Metal‐dependent protein phosphatase 1A functions as an extracellular signal‐regulated kinase phosphatase

Li, Rong; Gong, Zheng; Pan, Chang; Xie, Di-Dong; Tang, Junyi; Cui, Min; Xu, Yaping; Yao, Wei; Pang, Qi; Xu, Zhigang; Li, Minyong; Yu, Xiao; Sun, Junying

doi:10.1111/febs.12275

Cited by 28 publications

(21 citation statements)

References 32 publications

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“…Previous studies have demonstrated that PP2Cα is a potent negative regulator of nerve growth factor (NGF)- or epidermal growth factor (EGF)-induced extracellular regulated protein kinases (ERK) phosphorylation192223 and osmotic stress-induced c-Jun N-terminal kinases (JNK) phosphorylation24. ERK and JNK are members of mitogen-activated protein kinases, which are key regulators of multiple signaling pathways.…”

Section: Resultsmentioning

confidence: 99%

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The catalytic role of the M2 metal ion in PP2Cα

Pan

Tang

et al. 2015

Sci Rep

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PP2C family phosphatases (the type 2C family of protein phosphatases; or metal-dependent phosphatase, PPM) constitute an important class of signaling enzymes that regulate many fundamental life activities. All PP2C family members have a conserved binuclear metal ion active center that is essential for their catalysis. However, the catalytic role of each metal ion during catalysis remains elusive. In this study, we discovered that mutations in the structurally buried D38 residue of PP2Cα (PPM1A) redefined the water-mediated hydrogen network in the active site and selectively disrupted M2 metal ion binding. Using the D38A and D38K mutations of PP2Cα as specific tools in combination with enzymology analysis, our results demonstrated that the M2 metal ion determines the rate-limiting step of substrate hydrolysis, participates in dianion substrate binding and stabilizes the leaving group after P-O bond cleavage. The newly characterized catalytic role of the M2 metal ion in this family not only provides insight into how the binuclear metal centers of the PP2C phosphatases are organized for efficient catalysis but also helps increase our understanding of the function and substrate specificity of PP2C family members.

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Section: Resultsmentioning

confidence: 99%

“…The expression and purification of PP2Cα with an N-terminal His tag in addition to its mutants have been described previously1922. In brief, BL21 E. coli cells were transformed with PP2Cα plasmids and cultured at 37°C.…”

Section: Methodsmentioning

confidence: 99%

The catalytic role of the M2 metal ion in PP2Cα

Pan

Tang

et al. 2015

Sci Rep

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“…PPM1A has diverse substrates and generally functions as a negative regulator of stress response pathways. PPM1A directly dephosphorylates multiple members of the mitogen-activated protein kinase (MAPK) signaling pathway, including ERK, JNK and p38 MAPK (13,14). PPM1A is a tumor suppressor due to this negative regulation of MAPK signaling pathways, with consequent activation of p53, as well as its specific dephosphorylation of cyclin dependent kinases (14-16).…”

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confidence: 99%

A trapped human PPM1A–phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity

Debnath

Košek²,

Tagad

et al. 2018

Journal of Biological Chemistry

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Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg 2+ or Mn 2+ ions for activity in vitro. The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn 2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional, third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1A cat , complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1A cat D146E-c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1A cat toward a phosphopeptide substrate supported a randomorder, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.Reversible protein phosphorylation signaling pathways are shaped by opposing actions of protein kinases and phosphatases. These pathways regulate the response of cells and organisms to changing environmental and physiological circumstances; dysregulation of these pathways underlies many human diseases. Although phosphorylated protein residues are dominated by phosphoserine (pSer) Trapped PPM1A-phosphopeptide complex 2 of phosphatases involved in reversing the pSer/pThr and pTyr modifications are more evenly divided (1-3). Analysis of global dynamics of pSer/pThr and pTyr-based signaling suggests distinct patterns of regulation (4). The phosphoprotein phosphatases (PPP), with 13 members, and the metal-dependent phosphatases (PPM), with 18 members, provide most of the serine/threonine protein phosphatase activity in human cells (5,6).The activity, substrate specificity, and subcellular localization of PPP phosphatases are regulated primarily by binding of regulatory or inhibitor subunits, of which over 100 have been identified in human cells (7,8). PPM phosphatases generally are monomeric, but regulation of their activity remains incompletely understood (9-11). The evolutionarily distinct PPP and PPM phosphatases both feature tightly-bound bi-metal clusters in their active sites, but only the PPM phosphatases req...

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“…3,4 The PPM are characterized by their distinctive fold, the preponderance of highly conserved aspartate residues in the active site, and a requirement for supplementation with millimolar levels of Mg 2+ or Mn 2+ to support detectable activity with purified proteins. 5,6 Human PP2C α (PPM1A) exerts tumor suppressor activity through its activity on direct and indirect targets, such as p53, p38 MAPK, RelA, and ERK, 7–9 and was the first member of the PP2C family to be structurally characterized. 10 The PP2C fold consists of two antiparallel β -sheets surrounded by two sets of α -helices, with the active site located along one edge of the β -sheets.…”

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Conformational Changes in Active and Inactive States of Human PP2Cα Characterized by Hydrogen/Deuterium Exchange–Mass Spectrometry

et al. 2017

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PPM serine/threonine protein phosphatases function in signaling pathways and require millimolar concentrations of Mn2+ or Mg2+ ions for activity. Whereas the crystal structure of human PP2Cα displayed two tightly bound Mn2+ ions in the active site, recent investigations of PPM phosphatases have characterized the binding of a third, catalytically essential metal ion. The binding of the third Mg2+ to PP2Cα was reported to have millimolar affinity and to be entropically driven, suggesting it may be structurally and catalytically important. Here, we report the use of hydrogen/deuterium exchange–mass spectrometry and molecular dynamics to characterize conformational changes in PP2Cα between the active and inactive states. In the presence of millimolar concentrations of Mg2+, metal-coordinating residues in the PP2Cα active site are maintained in a more rigid state over the catalytically relevant time scale of 30–300 s. Submillimolar Mg2+ concentrations or introduction of the D146A mutation increased the conformational mobility in the Flap subdomain and in buttressing helices α1 and α2. Residues 192–200, located in the Flap subdomain, exhibited the greatest interplay between effects of Mg2+ concentration and the D146A mutation. Molecular dynamics simulations suggest that the presence of the third metal ion and the D146A mutation each produce distinct conformational realignments in the Flap subdomain. These observations suggest that the binding of Mg2+ to the D146/D239 binding site stabilizes the conformation of the active site and the Flap subdomain.

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Metal‐dependent protein phosphatase 1A functions as an extracellular signal‐regulated kinase phosphatase

Cited by 28 publications

References 32 publications

The catalytic role of the M2 metal ion in PP2Cα

The catalytic role of the M2 metal ion in PP2Cα

A trapped human PPM1A–phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity

Conformational Changes in Active and Inactive States of Human PP2Cα Characterized by Hydrogen/Deuterium Exchange–Mass Spectrometry

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