THE MITOGEN-ACTIVATED PROTEIN-KINASE PHOSPHATASE-3 N-TERMINAL NONCATALYTIC REGION IS RESPONSIBLE FOR TIGHT SUBSTRATE-BINDING AND ENZYMATIC SPECIFICITY

We have reported recently that the dual specificity mitogen-activated protein kinase phosphatase-3 (MKP-3) elicits highly selective inactiva tion of the extracellular signal-regulated kinase (ERK) class of mitog en-activated protein (MAP) kinases (Muda, M., Theodosiou, A., Rodrigue s, N., Boschert, Il., Camps, M., Gillieron, C., Davies, It, Ashworth, A., and Arkinstall, S. (1996) J. Biol. Chem, 271, 27205-27208). We now show that MKP-3 enzymatic specificity is paralleled by tight binding to both ERK1 and ERK2 while, in contrast, little or no interaction wit h either c-Jun N-terminal kinase/stress activated protein kinase (JNK/ SAPK) or p38 MAP kinases was detected. Further study revealed that the N-terminal noncatalytic domain of MKP-3 (MKP-3 Delta C) binds both ER K1 and ERK2, while the C-terminal MKP-3 catalytic core (MKP-3 Delta N) fails to precipitate either of these MAP kinases. A chimera consistin g of the N-terminal half of MKP-3 with the C-terminal catalytic core o f M3-6 also bound tightly to ERK1 but not to JNK3/SAPK beta. Consisten t with a role for N-terminal binding in determining MKP-3 specificity, at least 10-fold higher concentrations of purified MKP-3 Delta N than full-length MKP-3 is required to inhibit ERK2 activity. In contrast, both MKP-3 Delta N and full-length MKP-3 inactivate JNK/SAPK and p38 M AP kinases at similarly high concentrations. Also, a chimera of the M3 -6 N terminus with the MKP-3 catalytic core which fails to bind ERK el icits non selective inactivation of ERK1 and JNK3/SAPK beta. Together, these observations suggest that the physiological specificity of MKP- 3 for inactivation of ERK family MAP kinases reflects tight substrate binding by its N-terminal domain.