title: Binding of a third metal ion by the human phosphatases PP2Cα and Wip1 is required for phosphatase activity.
abstract: The PPM phosphatases require millimolar concentrations of Mg²⁺ or Mn²⁺ to activate phosphatase activity in vitro. The human phosphatases PP2Cα (PPM1A) and Wip1 (PPM1D) differ in their physiological function, substrate specificity, and apparent metal affinity. A crystallographic structure of PP2Cα shows only two metal ions in the active site. However, recent structural studies of several bacterial PP2C phosphatases have indicated three metal ions in the active site. Two residues that coordinate the third metal ion are highly conserved, suggesting that human PP2C phosphatases may also bind a third ion. Here, isothermal titration calorimetry analysis of Mg²⁺ binding to PP2Cα distinguished binding of two ions to high affinity sites from the binding of a third ion with a millimolar affinity, similar to the apparent metal affinity required for catalytic activity. Mutational analysis indicated that Asp239 and either Asp146 or Asp243 was required for low-affinity binding of Mg²⁺, but that both Asp146 and Asp239 were required for catalysis. Phosphatase activity assays in the presence of MgCl₂, MnCl₂, or mixtures of the two, demonstrate high phosphatase activity toward a phosphopeptide substrate when Mg²⁺ was bound to the low-affinity site, whether Mg²⁺ or Mn²⁺ ions were bound to the high affinity sites. Mutation of the corresponding putative third metal ion-coordinating residues of Wip1 affected catalytic activity similarly both in vitro and in human cells. These results suggest that phosphatase activity toward phosphopeptide substrates by PP2Cα and Wip1 requires the binding of a Mg²⁺ ion to the low-affinity site.
authors.affiliation: Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, United States.
authors.forename: Kan
authors.initials: K
authors.lastname: Tanoue
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Miller Jenkins
authors.forename: Stewart R
authors.initials: SR
authors.lastname: Durell
authors.forename: Subrata
authors.initials: S
authors.lastname: Debnath
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Sakai
authors.forename: Harichandra D
authors.initials: HD
authors.lastname: Tagad
authors.forename: Kazushige
authors.initials: K
authors.lastname: Ishida
authors.forename: Ettore
authors.initials: E
authors.lastname: Appella
authors.forename: Sharlyn J
authors.initials: SJ
authors.lastname: Mazur
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D057809:HEK293 Cells; D006801:Humans; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism; D010748:Phosphopeptides / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D000071636:Protein Phosphatase 2C; D016415:Sequence Alignment
chemical_list: D010748:Phosphopeptides; D001224:Aspartic Acid; D008345:Manganese; C000607456:PPM1A protein, human; C000607458:PPM1D protein, human; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C; D008274:Magnesium; D000409:Alanine
references.citation: J Lipid Res. 2012 Aug;53(8):1513-21
references.pmid: 22615346
references.citation: Trends Biochem Sci. 2010 Feb;35(2):109-14
references.pmid: 19879149
references.citation: Crit Rev Biochem Mol Biol. 2007 Nov-Dec;42(6):437-61
references.pmid: 18066953
references.citation: Biochemistry. 2011 May 31;50(21):4537-49
references.pmid: 21528848
references.citation: Protein Sci. 1996 Jul;5(7):1421-5
references.pmid: 8819174
references.citation: Mol Cell. 2007 Mar 23;25(6):917-31
references.pmid: 17386267
references.citation: Oncogene. 2010 Nov 11;29(45):5977-88
references.pmid: 20838380
references.citation: J Biol Chem. 2004 May 21;279(21):22715-26
references.pmid: 15016818
references.citation: Methods Enzymol. 2003;366:282-9
references.pmid: 14674255
references.citation: Biometals. 2009 Jun;22(3):469-77
references.pmid: 19262998
references.citation: Biochemistry. 2005 Apr 12;44(14):5285-94
references.pmid: 15807522
