Usage of tautomycetin, a novel inhibitor of protein phosphatase 1 (PP1), reveals that PP1 is a positive regulator of Raf-1 in vivo.
Protein phosphatase type 1 (PP1), together with protein phosphatase 2A (PP2A), is a major eukaryotic serine/threonine protein phosphatase involved in regulation of numerous cell functions. Although the roles of PP2A have been studied extensively using okadaic acid, a well known inhibitor of PP2A, biological analysis of PP1 has remained restricted ... because of lack of a specific inhibitor. Recently we reported that tautomycetin (TC) is a highly specific inhibitor of PP1. To elucidate the biological effects of TC, we demonstrated in preliminary experiments that treatment of COS-7 cells with 5 microm TC for 5 h inhibits endogenous PP1 by more than 90% without affecting PP2A activity. Therefore, using TC as a specific PP1 inhibitor, the biological effect of PP1 on MAPK signaling was examined. First, we found that inhibition of PP1 in COS-7 cells by TC specifically suppresses activation of ERK, among three MAPK kinases (ERK, JNK, and p38). TC-mediated inhibition of PP1 also suppressed activation of Raf-1, resulting in the inactivation of the MEK-ERK pathway. To examine the role of PP1 in regulation of Raf-1, we overexpressed the PP1 catalytic subunit (PP1C) in COS-7 cells and found that PP1C enhanced activation of Raf-1 activity, whereas phosphatase-dead PP1C blocked Raf-1 activation. Furthermore, a physical interaction between PP1C and Raf-1 was also observed. These data strongly suggest that PP1 positively regulates Raf-1 in vivo.
Mesh Terms:
Animals, Antifungal Agents, Bacterial Proteins, COS Cells, Dose-Response Relationship, Drug, Enzyme Activation, Enzyme Inhibitors, Furans, Humans, Lipids, MAP Kinase Signaling System, Mitogen-Activated Protein Kinases, Okadaic Acid, Phosphoprotein Phosphatases, Protein Phosphatase 1, Protein Phosphatase 2, Protein Subunits, Proto-Oncogene Proteins c-raf
Animals, Antifungal Agents, Bacterial Proteins, COS Cells, Dose-Response Relationship, Drug, Enzyme Activation, Enzyme Inhibitors, Furans, Humans, Lipids, MAP Kinase Signaling System, Mitogen-Activated Protein Kinases, Okadaic Acid, Phosphoprotein Phosphatases, Protein Phosphatase 1, Protein Phosphatase 2, Protein Subunits, Proto-Oncogene Proteins c-raf
J. Biol. Chem.
Date: Jan. 03, 2003
PubMed ID: 12374792
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