Molecular Cloning and Functional Characterization of a Novel Mitogen-activated Protein Kinase Phosphatase, MKP-4

doi:10.1074/jbc.272.8.5141

Molecular Cloning and Functional Characterization of a Novel Mitogen-activated Protein Kinase Phosphatase, MKP-4*

^‡ Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development S.A., CH-1228 Plan-les-Ouates, Geneva, Switzerland and the
^§ Cancer Research Campaign Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research, Fulham Road, London SW3 6JB, United Kingdom

^¶ To whom correspondence should be addressed. Tel.: 41-22-706-98-42; Fax: 41-22-794-69-65; E-mail: sa7182{at}ggr.co.uk

Abstract

Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38/RK/CSBP (p38) mitogen-activated protein (MAP) kinases are target enzymes activated by a wide range of cell-surface stimuli. Recently, a distinct class of dual specificity phosphatase has been shown to reverse activation of MAP kinases by dephosphorylating critical tyrosine and threonine residues. By searching the expressed sequence tag data base (dbEST) for homologues of known dual specificity phosphatases, we identified a novel partial human sequence for which we isolated a full-length cDNA (termed MKP-4). The deduced amino acid sequence of MKP-4 is most similar to MKP-X/PYST2 (61% identity) and MKP-3/PYST1 (57% identity), includes two N-terminal CH2 domains homologous to the cell cycle regulator Cdc25 phosphatase, and contains the extended active site sequence motif VXVHCXAGXSRSXTX₃AYLM (where X is any amino acid) conserved in dual specificity phosphatases. MKP-4 produced in Escherichia coli catalyzes vanadate-sensitive breakdown of p-nitrophenyl phosphate as well as in vitro inactivation of purified ERK2. When expressed in COS-7 cells, MKP-4 blocks activation of MAP kinases with the selectivity ERK > p38 = JNK/SAPK. This cellular specificity is similar to MKP-3/PYST1, although distinct from hVH-5/M3-6 (JNK/SAPK = p38 >>> ERK). Northern analysis reveals a highly restricted tissue distribution with a single MKP-4 mRNA species of approximately 2.5 kilobases detected only in placenta, kidney, and embryonic liver. Immunocytochemical analysis showed MKP-4 to be present within cytosol although punctate nuclear staining co-localizing with promyelocytic protein was also observed in a subpopulation (10-20%) of cells. Chromosomal localization by analysis of DNAs from human/rodent somatic cell hybrids and a panel of radiation hybrids assign the human gene for MKP-4 to Xq28. The identification and characterization of MKP-4 highlights the emergence of an expanding family of structurally homologous dual specificity phosphatases possessing distinct MAP kinase specificity and subcellular localization as well as diverse patterns of tissue expression.

Footnotes

↵* 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) Y08302[GenBank].
↵¹ The abbreviations used are:

ERK

extracellular signal-regulated kinase

MAP

mitogen-activated protein

JNK/SAPK

c-Jun N-terminal kinase/stress-activated protein kinase

MEK

MAP kinase or ERK kinase

MKP

MAP kinase phosphatase

EGF

epidermal growth factor

MBP

myelin basic protein

MAPKAP kinase-2

MAP kinase-activated protein kinase-2

ATF2

activating transcription factor 2

EST

expressed sequence tag

CH2 domain

Cdc25 homology domain 2

GST

glutathione S-transferase

HA

hemagglutinin

pNPP

p-nitrophenyl phosphate

SPA

scintillation proximity assay

PML

promyelocytic protein

FITC

fluorescein isothiocyanate

PCR

polymerase chain reaction

bp

base pair(s)

MOPS

4-morpholinepropanesulfonic acid.
↵² B. Antonsson, manuscript in preparation.
↵³ J. Staple, personal communication.
↵⁴ A. Smith and A. Ashworth, unpublished data.
- Received October 2, 1996.
- Revision received November 19, 1996.