Usage of Tautomycetin, a Novel Inhibitor of Protein Phosphatase 1 (PP1), Reveals That PP1 Is a Positive Regulator of Raf-1 in Vivo

Abstract

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 M 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.Protein phosphatases regulate numerous cellular functions and signal transduction pathways in cooperation with protein kinases (1, 2). Protein phosphatase types 1 and 2A, known as PP1 1 and PP2A, are two of four major protein serine/threonine phosphatases (PPs) that regulate diverse cellular events such as cell division, transcription, translation, muscle contraction, glycogen synthesis, and neuronal signaling (3-5).Okadaic acid (OA), a polyether fatty acid from the marine black sponge Halichondria okadai, was first identified as a small molecular weight inhibitor of PP and has been studied extensively (6). More than 40 compounds that inhibit PP1 as well as PP2A have been identified. Using these natural compounds, numerous experiments have been performed to analyze the roles of PPs in various cellular events (6, 7). The IC 50 values of such phosphatase inhibitors are almost identical for PP1 and PP2A, with the exception of compounds such as OA, TF-23A, and fostriecin (8 -10). PP2A is selectively inhibited by OA, TF-23A, and fostriecin, and this selectivity has made it possible to analyze PP2A function in living cells. However, no known inhibitor inhibits PP1 specifically. Oikawa et al. (11) reported the total chemical synthesis of tautomycin (TM), a small molecular weight PP inhibitor originally isolated from Streptomyces spiroverticillatus. Using the synthesized TM and related compounds, we previously examined the structure-function relationship of TM and found that the left-and right-hand moieties of TM are required for inhibition of PP and induction of apoptosis, respectively (12). We also re...

Regulation of ERK Kinase by MEK1 Kinase Inhibition in the Brain

“…Direct dephosphorylation of Thr-202/Tyr-204 of ERK is conducted by its dedicated phosphatases, such as striatal enriched phosphoprotein (56) and MKP-2 (11). However, consistent with the results presented here, both PP1 and PP2A are thought to indirectly regulate ERK (57,58).…”

Activation of p38 Mitogen-Activated Protein Kinase Is Required for Death Receptor–Independent Caspase-8 Activation and Cell Death in Response to Sphingosine

“…ERKs are activated by phosphorylation of both conserved threonine and tyrosine residues and inactivated on dephosphorylation by specific phosphatases (15)(16)(17)(18)(19)(20). Serine-threonine PP2A can dephosphorylate MEKand ERK-family kinases in vitro (21,22). In this study, we also found that inhibition of PP2A clearly restored sphingosineinduced suppression of MEK and ERK.…”

“…Serine-threonine protein phosphatase 2A (PP2A) dephosphorylates MAPK/ERK kinase (MEK) and ERK family kinases in vitro (19). Moreover, inhibition of PP2A leads to the activation of MEK and ERK (21,22). Recent studies show that ERK activity is down-regulated by serine-threonine phosphatase during p38 MAPK activation induced by arsenite in NIH 3T3 fibroblasts (23).…”

Inactivation of Host Akt/Protein Kinase B Signaling by Bacterial Pore-forming Toxins

“…Both of these phosphatases are inhibited by CA, whereas PP2A is more specifically inhibited by 100 nM OA, and PP1 is more specifically inhibited by tautomycin (TM) (Connor et al, 1999;Mitsuhashi et al, 2003;Chen et al, 2005). Calyculin A treatment of 5637 cells led to substantial increases in pAkt S473 levels, whereas okadaic acid and tautomycin had less dramatic effects ( Figure 4C).…”