA6H sequence was grown to mid-log phase in YES medium at 30°C. The culture was shifted to 48°C for 10 min (heat shock, HS) to induce Spc1 tyrosine phosphorylation and then shifted back to 30°C (Recovery). Aliquots of cells were harvested at the indicated time points, and Spc1 was purified by Ni–NTA chromatography. The level of Spc1 tyrosine phosphorylation was examined by immunoblotting with anti-phosphotyrosine antibodies. No tyrosine dephosphorylation of Spc1 was detected >2 hr after the temperature shift down. (B) Overexpression of the Pyp1 tyrosine phosphatase cannot suppress Spc1 activation induced by heat shock. Wild-type strain (KS1376) carrying chromosomal spc1⁺ tagged with the HA6H sequence was transformed with pREP1–pyp1HA6H, which expresses Pyp1 with a carboxy-terminal HA6H tag under the control of the thiamine-repressible nmt1 promoter. The transformant was grown in EMM2 medium at 30°C in the presence (+B₁) and absence (−B₁) of thiamine (vitamin B₁) for 18 hr and exposed to osmostress by 0.6 m KCl for 10 min (Os) or heat shock at 48°C for 5, 10, and 20 min. Spc1 and Pyp1 were purified by Ni–NTA chromatography under denaturing conditions and analyzed by immunoblotting with anti-phosphotyrosine and anti-HA antibodies. In the absence of thiamine, overexpressed Pyp1 completely suppressed Spc1 tyrosine phosphorylation upon osmostress but showed little activity in heat-shocked cells. (C) The Pyp1 tyrosine phosphatase rapidly loses its ability to interact with Spc1 after heat shock. Strain CA187 has chromosomal pyp1⁺ replaced with pyp1–C470S:myc to express the catalytically inactive Pyp1^C470S with a carboxy-terminal myc epitope tag. This strain was transformed with pREP1-GST–Spc1 and grown at 30°C in EMM2 medium without thiamine to induce expression of GST–Spc1 from the thiamine-repressible nmt1 promoter. Cells were exposed to heat shock at 48°C (left) or osmostress by 0.6 m KCl (right), and aliquots of cells were harvested at the indicated time points. Cell lysates were absorbed to GSH–Sepharose beads, and, after extensive washes, proteins bound to the beads (GSH-Beads) were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The amount of Pyp1^C470S detected in the crude lysates (Lysate) did not change significantly after the stress treatments.

Figure 2

Dephosphorylation of Spc1 Tyr-173 is inhibited in heat-shocked cells. (A) Wild-type strain (KS2096) carrying chromosomal spc1⁺ tagged with the HA6H sequence was grown to mid-log phase in YES medium at 30°C. The culture was shifted to 48°C for 10 min (heat shock, HS) to induce Spc1 tyrosine phosphorylation and then shifted back to 30°C (Recovery). Aliquots of cells were harvested at the indicated time points, and Spc1 was purified by Ni–NTA chromatography. The level of Spc1 tyrosine phosphorylation was examined by immunoblotting with anti-phosphotyrosine antibodies. No tyrosine dephosphorylation of Spc1 was detected >2 hr after the temperature shift down. (B) Overexpression of the Pyp1 tyrosine phosphatase cannot suppress Spc1 activation induced by heat shock. Wild-type strain (KS1376) carrying chromosomal spc1⁺ tagged with the HA6H sequence was transformed with pREP1–pyp1HA6H, which expresses Pyp1 with a carboxy-terminal HA6H tag under the control of the thiamine-repressible nmt1 promoter. The transformant was grown in EMM2 medium at 30°C in the presence (+B₁) and absence (−B₁) of thiamine (vitamin B₁) for 18 hr and exposed to osmostress by 0.6 m KCl for 10 min (Os) or heat shock at 48°C for 5, 10, and 20 min. Spc1 and Pyp1 were purified by Ni–NTA chromatography under denaturing conditions and analyzed by immunoblotting with anti-phosphotyrosine and anti-HA antibodies. In the absence of thiamine, overexpressed Pyp1 completely suppressed Spc1 tyrosine phosphorylation upon osmostress but showed little activity in heat-shocked cells. (C) The Pyp1 tyrosine phosphatase rapidly loses its ability to interact with Spc1 after heat shock. Strain CA187 has chromosomal pyp1⁺ replaced with pyp1–C470S:myc to express the catalytically inactive Pyp1^C470S with a carboxy-terminal myc epitope tag. This strain was transformed with pREP1-GST–Spc1 and grown at 30°C in EMM2 medium without thiamine to induce expression of GST–Spc1 from the thiamine-repressible nmt1 promoter. Cells were exposed to heat shock at 48°C (left) or osmostress by 0.6 m KCl (right), and aliquots of cells were harvested at the indicated time points. Cell lysates were absorbed to GSH–Sepharose beads, and, after extensive washes, proteins bound to the beads (GSH-Beads) were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The amount of Pyp1^C470S detected in the crude lysates (Lysate) did not change significantly after the stress treatments.

