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. 2009 Jul 28;106(30):12301-5.
doi: 10.1073/pnas.0901823106. Epub 2009 Jul 9.

Mitochondrial Phosphoglycerate Mutase 5 Uses Alternate Catalytic Activity as a Protein Serine/Threonine Phosphatase to Activate ASK1

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Mitochondrial Phosphoglycerate Mutase 5 Uses Alternate Catalytic Activity as a Protein Serine/Threonine Phosphatase to Activate ASK1

Kohsuke Takeda et al. Proc Natl Acad Sci U S A. .
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Abstract

Phosphoglycerate mutase (PGAM) is an enzyme of intermediary metabolism that converts 3-phosphoglycerate to 2-phosphoglycerate in glycolysis. Here, we discovered PGAM5 that is anchored in the mitochondrial membrane lacks PGAM activity and instead associates with the MAP kinase kinase kinase ASK1 and acts as a specific protein Ser/Thr phosphatase that activates ASK1 by dephosphorylation of inhibitory sites. Mutation of an active site His-105 in PGAM5 abolished phosphatase activity with ASK1 and phospho-Thr peptides as substrates. The Drosophila and Caenorhabditis elegans orthologs of PGAM5 also exhibit specific Ser/Thr phosphatase activity and activate the corresponding Drosophila and C. elegans ASK1 kinases. PGAM5 is unrelated to the other known Ser/Thr phosphatases of the PPP, MPP, and FCP families, and our results suggest that this member of the PGAM family has crossed over from small molecules to protein substrates and been adapted to serve as a specialized activator of ASK1.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
PGAM5 associates with and activates ASK1. (A) Endogenous interaction between PGAM5 and ASK1. ASK1 was immunoprecipitated (IP) from the 10,000× g pellet (P) and supernatant (S) fractions of B16 cells, and coimmunoprecipitated PGAM5 was detected by immunoblotting (IB). (B) PGAM5 activates ASK1. HA-ASK1 and/or PGAM5 were expressed in HEK293 cells. Cell lysates were subjected to IB. P-ASK antibody recognizes phosphorylation of human ASK1 Thr-838. (C) PGAM5 activates JNK and p38 but not ERK. PGAM5 were expressed in HEK293 cells. For a positive control for ERK activation, cells were treated with 1 mM H2O2 for 15 min (indicated as H). Cell lysates were subjected to IB.
Fig. 2.
Fig. 2.
PGAM5 is a protein Ser/Thr phosphatase that activates ASK1. (A) PGAM5 lacks PGAM activity. Flag-PGAM5 (amino acids 29–289) lacking the TM domain, Flag-PGAM1, and pcDNA3 were individually expressed in HEK293 cells and were subjected to immunoprecipitation with anti-Flag antibody, followed by phosphoglycerate mutase assay in vitro. As a positive control, recombinant human PGAM1 (rPGAM1) was used instead of immunoprecipitated proteins. An aliquot of cell lysate was subjected to immunoblotting (IB). (B) PGAM5 specifically dephosphorylates phospho-Ser and -Thr peptides. Bacterially generated GST-tagged PGAM5 lacking the TM domain (GST-PGAM5) and PTP1B were subjected to in vitro phosphatase assay using phosphopeptides. Results shown are the means of triplicate determinations ± SD. (C) PGAM5 and Sts-1 exhibit strict substrate specificity. Flag-PGAM5 and Flag-Sts-1 were individually expressed in HEK293 cells and were subjected to immunoprecipitation with anti-Flag antibody, followed by in vitro phosphatase assay. (D) His-105 is essential for phosphatase activity of PGAM5. GST-PGAM5 with intact His-105 (WT), His105Ala, and His105Phe were subjected to in vitro phosphatase assay using various concentrations of phospho-Thr peptides. Results shown are the means of triplicate determinations. (E) PGAM5 immunoprecipitated from HEK293 cells exhibits His-105-dependent phosphatase activity. Flag-PGAM5-WT, -His105Ala (H/A), and -His105Phe (H/F) were individually expressed in HEK293 cells and were subjected to immunoprecipitation with anti-Flag antibody, followed by in vitro phosphatase assay. An aliquot of cell lysate was subjected to IB.
Fig. 3.
Fig. 3.
Phosphatase activity of PGAM5 is required for activation of ASK1. (A) Phosphatase activity of PGAM5 is required for ASK1 activation. HA-ASK1-Δcoil was coexpressed with Flag-PGAM5-WT, -H/A, or -H/F in HEK293 cells. Cell lysates were subjected to immunoblotting (IB). P-ASK antibody recognizes phosphorylation of human ASK1 Thr-838. (B) Phosphatase activity of PGAM5 is required for activation of JNK and p38. PGAM5-WT, -H/A, and -H/F were individually expressed in HEK293 cells. Cell lysates were subjected to IB.
Fig. 4.
Fig. 4.
PGAM5 dephosphorylates ASK1. (A) PGAM5 induces a mobility shift of the band of ASK1. Deletion mutants of ASK1 [CT, amino acids (aa) 956–1374; ΔC, amino acid 1–940; ΔN, amino acid 649–1374] with or without Flag-PGAM5 were expressed in HEK293 cells. Cell lysates were subjected to immunoblotting (IB). Schematic illustrations of ASK1 and its deletion mutants are also shown. (B) ASK1-CT possesses phosphorylation site(s) targeted by PGAM5. Cell lysates from HEK293 cells expressing HA-ASK1-CT with or without PGAM5 were left untreated or treated with λPPase and subjected to IB. (C) PGAM5 dephosphorylates ASK1-CT in vitro. Flag-ASK1-CT was expressed in HEK293 cells and immunoprecipitated with anti-Flag antibody. After incubation with GST-PGAM5-WT, -H/A, -H/F, or λPPase, the immunoprecipitates were subjected to IB.
Fig. 5.
Fig. 5.
PGAM5 phosphatases are evolutionarily conserved activators of ASK1. (A) CePGAM5 activates NSY-1 in HEK293 cells. Flag-CePGAM5 and/or T7-NSY-1 were expressed in HEK293 cells. Cell lysates were subjected to immunoblotting (IB). P-ASK antibody recognizes phosphorylation of NSY-1 Thr-815. (B) DPGAM5 activates DASK1 in a His-94-dependent fashion in S2 cells. Flag-DPGAM5 [WT or His94Ala (H/A)] and/or Flag-DASK1 were expressed in S2 cells. Cell lysates were subjected to IB. P-ASK antibody recognizes phosphorylation of DASK1 Thr-747. (C) DPGAM5 specifically dephosphorylates phospho-Thr peptides in a His-94-dependent fashion. Flag-PGAM5-WT and Flag-DPGAM5-WT and -H/A were individually expressed in HEK293 cells and were subjected to immunoprecipitation with anti-Flag antibody, followed by in vitro phosphatase assay. An aliquot of cell lysate was subjected to IB.
Fig. 6.
Fig. 6.
PGAM5 is required for basal activity of ASK1. (A) DPGAM5 is required for basal activity of DASK1. Lysates of S2 cells treated with control double-stranded RNA (dsRNA) or either of 2 dsRNAs targeting different regions of the DPGAM5 mRNA (#1 and #2) were subjected to immunoblotting (IB). P-ASK antibody recognizes phosphorylation of DASK1 Thr-747. (B) PGAM5 is required for basal activity of ASK1. Lysates of Neuro2A cells transfected with control siRNA or either of 2 siRNAs targeting different regions of the PGAM5 mRNA (#1 and #2) were subjected to IB. P-ASK antibody recognizes phosphorylation of mouse ASK1 Thr-845.

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