references.citation: Protein Pept Lett. 2008;15(9):938-48
references.pmid: 18991770
references.citation: J Biol Chem. 2003 Apr 18;278(16):14299-305
references.pmid: 12514180
references.citation: Nat Rev Cancer. 2008 Dec;8(12):957-67
references.pmid: 19005492
references.citation: Oncogene. 2010 Apr 15;29(15):2281-91
references.pmid: 20101220
references.citation: Mol Cell. 2005 Apr 1;18(1):13-24
references.pmid: 15808505
references.citation: Biochem J. 2009 Jan 15;417(2):401-9
references.pmid: 19099538
references.citation: J Mol Biol. 2008 Feb 15;376(2):570-81
references.pmid: 18164312
references.citation: Biochem J. 1986 Aug 15;238(1):83-91
references.pmid: 3026347
references.citation: J Biol Chem. 2012 Mar 16;287(12):9178-92
references.pmid: 22291014
references.citation: Science. 2012 Jan 6;335(6064):85-8
references.pmid: 22116026
references.citation: Proc Natl Acad Sci U S A. 1997 Jun 10;94(12):6048-53
references.pmid: 9177166
references.citation: Chem Rev. 2001 Aug;101(8):2291-312
references.pmid: 11749374
references.citation: FEBS Lett. 1998 Oct 23;437(3):229-32
references.pmid: 9824296
references.citation: Rapid Commun Mass Spectrom. 2005;19(19):2747-56
references.pmid: 16136520
references.citation: Mol Cell. 2006 Sep 1;23(5):757-64
references.pmid: 16949371
references.citation: J Biol Chem. 1999 Jul 16;274(29):20336-43
references.pmid: 10400656
references.citation: Cell Cycle. 2010 Jun 1;9(11):2092-6
references.pmid: 20495376
references.citation: EMBO J. 2000 Dec 1;19(23):6517-26
references.pmid: 11101524
references.citation: EMBO J. 1996 Dec 16;15(24):6798-809
references.pmid: 9003755
references.citation: Front Biosci (Landmark Ed). 2012 Jan 01;17:1480-98
references.pmid: 22201816
references.citation: Structure. 2011 Jan 12;19(1):56-69
references.pmid: 21220116
references.citation: EMBO J. 1998 Aug 17;17(16):4744-52
references.pmid: 9707433
references.citation: Plant Physiol. 2011 May;156(1):106-16
references.pmid: 21357183
references.citation: Nature. 2009 Dec 3;462(7273):602-8
references.pmid: 19898420
references.citation: Structure. 2007 Jul;15(7):863-72
references.pmid: 17637345
references.citation: Science. 1990 Aug 24;249(4971):912-5
references.pmid: 2144057
references.citation: FEBS J. 2007 Jun;274(12):3128-37
references.pmid: 17521332
references.citation: Cell. 2009 Oct 30;139(3):468-84
references.pmid: 19879837
references.citation: Cancer Res. 2010 May 15;70(10):4112-22
references.pmid: 20460517
references.citation: Biochemistry. 2003 Jul 22;42(28):8513-21
references.pmid: 12859198
references.citation: Chem Rev. 2006 Aug;106(8):3338-63
references.pmid: 16895331
references.citation: BMC Bioinformatics. 2004 Aug 19;5:113
references.pmid: 15318951
references.citation: FEBS J. 2013 Jan;280(2):694-707
references.pmid: 22212593
references.citation: Nucleic Acids Res. 2013 Jan;41(Database issue):D348-52
references.pmid: 23197659
references.citation: J Biol Chem. 2011 Apr 15;286(15):13481-8
references.pmid: 21310952
references.citation: Cancer Cell. 2007 Oct;12(4):342-54
references.pmid: 17936559
references.citation: Cancer Metastasis Rev. 2008 Jun;27(2):123-35
references.pmid: 18265945
references.citation: J Mol Biol. 2007 Dec 7;374(4):890-8
references.pmid: 17961594
references.citation: J Endocrinol. 2006 Sep;190(3):711-8
references.pmid: 17003272
references.citation: Biochem J. 1996 Dec 15;320 ( Pt 3):801-6
references.pmid: 9003365
references.citation: Obes Res. 2005 Apr;13(4):678-86
references.pmid: 15897476
references.citation: Biochem J. 2006 Nov 1;399(3):427-34
references.pmid: 16879102
references.citation: Protein Sci. 2007 Jan;16(1):30-42
references.pmid: 17192587
references.citation: Structure. 2004 Nov;12(11):1947-54
references.pmid: 15530359
references.citation: FEBS Lett. 1999 Sep 24;458(3):363-9
references.pmid: 10570941
references.citation: Genes Dev. 2005 May 15;19(10):1162-74
references.pmid: 15870257
references.citation: J Biol Chem. 2010 Apr 23;285(17):12935-47
references.pmid: 20118229