Figure 2

Dephosphorylation of Spc1 Tyr-173 is inhibited in heat-shocked cells. (A) Wild-type strain (KS2096) carrying chromosomal spc1⁺ tagged with the HA6H sequence was grown to mid-log phase in YES medium at 30°C. The culture was shifted to 48°C for 10 min (heat shock, HS) to induce Spc1 tyrosine phosphorylation and then shifted back to 30°C (Recovery). Aliquots of cells were harvested at the indicated time points, and Spc1 was purified by Ni–NTA chromatography. The level of Spc1 tyrosine phosphorylation was examined by immunoblotting with anti-phosphotyrosine antibodies. No tyrosine dephosphorylation of Spc1 was detected >2 hr after the temperature shift down. (B) Overexpression of the Pyp1 tyrosine phosphatase cannot suppress Spc1 activation induced by heat shock. Wild-type strain (KS1376) carrying chromosomal spc1⁺ tagged with the HA6H sequence was transformed with pREP1–pyp1HA6H, which expresses Pyp1 with a carboxy-terminal HA6H tag under the control of the thiamine-repressible nmt1 promoter. The transformant was grown in EMM2 medium at 30°C in the presence (+B₁) and absence (−B₁) of thiamine (vitamin B₁) for 18 hr and exposed to osmostress by 0.6 m KCl for 10 min (Os) or heat shock at 48°C for 5, 10, and 20 min. Spc1 and Pyp1 were purified by Ni–NTA chromatography under denaturing conditions and analyzed by immunoblotting with anti-phosphotyrosine and anti-HA antibodies. In the absence of thiamine, overexpressed Pyp1 completely suppressed Spc1 tyrosine phosphorylation upon osmostress but showed little activity in heat-shocked cells. (C) The Pyp1 tyrosine phosphatase rapidly loses its ability to interact with Spc1 after heat shock. Strain CA187 has chromosomal pyp1⁺ replaced with pyp1–C470S:myc to express the catalytically inactive Pyp1^C470S with a carboxy-terminal myc epitope tag. This strain was transformed with pREP1-GST–Spc1 and grown at 30°C in EMM2 medium without thiamine to induce expression of GST–Spc1 from the thiamine-repressible nmt1 promoter. Cells were exposed to heat shock at 48°C (left) or osmostress by 0.6 m KCl (right), and aliquots of cells were harvested at the indicated time points. Cell lysates were absorbed to GSH–Sepharose beads, and, after extensive washes, proteins bound to the beads (GSH-Beads) were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The amount of Pyp1^C470S detected in the crude lysates (Lysate) did not change significantly after the stress treatments.

Figure 3

The dual-phosphorylated, active form of Spc1 accumulates only transiently in response to heat shock. (A) Transient activation of Spc1 after heat shock. Strain GD1942, in which chromosomal spc1⁺ is tagged with the sequence encoding the myc epitope, was grown to mid-log phase in YES medium at 30°C and then shifted to 48°C at time 0. Aliquots of cells were harvested at the indicated time points, and Spc1 was purified by anti-myc immunoprecipitation. Activity of Spc1 was assayed by incubation with the substrate GST–Atf1 in the presence of Mg²⁺ and [γ-³²P]ATP, and was analyzed by SDS-PAGE and autoradiography (top). Anti-myc immunoblotting shows the amount of immunoprecipitated Spc1 in the assays (bottom). (B) Antibodies against dual-phosphorylated mammalian p38 cross react with Spc1 phosphorylated on both Thr-171 and Tyr-173. Strains expressing the myc-tagged wild-type Spc1 (GD1942) or Spc1AY (CA140), which has Thr-171 substituted with unphosphorylatable alanine, were grown in YES medium at 30°C and then exposed to osmostress by 0.6 m KCl. The wild-type and mutant Spc1 in the cell lysates was probed with anti-phospho-p38 (top) and anti-myc (bottom) antibodies. (C) Spc1 phosphorylated on both Thr-171 and Tyr-173 accumulates only transiently after heat shock in wild-type and wis1DD cells. KS2096 (wild type) and KS2086 (wis1DD) strains carrying the spc1:HA6H allele were grown to mid-log phase at 30°C in YES medium and shifted to 48°C at time 0. Aliquots of cells were harvested at the indicated time points, and Spc1 was purified by Ni–NTA chromatography, which was followed by immunoblotting with anti-phospho-p38, anti-phosphotyrosine, and anti-HA antibodies.

