Catalytic Activation of the Phosphatase MKP-3 by ERK2 Mitogen-Activated Protein Kinase

Abstract

MAP kinase phosphatase-3 (MKP-3) dephosphorylates phosphotyrosine and phosphothreonine and inactivates selectively ERK family mitogen-activated protein (MAP) kinases. MKP-3 was activated by direct binding to purified ERK2. Activation was independent of protein kinase activity and required binding of ERK2 to the noncatalytic amino-terminus of MKP-3. Neither the gain-of-function Sevenmaker ERK2 mutant D319N nor c-Jun amino-terminal kinase-stress-activated protein kinase (JNK/SAPK) or p38 MAP kinases bound MKP-3 or caused its catalytic activation. These kinases were also resistant to enzymatic inactivation by MKP-3. Another homologous but nonselective phosphatase, MKP-4, bound and was activated by ERK2, JNK/SAPK, and p38 MAP kinases. Catalytic activation of MAP kinase phosphatases through substrate binding may regulate MAP kinase activation by a large number of receptor systems.Signal transduction pathways that lead to activation of MAP kinases control many diverse and essential functions in yeast, worms, flies, and mammals. Extracellular signal-regulated kinase-1 (ERK1) and ERK2 exemplify one class of MAP kinase that undergoes activation by a range of stimuli including growth factors, cytokines, cell adhesion, tumor-promoting phorbol esters, and oncogenes (1). Specific functions assigned to ERK activity include chemotaxis, neuronal differentiation, and synaptic changes underlying memory and learning, as well as cellular mitogenesis and oncogenic transformation (1, 2).Full activation of ERK requires phosphorylation of threonine and tyrosine residues by a class of MAP kinase/ERK kinase (MEK) exemplified by MEK-1 (1, 3). Conversely, an emerging family of dual-specificity phosphatases that act on both phosphotyrosine and phosphothreonine reverse this process and also appear to be critical regulators of MAP kinase activity. CL100/3CH134 or MAP kinase phosphatase-1 (MKP-1) is the archetypal member of this gene family and has high substrate specificity for MAP kinases (4). Up to nine other mammalian dual-specificity phosphatases have been identified, and several of these are under tight transcriptional control and display distinct tissue, cell, and subcellular expression patterns (5, 6). MKP-3 appears exceptional in that it specifically inactivates ERK as compared with c-Jun NH 2 -terminal kinases/stress-activated protein kinases (JNK/SAPK) or p38 MAP kinases (7). We now show that ERK, but not other MAP kinases, cause substratetriggered activation of MKP-3.We purified various MKP-3 deletion mutants expressed in Escherichia coli and found that the NH 2 -terminal noncatalytic domain (amino acids 1 to 221) binds tightly to its target MAP kinases p44 ERK1 and p42 ERK2 (8). Binding to purified ERK2 (9) stimulates p-nitrophenyl phosphate (p-NPP) phosphatase activity of full-length MKP-3 by up to 30-fold (Fig. 1A) (10). Both glutathione-S-transferase (GST)-ERK2 and ERK2 caused similar activation of either the fusion protein GST-MKP-3, His-tagged MKP-3, or free MKP-3 (11). Activation of MKP-3 was dose-dependent and saturable wit...

Activation of p42 Mitogen-activated Protein Kinase (MAPK), but not c-Jun NH2-Terminal Kinase, Induces Phosphorylation and Stabilization of MAPK PhosphataseXCL100 inXenopusOocytes

“…Jacobs et al (1999) demonstrated that FXFP is an evolutionarily conserved motif that mediates binding of p42 MAPK to various substrate proteins. The discovery that both substrate specificity and tight substrate binding are conferred by the amino terminus of the related phosphatase MKP-3 (Camps et al, 1998;Muda et al, 1998) supports the hypothesis that the FNFP motif within XCL100 mediates its role as a p42 MAPK substrate. In addition, the absence of an analogous experimentally defined JNK docking site (Yang et al, 1998a,b;Jacobs et al, 1999) in XCL100 is consistent with our conclusion that this phosphatase is a poor substrate for JNK.…”

“…Moreover, p42 MAPK may positively regulate XCL100 through at least two other mechanisms: activation of p42 MAPK can induce expression of the closely related phosphatases MKP-1 and MKP-2 (Brondello et al, 1997), and, by analogy to other MKP family members, p42 MAPK may catalytically activate XCL100 by binding to its amino terminus (Camps et al, 1998;Dowd et al, 1998;Fjeld et al, 2000;Chen et al, 2001;Slack et al, 2001). The presence of multiple activation mechanisms may increase the robustness of the p42 MAPK/XCL100 system.…”

Activation and signaling of the p38 MAP kinase pathway

“…Many dual-specificity phosphatases have been identified that act upon various members of the MAP kinase pathway and are grouped as the MAP kinase phosphatase (MKP) family [45]. Several members can efficiently dephosphorylate p38α and p38β [46,47]; however, p38γ and p38δ are resistant to all known MKP family members. In addition, other types of phosphatases such as serine/ threonine protein phosphotase type 2C (PP2C) has been shown to have a role in downregulating the MAP kinase HOG1 pathway as well as negatively regulating human MKK6 and MKK4 levels in vitro and in vivo [48][49][50][51].…”

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

“…Recent studies suggest that the N-terminal noncatalytic domain of MKP-3 is responsible for tight binding to its substrates p44-ERK1 and p42-ERK2 . Moreover, MKP-3 is activated by binding to puri®ed ERK2 and this activation is independent of protein kinase activity (Camps et al, 1998). Whether this is a common mechanism for the regulation of other members of the MAP kinase phosphatase family remains to be determined.…”

“…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).…”