MKP-3, a novel cytosolic protein-tyrosine phosphatase that exemplifies a new class of mitogen-activated protein kinase phosphatase.
MKP-1 (also known as CL100, 3CH134, Erp, and hVH-1) exemplifies a class of dual-specificity phosphatase able to reverse the activation of mitogen-activated protein (MAP) kinase family members by dephosphorylating critical tyrosine and threonine residues. We now report the cloning of MKP-3, a novel protein phosphatase that also suppresses MAP kinase activation state. The deduced amino acid sequence of MKP-3 is 36% identical to MKP-1 and contains the characteristic extended active-site sequence motif VXVHCXXGXSRSXTXXXAYLM (where X is any amino acid) as well as two N-terminal CH2 domains displaying homology to the cell cycle regulator Cdc25 phosphatase. When expressed in COS-7 cells, MKP-3 blocks both the phosphorylation and enzymatic activation of ERK2 by mitogens. Northern analysis reveals a single mRNA species of 2.7 kilobases with an expression pattern distinct from other dual-specificity phosphatases. MKP-3 is expressed in lung, heart, brain, and kidney, but not significantly in skeletal muscle or testis. In situ hybridization studies of MKP-3 in brain reveal enrichment within the CA1, CA3, and CA4 layers of the hippocampus. Metrazole-stimulated seizure activity triggers rapid (<1 h) but transient up-regulation of MKP-3 mRNA in the cortex, piriform cortex, and some amygdala nuclei. Metrazole stimulated similar regional up-regulation of MKP-1, although this was additionally induced within the thalamus. MKP-3 mRNA also undergoes powerful induction in PC12 cells after 3 h of nerve growth factor treatment. This response appears specific insofar as epidermal growth factor and dibutyryl cyclic AMP fail to induce significant MKP-3 expression. Subcellular localization of epitope-tagged MKP-3 in sympathetic neurons reveals expression in the cytosol with exclusion from the nucleus. Together, these observations indicate that MKP-3 is a novel dual-specificity phosphatase that displays a distinct tissue distribution, subcellular localization, and regulated expression, suggesting a unique function in controlling MAP kinase family members. Identification of a second partial cDNA clone (MKP-X) encoding the C-terminal 280 amino acids of an additional phosphatase that is 76% identical to MKP-3 suggests the existence of a distinct structurally homologous subfamily of MAP kinase phosphatases.
Muda
M
M
Glaxo Institute for Molecular Biology, CH-1228 Plan-les-Ouates, Geneva, Switzerland.
Boschert
U
U
Dickinson
R
R
Martinou
J C
JC
Martinou
I
I
Camps
M
M
Schlegel
W
W
Arkinstall
S
S
D000375:Aging; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology* / Q000254:growth & development; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers; D054642:Dual Specificity Phosphatase 6; D015723:Gene Library; D007668:Kidney; D008297:Male; D008969:Molecular Sequence Data; D009474:Neurons / Q000201:enzymology*; D016716:PC12 Cells; D016133:Polymerase Chain Reaction; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017783:Superior Cervical Ganglion / Q000201:enzymology*; D014158:Transcription, Genetic; D014162:Transfection
D017931:DNA Primers; D012333:RNA, Messenger; D011994:Recombinant Proteins; D054642:Dual Specificity Phosphatase 6; C517037:Dusp6 protein, rat; D017027:Protein Tyrosine Phosphatases