Catalytic activation of plant MAP kinase phosphatase NtMKP1 by its physiological substrate SIPK, but not by calmodulins

Abstract

Calmodulin (CaM), a ubiquitous Ca²⁺ sensor in eukaryotes, regulates a number of target proteins in a Ca²⁺-dependent manner. Previously, we identified NtMKP1, a tobacco MAP kinase phosphatase, as a novel CaM-binding protein. NtMKP1, produced by in vitro transcription/translation, dephosphorylated and inactivated SIPK, a tobacco stress-responsive MAP kinase. Deletion and site-directed mutagenesis revealed that Lys⁴¹ and Arg⁴³ in the N-terminal noncatalytic region of NtMKP1 are important to bind and inactivate SIPK, whereas the CaM-binding domain and C-terminal domain were dispensable. Moreover, hydrolysis of an artificial substrate by NtMKP1 was significantly stimulated by the SIPK-binding, but not by CaM-binding. Transient expression analysis in Nicotiana benthamiana indicated that Lys⁴¹ and Arg⁴³ in N-terminal noncatalytic region of NtMKP1, but not CaM-binding are important for the function of NtMKP1 in vivo. These results indicate that NtMKP1 activity is regulated by the binding of its physiological substrate SIPK, and CaM-binding to NtMKP1 might have other effect.