Two Adjacent Docking Sites in the Yeast Hog1 Mitogen-Activated Protein (MAP) Kinase Differentially Interact with the Pbs2 MAP Kinase Kinase and the Ptp2 Protein Tyrosine Phosphatase

Abstract

Functional interactions between a mitogen-activated protein kinase (MAPK) and its regulators require specific docking interactions. Here, we investigated the mechanism by which the yeast osmoregulatory Hog1 MAPK specifically interacts with its activator, the MAPK kinase Pbs2, and its major inactivator, the protein phosphatase Ptp2. We found, in the N-terminal noncatalytic region of Pbs2, a specific Hog1-binding domain, termed HBD-1. We also defined two adjacent Pbs2-binding sites in Hog1, namely, the common docking (CD) domain and Pbs2-binding domain 2 (PBD-2). The PBD-2 docking site appears to be sterically blocked in the intact Hog1 molecule, but its affinity to Pbs2 is apparent in shorter fragments of Hog1. Both the CD and the PBD-2 docking sites are required for the optimal activation of Hog1 by Pbs2, and in the absence of both sites, Hog1 cannot be activated by Pbs2. These data suggest that the initial interaction of Pbs2 with the CD site might induce a conformational change in Hog1 so that the PBD-2 site becomes accessible. The CD and PBD-2 docking sites are also involved in the specific interaction between Hog1 and Ptp2 and govern the dynamic dephosphorylation of activated Hog1. Thus, the CD and the PBD-2 docking sites play critical roles in both the activation and inactivation of Hog1.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We thank P. O'Grady for critical reading of the manuscript, M. Takekawa and Z. Miyake for technical advice, and V. Reiser, M. Takekawa, K. Yamamoto, T. Maeda, Q. Ge, and S. Wurgler-Murphy for unpublished plasmids and yeast mutants.

This work was supported in part by several grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to H.S. and K.T. and a grant from the Salt Science Research Foundation (no. 0715) to K.T.