Figure 3

The dual-phosphorylated, active form of Spc1 accumulates only transiently in response to heat shock. (A) Transient activation of Spc1 after heat shock. Strain GD1942, in which chromosomal spc1⁺ is tagged with the sequence encoding the myc epitope, was grown to mid-log phase in YES medium at 30°C and then shifted to 48°C at time 0. Aliquots of cells were harvested at the indicated time points, and Spc1 was purified by anti-myc immunoprecipitation. Activity of Spc1 was assayed by incubation with the substrate GST–Atf1 in the presence of Mg²⁺ and [γ-³²P]ATP, and was analyzed by SDS-PAGE and autoradiography (top). Anti-myc immunoblotting shows the amount of immunoprecipitated Spc1 in the assays (bottom). (B) Antibodies against dual-phosphorylated mammalian p38 cross react with Spc1 phosphorylated on both Thr-171 and Tyr-173. Strains expressing the myc-tagged wild-type Spc1 (GD1942) or Spc1AY (CA140), which has Thr-171 substituted with unphosphorylatable alanine, were grown in YES medium at 30°C and then exposed to osmostress by 0.6 m KCl. The wild-type and mutant Spc1 in the cell lysates was probed with anti-phospho-p38 (top) and anti-myc (bottom) antibodies. (C) Spc1 phosphorylated on both Thr-171 and Tyr-173 accumulates only transiently after heat shock in wild-type and wis1DD cells. KS2096 (wild type) and KS2086 (wis1DD) strains carrying the spc1:HA6H allele were grown to mid-log phase at 30°C in YES medium and shifted to 48°C at time 0. Aliquots of cells were harvested at the indicated time points, and Spc1 was purified by Ni–NTA chromatography, which was followed by immunoblotting with anti-phospho-p38, anti-phosphotyrosine, and anti-HA antibodies.

Figure 3

The dual-phosphorylated, active form of Spc1 accumulates only transiently in response to heat shock. (A) Transient activation of Spc1 after heat shock. Strain GD1942, in which chromosomal spc1⁺ is tagged with the sequence encoding the myc epitope, was grown to mid-log phase in YES medium at 30°C and then shifted to 48°C at time 0. Aliquots of cells were harvested at the indicated time points, and Spc1 was purified by anti-myc immunoprecipitation. Activity of Spc1 was assayed by incubation with the substrate GST–Atf1 in the presence of Mg²⁺ and [γ-³²P]ATP, and was analyzed by SDS-PAGE and autoradiography (top). Anti-myc immunoblotting shows the amount of immunoprecipitated Spc1 in the assays (bottom). (B) Antibodies against dual-phosphorylated mammalian p38 cross react with Spc1 phosphorylated on both Thr-171 and Tyr-173. Strains expressing the myc-tagged wild-type Spc1 (GD1942) or Spc1AY (CA140), which has Thr-171 substituted with unphosphorylatable alanine, were grown in YES medium at 30°C and then exposed to osmostress by 0.6 m KCl. The wild-type and mutant Spc1 in the cell lysates was probed with anti-phospho-p38 (top) and anti-myc (bottom) antibodies. (C) Spc1 phosphorylated on both Thr-171 and Tyr-173 accumulates only transiently after heat shock in wild-type and wis1DD cells. KS2096 (wild type) and KS2086 (wis1DD) strains carrying the spc1:HA6H allele were grown to mid-log phase at 30°C in YES medium and shifted to 48°C at time 0. Aliquots of cells were harvested at the indicated time points, and Spc1 was purified by Ni–NTA chromatography, which was followed by immunoblotting with anti-phospho-p38, anti-phosphotyrosine, and anti-HA antibodies.

Figure 4

Ptc1 and Ptc3 phosphatases regulate phosphorylation of Spc1 Thr-171. (A) Wild-type (KS1376) and Δptc1Δptc3 mutant (CA135) strains carrying the spc1:HA6H allele were grown at 30°C to mid-log phase and shifted to 48°C at time 0. Cells were harvested at the indicated time points and Spc1 was purified on Ni–NTA beads for immunoblotting with anti-phospho-p38, anti-phosphotyrosine, and anti-HA antibodies. After the initial Spc1 activation, dual-phosphorylated Spc1 decreased more slowly in Δptc1 Δptc3 cells than in wild-type cells, a result that was reproduced in repeated experiments. (B) Wild-type spc1:HA6H strain (KS1376) was transformed with the pREP vector and the pREP-ptc3⁺ plasmid to express ptc3⁺ under the regulation of the thiamine-repressible nmt1 promoter. The transformants were grown at 30°C for 18 hr in EMM2 medium without thiamine to induce expression from the nmt1 promoter before the temperature shift to 48°C at time 0. Cells harvested at the indicated time points were subjected to analyses as described in A.

