The mitogen-activated protein (MAP) kinase family includes extracellular signal-regulated kinase (ERK), c-Jun NH 2 -terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38/RK/CSBP (p38) as structurally and functionally distinct enzyme classes. Here we describe two new dual specificity phosphatases of the CL100/MKP-1 family that are selective for inactivating ERK or JNK/SAPK and p38 MAP kinases when expressed in COS-7 cells. M3/6 is the first phosphatase of this family to display highly specific inactivation of JNK/SAPK and p38 MAP kinases. Although stress-induced activation of p54 SAPK␤, p46 SAPK␥ (JNK1) or p38 MAP kinases is abolished upon co-transfection with increasing amounts of M3/6 plasmid, epidermal growth factor-stimulated ERK1 is remarkably insensitive even to the highest levels of M3/6 expression obtained. In contrast to M3/6, the dual specificity phosphatase MKP-3 is selective for inactivation of ERK family MAP kinases. Low level expression of MKP-3 blocks totally epidermal growth factor-stimulated ERK1, whereas stress-induced activation of p54 SAPK␤ and p38 MAP kinases is inhibited only partially under identical conditions. Selective regulation by M3/6 and MKP-3 was also observed upon chronic MAP kinase activation by constitutive p21 ras GTPases. Hence, although M3/6 expression effectively blocked p54 SAPK␤ activation by p21 rac (G12V), ERK1 activated by p21 ras (G12V) was insensitive to this phosphatase. ERK1 activation by oncogenic p21 ras was, however, blocked totally by co-expression of MKP-3. This is the first report demonstrating reciprocally selective inhibition of different MAP kinases by two distinct dual specificity phosphatases.The mitogen activated protein (MAP) kinase 1 family comprises the extracellular signal-regulated kinase (ERK), c-Jun NH 2 -terminal kinase/stress-activated protein kinase (JNK/ SAPK), and p38/RK/CSBP (p38) as three structurally and functionally distinct enzyme classes (1-7). ERK family members are activated by a variety of growth and differentiation factors, while MAP kinases of the JNK/SAPK, and p38 class are activated preferentially by cellular stresses, and inflammatory cytokines (1)(2)(3)(4)(5)(7)(8)(9)(10)(11)(12). Activated MAP kinases phosphorylate a range of cellular substrates, including additional kinases and several transcription factors (7,(13)(14)(15)(16). MAP kinase-dependent regulation of diverse targets indicates a critical role orchestrating many varied and important cellular processes. Likely functions include a pivotal role for ERK in mediating neuronal differentiation in PC12 cells as well as growth factor-stimulated proliferation and oncogenic transformation in fibroblasts (17-21). Recent investigations also support the view that activation of JNK/SAPK and p38 MAP kinases are critical in processes mediating platelet aggregation and secretion, in generation of inflammatory cytokines as well as in triggering of apoptotic death in a range of cell types (6,12,(22)(23)(24)(25).Full activation of MAP kinases requires dual phosphorylation on...

