MOLECULAR-CLONING AND FUNCTIONAL-CHARACTERIZATION OF A NOVEL MITOGEN-ACTIVATED PROTEIN-KINASE PHOSPHATASE, MKP-4

Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/s tress-activated protein kinase (JNK/SAPK), and p38/RK/CSBP (p38) mitog en-activated protein (MAP) kinases are target enzymes activated by a w ide range of cell-surface stimuli, Recently, a distinct class of dual specificity phosphatase has been shown to reverse activation of MAP ki nases by dephosphorylating critical tyrosine and threonine residues, B y searching the expressed sequence tag data base (dbEST) for homologue s of known dual specificity phosphatases, we identified a novel partia l 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-te rminal CH2 domains homologous to the cell cycle regulator Cdc25 phosph atase, and contains the extended active site sequence motif VXVHCXAGXS RSXTX(3)AYLM (where X is any amino acid) conserved in dual specificity phosphatases, MKP-4 produced in Escherichia coil catalyzes vanadate-s ensitive breakdown of p-nitrophenyl phosphate as well as in vitro inac tivation 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 specie s of approximately 2.5 kilobases detected only in placenta, kidney, an d embryonic liver, Immunocytochemical analysis showed MKP-4 to be pres ent within cytosol although punctate nuclear staining co-localizing wi th promyelocytic protein was also observed in a subpopulation (10-20%) of cells. Chromosomal localization by analysis of DNAs from human/rod ent somatic cell hybrids and a panel of radiation hybrids assign the h uman gene for MKP-4 to Xq28., The identification and characterization of MKP-4 highlights the emergence of an expanding family of structural ly homologous dual specificity phosphatases possessing distinct MAP ki nase specificity and subcellular localization as well as diverse patte rns of tissue expression.