Phosphorylation of the Kinase Interaction Motif in Mitogen-activated Protein (MAP) Kinase Phosphatase-4 Mediates Cross-talk between Protein Kinase A and MAP Kinase Signaling Pathways

Robin J Dickinson,Laurent Delavaine,Rocío Cejudo-Marín,Graeme Stewart,Christopher J Staples,Mark P Didmon,Antonio Garcia Trinidad,Andrés Alonso,Rafael Pulido,Stephen M Keyse

doi:10.1074/jbc.m111.255844

Abstract

MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site (55)RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo.

Highlights

Mutation of Arg-52, -53, and -55 to alanine does not affect the intrinsic catalytic activity of DUSP9/MKP-4 toward para-nitrophenyl phosphate (p-NPP) in vitro, it does prevent the catalytic activation of DUSP9/MKP-4 by recombinant ERK2 and p38␣ MAPKs (Fig. 1, B and C), indicating that the kinase interaction motif (KIM) mediates a functional interaction with these kinases
The R52A/ R53A/R55A mutant (DUSP9-KIM) shows no significant level of interaction with all three MAPKs (Fig. 1, D and E). These results are consistent with our previous data, which demonstrated that DUSP9/MKP-4 could dephosphorylate both ERK1/2 and p38␣ but not JNK when expressed in mammalian cells (6)
We demonstrate that DUSP9/MKP-4 recognizes its cognate MAPK substrates ERK1/2 and p38␣ via a conserved KIM

Summary

Background

We demonstrate that inactivation of both ERK1/2 and p38␣ by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. DUSP9/ MKP-4 is unique among the cytoplasmic MKPs in containing a conserved cAMP-dependent protein kinase (PKA) consensus phosphorylation site (Ser-58) immediately carboxyl-terminal to the KIM. This site can be modified by PKA in vitro, and phosphorylation abolishes the interaction of the amino-terminal domain of DUSP9/MKP-4 with both ERK2 and p38␣ in glutathione S-transferase (GST) pulldown assays. Using a phospho-specific antibody raised against this site, we demonstrate that endogenous DUSP9/MKP-4 is modified on Ser-58 in response to the PKA agonist forskolin and in murine placental tissue in vivo. We conclude that DUSP9/ MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and both mitogen- and stressactivated MAPK signaling

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION