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 activesite 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.A wide range of cell-surface stimuli, including growth and differentiation factors and cytokines as well as ultraviolet radiation and osmotic shock, trigger rapid and powerful activation of mitogen-activated protein (MAP) 1 kinase family members (1-5). Currently, three major subclasses of MAP kinase can be identified, and these comprise the ERK, SAPK/JNK, and p38/HOG1 families (2, 3, 6). Full activation of MAP kinase requires phosphorylation on critical tyrosine and threonine residues, and several upstream dual-specificity kinases catalyzing this modification have now been identified (1-3, 6). Once activated, MAP kinases phosphorylate and regulate several cellular proteins, including additional protein kinases, cytoskeletal elements, stathmin,...

A structurally distinct subfamily of ten dual-specificity (Thr/Tyr) protein phosphatases is responsible for the regulated dephosphorylation and inactivation of mitogen-activated protein kinase (MAPK) family members in mammals. These MAPK phosphatases (MKPs) interact specifically with their substrates through a modular kinase-interaction motif (KIM) located within the N-terminal non-catalytic domain of the protein. In addition, MAPK binding is often accompanied by enzymatic activation of the C-terminal catalytic domain, thus ensuring specificity of action. Despite our knowledge of the biochemical and structural basis for the catalytic mechanism of the MKPs, we know much less about their regulation and physiological functions in mammalian cells and tissues. However, recent studies employing a range of model systems have begun to reveal essential non-redundant roles for the MKPs in determining the outcome of MAPK signalling in a variety of physiological contexts. These include development, immune system function, metabolic homeostasis and the regulation of cellular stress responses. Interestingly, these functions may reflect both restricted subcellular MKP activity and changes in the levels of signalling through multiple MAPK pathways.

Pyst1 is an inducible antagonist of FGF signaling in embryos and acts in a negative feedback loop to regulate the activity of MAPK. Our results demonstrate both the importance of MAPK signaling in neural induction and limb bud outgrowth and the critical role played by dual specificity MAP kinase phosphatases in regulating developmental outcomes in vertebrates.