MKP-3, a Novel Cytosolic Protein-tyrosine Phosphatase That Exemplifies a New Class of Mitogen-activated Protein Kinase Phosphatase

Abstract

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,...

MKP5, a new member of the MAP kinase phosphatase family, which selectively dephosphorylates stress-activated kinases

“…Importantly, these results would suggest that MKP5 selectivity is similar to that of M3/6, which is also more speci®c towards the stress-activated kinases (JNK/SAPK&p38444ERK) (Muda et al, 1996b). It would also appear that the enzymatic speci®city of these two members (MKP5 and M3/6), is quite di erent from MKP-4 (ERK4p38=JNK/SAPK) , MKP-3 (ERK44JNK/SAPK &p38) (Muda et al, 1996a;Groom et al, 1996), MKP-2 (ERK=JNK/SAPK4p38) (Chu et al, 1996) and PAC-1 (ERK=p384JNK/SAPK) (Chu et al, 1996).…”

“…The subcellular localization of MKP5 di ers from that of the other dual speci®city phosphatases. MKP-1/ CL100, PAC-1, hVH-2/MKP-2/TYP-1 and hVH-3/B23 are localized entirely within the nucleus (Rohan et al, 1993;Guan and Butch, 1995;Kwak and Dixon, 1995;Brondello et al, 1995), MKP-3/PYST1 is entirely cytoplasmic (Muda et al, 1996a;Groom et al, 1996), MKP-4 is mainly cytoplasmic with some punctate staining in the nucleus , while hVH-5/M3/6 is cytosolic or nuclear depending on cell type (Theodosiou et al, 1996). Two N-terminal charged clusters of amino acids, RRAR-(15)-RAR, have been identi®ed in hVH-3/B23 (Kwak and Dixon, 1995; Ishibashi et al, 1994).…”

“…MKP5 was subcloned into pMT-SM III, a modi®ed version of the eukaryotic expression vector pMT-SM (Muda et al, 1996a;Schaap et al, 1993), in which the 9E10 Myc epitope coding sequence is incorporated, in-frame, into the multiple cloning site. Primers TTAGGTACCTGT-CTTGAGTTCTT-CTTGAATTGC (nucleotides 74 ± 106) and CCATCTAGA-CCACGCCCATCAGC (nucleotides 1579 ± 1545) were used to amplify MKP5 ORF from human breast cDNA (the underlined nucleotides correspond to the engineered Asp718 and XbaI sites).…”

“…Endogenous MAP kinases were detected using the polyclonal anti-p38 MAPK, anti-SAPK/JNK and anti-ERK2 antibodies. Immunoprecipitation of HA-tagged MAP kinases and Myc-tagged MKP5, immune complex kinase assays and Western blotting were all performed as described previously (Muda et al, 1996a.…”

“…However, it has since been demonstrated that MKP-1 also catalyzes the dephosphorylation of both JNK/SAPK and p38 (Chu et al, 1996;Hirsch and Stork, 1997;Franklin and Kraft, 1997). Nevertheless, recent in vitro studies suggest that MKP-3/PYST1 (Muda et al, 1996a;Mourey et al, 1996;Groom et al, 1996) and M3/6/ hVH-5 (Theodosiou et al, 1996;Martell et al, 1995) are highly selective in inactivating either ERK, or JNK/SAPK and p38 MAP kinases, respectively (Groom et al, 1996;Muda et al, 1996bMuda et al, , 1998. The N-terminal non-catalytic domain of MKP-3 appears to be responsible for binding the ERK2 MAP kinase, thus directing the speci®city of this phosphatase Camps et al, 1998).…”