Dual specificity phosphatases target MAPK

The mitogen-activated protein kinase (MAPK), also known as extracellular signal-regulated kinase (ERK), plays a crucial role in various signal transduction pathways. ERK is activated by its upstream activator, MEK, via threonine and tyrosine phosphorylation. ERK activity in the cell is tightly regulated by phosphorylation and dephosphorylation. A novel dual specific phosphatase, HVH2, has been characterized that may function in vivo as a MAP kinase phosphatase. The deduced amino acid sequence of HVH2 shows significant identity to the VH1-related dual specific phosphatase family. In addition, the N-terminal region of HVH2 also displays sequence identity to the cell cycle regulator, Cdc25 phosphatase. Recombinant HVH2 phosphatase exhibits a high substrate specificity toward activated ERK and dephosphorylates both threonine and tyrosine residues of activated ERK1 and ERK2. Immunofluorescence studies with an epitope-tagged HVH2 show that the enzyme is localized in the cell nucleus. Transfection of HVH2 into NIH3T3 cells inhibits the v-src and MEK-induced transcriptional activation of a serum-responsive element containing promoter, consistent with the notion that HVH2 promotes the inactivation of MAP kinase. HVH2 mRNA shows an expression pattern distinct from CL100 (the human homolog of mouse MKP1) and PAC1, two previously identified MAP kinase phosphatases. These data suggest a possible role of HVH2 in MAP kinase regulation (Guan, 1995).

Dual specificity protein tyrosine phosphatases (dsPTPs) are a subfamily of protein tyrosine phosphatases implicated in the regulation of mitogen-activated protein kinase (MAPK). In addition to hydrolyzing phosphotyrosine, dsPTPs can hydrolyze phosphoserine/threonine-containing substrates and have been shown to dephosphorylate activated MAPK. rVH6, a novel dsPTP from rat hippocampus has been identified. rVH6 contains the conserved dsPTP active site sequence, VXVHCX2GX2RSX5AY(L/I)M, and exhibits phosphatase activity against activated MAPK. In PC12 cells, rVH6 mRNA is induced during nerve growth factor-mediated differentiation but not during insulin or epidermal growth factor mitogenic stimulation. In MM14 muscle cells, rVH6 mRNA is highly expressed in proliferating cells and declines rapidly during differentiation. rVH6 expression correlates with the inability of fibroblast growth factor to stimulate MAPK activity in proliferating (but not in differentiating) MM14 cells. rVH6 protein localizes to the cytoplasm and is the first dsPTP to be localized outside the nucleus. This novel subcellular localization may expose rVH6 to potential substrates that differ from nuclear dsPTPs substrates (Mourey, 1996).

The MAPK family includes extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38/RK/CSBP (p38) as structurally and functionally distinct enzyme classes. This study describes 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 SAPKbeta, p46 SAPKgamma (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 obtained of M3/6 expression. 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 totally blocks epidermal growth factor-stimulated ERK1, whereas stress-induced activation of p54 SAPKbeta and p38 MAP kinases are inhibited only partially under identical conditions. Selective regulation by M3/6 and MKP-3 is also observed upon chronic MAP kinase activation by constitutive p21(ras) GTPases. Hence, although M3/6 expression effectively blocks p54 SAPKbeta activation by p21(rac) (G12V), when activated by p21(ras) (G12V), ERK1 is insensitive to this phosphatase. However, ERK1 activation by oncogenic p21(ras) is 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 (Muda, 1996).

The Pyst1 and Pyst2 mRNAs encode closely related proteins that are novel members of a family of dual-specificity MAP kinase phosphatases typified by CL100/MKP-1. Pyst1 is expressed constitutively in human skin fibroblasts; in contrast to other members of this family of enzymes, its mRNA is not inducible by either stress or mitogens. Unlike the nuclear CL100 protein, Pyst1 is localized in the cytoplasm of transfected Cos-1 cells. Like CL100/ MKP-1, Pyst1 dephosphorylates and inactivates MAP kinase in vitro and in vivo. In addition, Pyst1 is able to form a physical complex with endogenous MAP kinase in Cos-1 cells. However, unlike CL100, Pyst1 displays very low activity toward the stress-activated protein kinases (SAPKs) or RK/p38 in vitro, indicating that these kinases are not physiological substrates for Pyst1. This specificity is underlined by the inability of Pyst1 to block either the stress-mediated activation of the JNK-1 SAP kinase or RK/p38 in vivo, or to inhibit nuclear signaling events mediated by the SAP kinases in response to UV radiation. These results provide the first evidence that the members of the MAP kinase family of enzymes are differentially regulated by dual-specificity phosphatases and also indicate that the MAP kinases may be regulated by different members of this family of enzymes depending on their subcellular location (Groom, 1996).

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 homologs of known dual specificity phosphatases, a novel, partial sequence in humans has been identified for a full-length cDNA (termed MKP-4) and isolated. The deduced amino acid sequence of MKP-4 is most similar to MKP-X/PYST2 (61% identity) and MKP-3/PYST1 (57% identity); it 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 shows MKP-4 to be present within cytosol, although punctate nuclear staining co-localizing with promyelocytic protein is 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 (Muda, 1997).

Transcriptional activation of dual specificity phosphatases: a feedback loop