Identification of tyrosine autophosphorylation sites of Arabidopsis MEKK1 and their involvement in the regulation of kinase activity

Abstract

The MEKK1 kinase is a key regulator of stress signaling in Arabidopsis; however, little is known about the regulation of its kinase activity. Here, we found that recombinant MEKK1, expressed in both mammalian HEK293 cells and Escherichia coli, shows a mobility shift in SDS-PAGE, and immunoblotting detected phosphorylation of serine, threonine, and tyrosine residues. N-terminal deletions, site-directed mutagenesis, and protein phosphatase treatment revealed that the mobility shift results from autophosphorylation of the kinase domain. We identified the tyrosine autophosphorylation sites in the N-terminal region of MEKK1. Tyrosine to phenylalanine mutations decrease phosphorylation of the substrate MKK1, suggesting the important role of this residue in the regulation of MEKK1 kinase activity. The present study is the first to show that plant MAPKKKs are regulated by tyrosine phosphorylation.