New insights into the catalytic activation of the MAPK phosphatase PAC-1 induced by its substrate MAPK ERK2 binding.

PAC-1 is an inducible, nuclear-specific, dual-specificity mitogen-activated protein (MAP) kinase phosphatase that has been shown recently to be a transcription target of the human tumor-suppressor protein p53 in signaling apoptosis and growth suppression. However, its substrate specificity and regulation of catalytic activity thus far remain elusive. Here, we report in ...
vitro characterization of PAC-1 phosphatase activity with three distinct MAP kinase subfamilies. We show that the recombinant PAC-1 exists in a virtually inactive state when alone in vitro, and dephosphorylates extracellular signal-regulated kinase 2 (ERK2) but not p38alpha or c-Jun NH(2)-terminal kinase 2 (JNK2). ERK2 dephosphorylation by PAC-1 requires association of its amino-terminal domain with ERK2 that results in catalytic activation of the phosphatase. p38alpha also interacts with but does not activate PAC-1, whereas JNK2 does not bind to or cause catalytic activation by PAC-1. Moreover, our structure-based analysis reveals that individual mutation of the conserved Arg294 and Arg295 that likely comprise the phosphothreonine-binding pocket in PAC-1 to either alanine or lysine results in a nearly complete loss of its phosphatase activity even in the presence of ERK2. These results suggest that Arg294 and Arg295 play an important role in PAC-1 catalytic activation induced by ERK2 binding.
Mesh Terms:
Animals, Arginine, Binding Sites, Catalysis, Cloning, Molecular, Dual Specificity Phosphatase 2, Enzyme Activation, Mice, Mitogen-Activated Protein Kinase 1, Mitogen-Activated Protein Kinases, Mutagenesis, Site-Directed, Phosphorylation, Protein Phosphatase 2, Protein Tyrosine Phosphatases, Substrate Specificity
J. Mol. Biol.
Date: Dec. 09, 2005
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