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.
Zhang
Qiang
Q
Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York University, One Gustave L. Levy Place, New York, NY 10029, USA.
Muller
Michaela
M
Chen
Can Hao
CH
Zeng
Lei
L
Farooq
Amjad
A
Zhou
Ming-Ming
MM
D000818:Animals; D001120:Arginine; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003001:Cloning, Molecular; D054641:Dual Specificity Phosphatase 2; D004789:Enzyme Activation; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D001120:Arginine; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases; D054648:Protein Phosphatase 2; C517032:DUSP2 protein, human; D054641:Dual Specificity Phosphatase 2; C517033:Dusp2 protein, mouse; D017027:Protein Tyrosine Phosphatases