ATMPK4, an arabidopsis homolog of mitogen-activated protein kinase, is activated in vitro by AtMEK1 through threonine phosphorylation

The modulation of mitogen-activated protein kinase (MAPK) activity regulate s many intracellular signaling processes. In animal and yeast cells, MAP ki nases are activated via phosphorylation by the dual-specificity kinase MEK (MAP kinase kinase). Several plant homologs of MEK and MAPK have been ident ified, but the biochemical events underlying the activation of plant MAPKs remain unknown. We describe the in vitro activation of an Arabidopsis homol og of MAP kinase, ATMPK4. ATMPK4 was phosphorylated in vitro by an Arabidop sis MEK homolog, AtMEK1. This phosphorylation occurred principally on threo nine (Thr) residues and resulted in elevated ATMPK4 kinase activity. A seco nd Arabidopsis MEK isoform, ATMAP2K alpha, failed to phosphorylate ATMPK4 i n vitro. Tyr dephosphorylation by the Arabidopsis Tyr-specific phosphatase AtPTP1 resulted in an almost complete loss of ATMPK4 activity. Immunoprecip itates of Arabidopsis extracts with anti-ATMPK4 antibodies displayed myelin basic protein kinase activity that was sensitive to treatment with AtPTP1. These results demonstrate that a plant MEK can phosphorylate and activate MAPK, and that Tyr phosphorylation is critical for the catalytic activity o f MAPK in plants. Surprisingly, in contrast to the animal enzymes, AtMEK1 m ay not be a dual-specificity kinase but, rather, the required Tyr phosphory lation on ATMPK4 may result from autophosphorylation.