Structure-function analysis of stress-induced phosphatase

Research project P 14631 Structure-Funktion Analysis of Stress-induced Phosphatase Heribert HIRT 09.10.2000 In yeast, animals, and plants, many stresses are signaled through highly conserved MAPK (mitogen-activated protein kinase) cascades. We have recently identified MP2C, encoding a Medicago PP2C (protein phosphatase 2C), as a negative regulator of yeast and plant MAPK pathways. Biochemical analysis showed that MP2C but not several other plant PP2Cs functions as a NLAYK phosphatase. Structural analysis revealed that the unique noncatalytic N-terminus of MP2C contains a MAPK docking site that is required for substrate interaction and specificity. In this work, the analysis of MP2C will be extended to a genetic investigation of the biological function of AP2C, the Arabidopsis homologue of MP2C. Besides performing a structure-function genetic analysis of AP2C, interacting partners of AP2C will be isolated and analysed by biochemical methods.

Protein phosphatases of type 2C (PP2Cs) play important roles in eukaryotic signal transduction. In contrast to protein phosphatases of type 1, 2A and 2B, PP2Cs act as monomers, indicating that this class of phosphatases must contain all the information for substrate specificity and regulation. In plants, several classes of PP2Cs have been isolated showing a common structure with a conserved phosphatase domain and unique N-terminal extensions of variable lenghts. In a previous project, we had isolated MP2C, a PP2C that acts as a negative regulator of a mitogen-activated protein kinase (MAPK) pathway in yeast. In this project, we showed that MP2C is a MAPK phosphatase that inactivates MAPKs through direct physical interaction and threonine dephosphorylation of the pTEpY motif that is necessary for activity of MAPKs. Binding studies demonstrated that the N-terminus but not the catalytic domain of MP2C is necessary and sufficient for MAPK interaction. The N-terminus of MP2C contains a consensus MAPK docking motif that is conserved in several mammalian MAPK-interacting proteins, including the transcription factor Elk1, the protein kinases Rsk1 and MEK1, and the tyrosine phosphatase PTP-SL. Structure- function analysis of MP2C with two other plant PP2Cs, ABI2 and AtP2C-HA, revealed that the N-terminus is involved in determining the substrate specificity of the phosphatases. Considering the structural similarity of eukaryotic PP2Cs, these data suggest that the unique domains of PP2Cs play an essential role in determining substrate interaction and specificity.