A novel mitogen-activated protein kinase phosphatase is an important negative regulator of lipopolysaccharide-mediated c-Jun N-terminal kinase activation in mouse macrophage cell lines

We have isolated a cDNA homologous to known dual-specificity phosphatases f rom a mouse macrophage cDNA library and termed it MKP-M (for mitogen-activa ted protein kinase phosphatase isolated from macrophages). Three other pres umed splice variant isoforms have also been identified for MKP-M. The longe st and most abundant mRNA contains an open reading frame corresponding to 6 77 amino acids and produces an 80-kDa protein. The deduced amino acid seque nce of MKP-M is most similar to those of hVH-5 (or mouse M3/6) and VHP1, a Caenorhabditis elegans tyrosine phosphatase. It includes an N-terminal rhod anase homology domain, the extended active-site sequence motif (V/L)X(V/I)H CXAG(I/V)SRSXT(I/V)XXAY(L/I)M (where X is any amino acid), and a C-terminal PEST sequence. Northern blot analysis revealed a dominant MKP-M mRNA speci es of approximately 5.5 kb detected ubiquitously among all tissues examined . MKP-M was constitutively expressed in mouse macrophage cell lines, and it s expression levels were rapidly increased by lipopolysaccharide (LPS) stim ulation but not by tumor necrosis factor alpha (TNF-alpha), gamma interfero n, interleukin-2 (IL-2), or IL-15 stimulation. Immunocytochemical analysis showed MKP-M to be present within cytosol. When expressed in COS7 cells, MK P-M blocks activation of mitogen-activated protein kinases with the selecti vity c-jun N-terminal kinase (JNK) much greater than p38 = extracellular si gnal-regulated kinase. Furthermore, expression of a catalytically inactive form of MKP-M in a mouse macrophage cell line increased the intensity and d uration of JNK activation and TNF-alpha secretion after LPS stimulation, su ggesting that MKP-M is at least partially responsible for the desensitizati on of LPS-mediated JNK activation and cytokine secretion in macrophages.