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.
Mitsuhashi
Shinya
S
Division of Biochemical Oncology and Immunology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan.
Shima
Hiroshi
H
Tanuma
Nobuhiro
N
Matsuura
Nobuyasu
N
Takekawa
Mutsuhiro
M
Urano
Takeshi
T
Kataoka
Tohru
T
Ubukata
Makoto
M
Kikuchi
Kunimi
K
D000818:Animals; D000935:Antifungal Agents / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism / Q000494:pharmacology; D019556:COS Cells; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005663:Furans; D006801:Humans; D008055:Lipids; D020935:MAP Kinase Signaling System / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*
D000935:Antifungal Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D005663:Furans; D008055:Lipids; D021122:Protein Subunits; C058744:tautomycetin; D019319:Okadaic Acid; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2