We have reported recently that the dual specificity mitogen-activated protein kinase phosphatase-3 (MKP-3) elicits highly selective inactivation of the extracellular signal-regulated kinase (ERK) class of mitogen-activated protein (MAP) kinases (Muda, M., Theodosiou, A., Rodrigues, N., Boschert, U., Camps, M., Gillieron, C., Davies, K., Ashworth, A., and Arkinstall, S. (1996) J. Biol. Chem. 271, 27205-27208). We now show that MKP-3 enzymatic specificity is paralleled by tight binding to both ERK1 and ERK2 while, in contrast, little or no interaction with either c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK) or p38 MAP kinases was detected. Further study revealed that the N-terminal noncatalytic domain of MKP-3 (MKP-3⌬C) binds both ERK1 and ERK2, while the C-terminal MKP-3 catalytic core (MKP-3⌬N) fails to precipitate either of these MAP kinases. A chimera consisting of the N-terminal half of MKP-3 with the C-terminal catalytic core of M3-6 also bound tightly to ERK1 but not to JNK3/SAPK␤. Consistent with a role for N-terminal binding in determining MKP-3 specificity, at least 10-fold higher concentrations of purified MKP-3⌬N than full-length MKP-3 is required to inhibit ERK2 activity. In contrast, both MKP-3⌬N and full-length MKP-3 inactivate JNK/SAPK and p38 MAP kinases at similarly high concentrations. Also, a chimera of the M3-6 N terminus with the MKP-3 catalytic core which fails to bind ERK elicits non selective inactivation of ERK1 and JNK3/SAPK␤. Together, these observations suggest that the physiological specificity of MKP-3 for inactivation of ERK family MAP kinases reflects tight substrate binding by its N-terminal domain.The extracellular signal-regulated kinase (ERK), 1 c-Jun Nterminal kinase/stress-activated protein kinase (JNK/SAPK) and p38/RK/CSBP (p38) represent three major classes of mitogen-activated protein (MAP) kinase (1-4). Different cell stimuli appear to activate distinct MAP kinases preferentially. Hence, while growth factors and some oncogenes are linked to activation of ERK, inflammatory cytokines and cell stresses lead to activation of JNK/SAPK and p38 (1-4). MAP kinases are known to phosphorylate several key regulatory proteins including additional kinases, cytoskeletal proteins, nuclear receptors, as well as several transcription factors, indicating a central role in controlling cell function (1, 5-10). Indeed, ERK has been shown to be important in processes leading to neuronal differentiation, mitogenesis, and oncogenic transformation (1, 11-15), while JNK/SAPK and p38 MAP kinases play critical roles in pathways leading to the generation of inflammatory cytokines and apoptotic death (1, 16 -20). MAP kinase activation requires phosphorylation on Thr and Tyr residues located within the motif TXY of kinase domain VIII (1, 2, 4, 7). While several upstream kinases catalyze this modification on specific MAP kinases, an emerging family of dual-specificity phosphatase dephosphorylate both threonine and tyrosine residues and appear likely to inactivate MAP kinases effect...

Dual-speci®city protein tyrosine phosphatases are a burgeoning family of enzymes, some of which, the MKPs, are implicated in the regulation of mitogenactivated protein (MAP) kinases. MKPs have been shown to reverse the activation of the MAP kinases by hydrolyzing phosphothreonine and phosphotyrosine residues present in the substrates. Here we describe the characterization of a novel member of the MKP family, MKP5. The MKP5 gene, which maps to human chromosome 1q32, is expressed tissue-speci®cally as two transcripts of approximately 3.4 and 2.4 kb in human liver and skeletal muscle. When expressed in mammalian cells, MKP5 blocks the enzymatic activation of MAP kinases with the selectivity p38&JNK/ SAPK44ERK. Immunoprecipitation of endogenous MAP kinases by the catalytically inactive transfected MKP5 demonstrates that it preferentially binds to the p38 and JNK/SAPK kinases. These ®ndings suggest that the selectivity of this phosphatase may be determined at least in part at the level of substrate binding.

An emerging family of structurally distinct dual-specificity serine, threonine and tyrosine phosphatases that act on MAP kinases consists of ten members in mammals, and members have been found in animals, plants and yeast. Three subgroups have been identified that differ in exon structure, sequence and substrate specificity.

The Survival Motor Neuron (SMN) gene shows deletions in the majority of patients with Spinal Muscular Atrophy (SMA), a disease of motor neuron degeneration. To date only two missense mutations have been reported in SMN in patients with SMA. The fact that no SMN-homologues have been forthcoming from data-base searching has resulted in a lack of hypotheses concerning the structural and functional consequences of these mutations. Recently SMN has been shown to interact with heterogeneous nuclear ribonucleoproteins (hnRNPs) suggesting a role in mRNA metabolism. We describe a novel missense mutation and the subsequent identification of a triplicated tyrosine-glycine (Y-G) peptide sequence at the C-terminal of SMN which encompasses each of the three predicted amino acid sequence substitutions. We have identified apparent orthologues of SMN in Caenorhabditis elegans and Schizosaccharomyces pombe. These sequences retain the highly conserved Y-G motif and provide additional support for a role of SMN in mRNA metabolism.