Mitochondrial phosphoglycerate mutase 5 uses alternate catalytic activity as a protein serine/threonine phosphatase to activate ASK1

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.MAP kinase ͉ protein phosphatase P hosphoglycerate mutase (PGAM) is an evolutionarily conserved enzyme of intermediary metabolism that converts 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG) in glycolysis and the founder member of the PGAM protein family (1). Members of this family share a common catalytic domain, designated as the PGAM domain, and function as phosphotransferases and/or phosphohydrolases with small molecule substrates. For instance, 1,3-and 1,2-bisphosphoglycerate and fructose-2,6-bisphosphate are the substrates of well-characterized members of this family, the bisphosphoglycerate mutase (BPGM) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB), respectively. The catalytic core of the PGAM domain centers on a histidine residue that acts as a phospho accepter during catalysis. These biochemical and structural features are also shared with the histidine acid phosphatase family of enzymes, and it has recently been proposed that these 2 families can be integrated into a histidine phosphatase superfamily (2). Whereas members of this superfamily predominantly target small molecule substrates, prostatic acid phosphatase has been shown to catalyze hydrolysis of high energy Phospho-Tyr bonds in substrates such as ErbB2 and EGF receptor (3, 4). In addition, Sts-1/TULA-2 in the PGAM family has been shown to function as a protein tyrosine phosphatase, dephosphorylating tyrosine kinases ZAP-70, Syk, and EGF receptor (5-7). These results open the possibility that enzymes in the PGAM family might regulate cellular functions beyond those in glucose metabolism.PGAM5 is a member of the PGAM family that has been shown to interact with Bcl-X L , an apoptosis regulator (8), and Keap1, a redox-regulated substrate adaptor for a cullindependent ubiquitin ligase complex (9). Although it has been suggested that the latter interaction is involved in the regulation of gene expression by the transcription factor Nrf2 (10), molecular functions of PGAM5 have not been elucidated. In this study, we identified PGAM5 as an interacting p...

Rhomboid Protease PARL Mediates the Mitochondrial Membrane Potential Loss-induced Cleavage of PGAM5

“…Cell Culture and Transfection-Cells were cultured as previously described (12). The transfection of expression plasmids was performed using FuGENE6 (Roche Applied Science) or PEI-Max (Polysciences).…”

“…Immunoblotting and Immunocytochemistry-Immunoblotting was performed as previously described (12). For immunocytochemistry, cultured cells were fixed with 2% formaldehyde in PBS and permeabilized with 0.2% Triton X-100.…”

“…Antibodies-The generation of the rabbit polyclonal anti-PGAM5 antibody (RTL) was described previously (12). The rat monoclonal anti-PGAM5 antibody (K1B6) was raised against purified GST-tagged PGAM5 lacking the TM domain (amino acids 30 -289) as previously described (21).…”

“…Although all the amino acid residues composing the catalytic core of the other family members are conserved in PGAM5, we have previously reported that PGAM5 lacks mutase activity and instead acts as a Ser/Thr protein phosphatase that activates the stress-activated MAP kinase pathways (12). PGAM5 has also been reported to be targeted to mitochondria through its N-terminal TM domain (13), indicating that PGAM5 is a unique mitochondrial resident phosphatase involved in cellular stress responses.…”

A Conserved Motif Mediates both Multimer Formation and Allosteric Activation of Phosphoglycerate Mutase 5

“…Although most PGAM members catalyze the dephosphorylation/phosphorylation of small metabolites, three members of the PGAM family, STS1, STS2 and PGAM5, are protein phosphatases (1)(2)(3). The tyrosine phosphatase activity of the closely related STS1 and STS2 proteins underlies their involvement in T-cell receptor signaling (2), and the serine/threonine phosphatase activity of PGAM5 has been implicated in mitochondrial fragmentation and cell death (4).…”