A Protein Phosphatase Methylesterase (PME-1) Is One of Several Novel Proteins Stably Associating with Two Inactive Mutants of Protein Phosphatase 2A

doi:10.1074/jbc.274.20.14382

A Protein Phosphatase Methylesterase (PME-1) Is One of Several Novel Proteins Stably Associating with Two Inactive Mutants of Protein Phosphatase 2A*

From the ^‡Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115, the ^§Institute of Molecular Biology, University of Vienna, A-1030 Vienna, Austria, the ^**Department of Biochemistry and Winship Cancer Center, Emory University School of Medicine, Atlanta, Georgia 30322, and the ^§§Harvard Microchemistry Facility, Harvard Biological Laboratories, Cambridge, Massachusetts 02138

Abstract

Carboxymethylation of proteins is a highly conserved means of regulation in eukaryotic cells. The protein phosphatase 2A (PP2A) catalytic (C) subunit is reversibly methylated at its carboxyl terminus by specific methyltransferase and methylesterase enzymes which have been purified, but not cloned. Carboxymethylation affects PP2A activity and varies during the cell cycle. Here, we report that substitution of glutamine for either of two putative active site histidines in the PP2A C subunit results in inactivation of PP2A and formation of stable complexes between PP2A and several cellular proteins. One of these cellular proteins, herein named protein phosphatase methylesterase-1 (PME-1), was purified and microsequenced, and its cDNA was cloned. PME-1 is conserved from yeast to human and contains a motif found in lipases having a catalytic triad-activated serine as their active site nucleophile. Bacterially expressed PME-1 demethylated PP2A C subunit in vitro, and okadaic acid, a known inhibitor of the PP2A methylesterase, inhibited this reaction. To our knowledge, PME-1 represents the first mammalian protein methylesterase to be cloned. Several lines of evidence indicate that, although there appears to be a role for C subunit carboxyl-terminal amino acids in PME-1 binding, amino acids other than those at the extreme carboxyl terminus of the C subunit also play an important role in PME-1 binding to a catalytically inactive mutant.

Footnotes

↵* This work was supported in part by National Institutes of Health Grant CA57327 (to D. C. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ Supported by an Erwin Schrödinger Postdoctoral Fellowship and Austrian Science Foundation Grant FWF, P12523-MOB.
↵‖ Contributed equally to the results of this work.
↵‡ Present address: Cubist Pharmaceuticals, Inc., Cambridge, MA 02139.
↵¶¶ To whom correspondence should be addressed: Dept. of Biochemistry, Emory University School of Medicine, 1510 Clifton Rd., Atlanta, GA 30322. Tel.: 404-727-5620; Fax: 404-727-3231; E-mail:dpallas{at}emory.edu.
↵2 L. Haehnel and D. C. Pallas, unpublished data.
↵3 X. X. Yu, X. Du, E. Ogris, R. E. Green, Q. Feng, L. Clon, and David Pallas, submitted for publication.
↵4 E. Ogris, I. Mudrak, E. Mak, D. Gibson, and D. C. Pallas, submitted for publication.
↵5 D. C. Pallas, unpublished data.
↵6 X. Du and D. C. Pallas, unpublished data.
↵7 K. C. Nelson and D. C. Pallas, unpublished data.
Abbreviations:

PP2A

protein phosphatase 2A

MT

middle tumor antigen

ST

small tumor antigen

EST

expressed sequence tag

RT-PCR

reverse transcriptase-polymerase chain reaction

PAGE

polyacrylamide gel electrophoresis

UTR

untranslated region

wt

wild type

ORF

open reading frame

PMSF

phenylmethylsulfonyl fluoride
- Received October 14, 1998.
- Revision received January 29, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.