Figure 4

Ptc1 and Ptc3 phosphatases regulate phosphorylation of Spc1 Thr-171. (A) Wild-type (KS1376) and Δptc1Δptc3 mutant (CA135) strains carrying the spc1:HA6H allele were grown at 30°C to mid-log phase and shifted to 48°C at time 0. Cells were harvested at the indicated time points and Spc1 was purified on Ni–NTA beads for immunoblotting with anti-phospho-p38, anti-phosphotyrosine, and anti-HA antibodies. After the initial Spc1 activation, dual-phosphorylated Spc1 decreased more slowly in Δptc1 Δptc3 cells than in wild-type cells, a result that was reproduced in repeated experiments. (B) Wild-type spc1:HA6H strain (KS1376) was transformed with the pREP vector and the pREP-ptc3⁺ plasmid to express ptc3⁺ under the regulation of the thiamine-repressible nmt1 promoter. The transformants were grown at 30°C for 18 hr in EMM2 medium without thiamine to induce expression from the nmt1 promoter before the temperature shift to 48°C at time 0. Cells harvested at the indicated time points were subjected to analyses as described in A.

Figure 5

(A) In vitro dephosphorylation of Spc1 Thr-171 by Ptc1. GST–Ptc1 was expressed in E. coli DH5α and purified by GSH–Sepharose chromatography. Wild-type (PR109) S. pombe strain carrying the pREP1-GST–Spc1 plasmid was grown in EMM2 medium without thiamine to induce expression of GST–Spc1. After a 10-min osmostress by 0.6 m KCl, cells were harvested, and phosphorylated GST–Spc1 was purified on GSH–Sepharose beads. GST–Ptc1 and GST–Spc1 were incubated in the presence (+) or absence (−) of 20 mm MgCl₂, and the phosphorylation state of GST–Spc1 was examined by immunoblotting with anti-phospho-p38 and anti-phosphotyrosine antibodies. (B) Physical interaction between Spc1 and the Ptc1 phosphatase. Strain GD1942 expressing the myc-tagged Spc1 was grown to mid-log phase in YES medium and harvested before and after a 5-min heat shock at 48°C. Cell lysates were incubated with bacterially produced GST and GST–Ptc1, which were immobilized on GSH–Sepharose beads. After extensive washes, proteins bound to the beads (top and middle) were detected by immunoblotting with anti-GST and anti-myc antibodies. (bottom) Anti-myc immunoblotting of the total lysates.

Figure 5

(A) In vitro dephosphorylation of Spc1 Thr-171 by Ptc1. GST–Ptc1 was expressed in E. coli DH5α and purified by GSH–Sepharose chromatography. Wild-type (PR109) S. pombe strain carrying the pREP1-GST–Spc1 plasmid was grown in EMM2 medium without thiamine to induce expression of GST–Spc1. After a 10-min osmostress by 0.6 m KCl, cells were harvested, and phosphorylated GST–Spc1 was purified on GSH–Sepharose beads. GST–Ptc1 and GST–Spc1 were incubated in the presence (+) or absence (−) of 20 mm MgCl₂, and the phosphorylation state of GST–Spc1 was examined by immunoblotting with anti-phospho-p38 and anti-phosphotyrosine antibodies. (B) Physical interaction between Spc1 and the Ptc1 phosphatase. Strain GD1942 expressing the myc-tagged Spc1 was grown to mid-log phase in YES medium and harvested before and after a 5-min heat shock at 48°C. Cell lysates were incubated with bacterially produced GST and GST–Ptc1, which were immobilized on GSH–Sepharose beads. After extensive washes, proteins bound to the beads (top and middle) were detected by immunoblotting with anti-GST and anti-myc antibodies. (bottom) Anti-myc immunoblotting of the total lysates.

Figure 6

Regulation of the Spc1 SAPK by PP2C and the Pyp tyrosine phosphatases. Spc1 is activated by various forms of stress including high osmolarity stress, oxidative stress, and heat shock. The Wis1 MEK activates Spc1 by phosphorylating Thr-171 and Tyr-173. Osmostress and oxidative stress activate Wis1 through phosphorylation of Ser-469/Thr-473, which is carried out by MEKKs, Wis4 and Win1. Heat shock brings about weak activation of Wis1 in a MEKK-dependent manner as well as inhibition of Pyp1 and presumably Pyp2, the Spc1 Tyr-173 phosphatases, which results in strong activation of Spc1. Although Tyr-173 remains phosphorylated in heat-shocked cells because of inhibition of the Pyp phosphatases, Spc1 activity is attenuated by Thr-171 dephosphorylation, which is carried out by Ptc1, Ptc3, and other threonine phosphatases. Transcription of pyp2⁺ and ptc1⁺ is induced by the Spc1–Atf1 pathway in response to stress stimuli, which constitutes dual loops of negative feedback. Expression of pyp1⁺ and ptc3⁺ is constitutive; however, they might be subjected to post-transcriptional regulation.