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Protein phosphatase 2CK K (PP2CK K) or PP2CL L-1 expressed in COS7 cells suppressed anisomycin-and NaClenhanced phosphorylations of p38 co-expressed in the cells. PP2CK K or PP2CL L-1 expression also suppressed both basal and stress-enhanced phosphorylations of MKK3b and MKK6b, which are upstream protein kinases of p38, and of MKK4, which is one of the major upstream protein kinases of JNK. Basal activity of MKK7, another upstream protein kinase of JNK, was also suppressed by PP2CK K or PP2CL L-1 expression. However, basal as well as serum-activated phosphorylation of MKK1a, an upstream protein kinase of ERKs, was not affected by PP2CL L or PP2CL L-1. A catalytically inactive mutant of PP2CL L-1 further enhanced the NaCl-stimulated phosphorylations of MMK3b, MKK4 and MKK6b, suggesting that this mutant PP2CL L-1 works as a dominant negative form. These results suggest that PP2C selectively inhibits the SAPK pathways through suppression of MKK3b, MKK4, MKK6b and MKK7 activities in mammalian cells.z 1998 Federation of European Biochemical Societies.

Stress-activated protein kinase (SAPK) signaling plays essential roles in eliciting adequate cellular responses to stresses and proinflammatory cytokines. SAPK pathways are composed of three successive protein kinase reactions. The phosphorylation of SAPK signaling components on Ser/Thr or Thr/Tyr residues suggests the involvement of various protein phosphatases in the negative regulation of these systems. Accumulating evidence indicates that three families of protein phosphatases, namely the Ser/Thr phosphatases, the Tyr phosphatases and the dual specificity Ser/Thr/Tyr phosphatases regulate these pathways, each mediating a distinct function. Differences in substrate specificities and regulatory mechanisms for these phosphatases form the molecular basis for the complex regulation of SAPK signaling. Here we describe the properties of the protein phosphatases responsible for the regulation of SAPK signaling pathways.Keywords: stress response; stress-activated protein kinase; protein phosphatase. I N T R O D U C T I O NStress-activated protein kinases (SAPKs), a subfamily of the mitogen-activated protein kinase (MAPK) superfamily, are highly conserved from yeast to mammals. SAPKs relay signals in response to various extracellular stimuli, including environmental stresses and proinflammatory cytokines. In mammalian cells, two distinct classes of SAPKs have been identified, the c-Jun N-terminal kinases (JNK) and the p38 MAPKs [1,2] (Fig. 1).The activation of SAPKs requires phosphorylation of conserved tyrosine and threonine residues within the catalytic domain. This phosphorylation is mediated by dual specificity protein kinases, members of the MAPK kinase (MKK) family. MKK3 and MKK6 are specific for p38, MKK7 selectively phosphorylates JNK, and MKK4 recognizes either class of the stress actived kinases (Fig. 1). The MKKs are also activated by the phosphorylation of conserved serine and threonine residues [1,2]. Several MKK-activating MKK kinases (MKKKs) have been identified, some of which are activated again by phosphorylation [3,4]. In the absence of a signal, the constituents of the SAPK cascade return to their inactive, dephosphorylated state, suggesting an essential role for phosphatases in SAPK regulation.Protein phosphatases are classified into three groups, Ser/Thr phosphatases, Ser/Thr/Tyr phosphatases and Tyr phosphatases, depending on their phosphoamino-acid specificity. The dephosphorylation of SAPK signal pathway components on either Ser/Thr or Thr/Tyr residues requires the participation of various phosphatases. In this article, we first review the roles of protein phosphatases in the regulation of yeast SAPK pathways, then focus on the properties of the protein phosphatases implicated in the mammalian SAPK systems. R E G U L A T I O N O F S A P K S I G N A L P A T H W A Y S B Y P R O T E I N P H O S P H A T A S E S I N Y E A S T C E L L SA molecular genetic analysis of yeast cells indicated that two distinct protein phosphatase groups, protein Tyr phosphatases (PTP) and protein Ser/Thr phosphatases of ...

Osteoclasts are multinucleated cells that differentiate from hematopoietic precursors 1) and possess characteristics to resorb mineralized bone. Osteoclast-like multinucleated cells (OCLs) can be differentiated in vitro from cocultures of mouse bone marrow cells and calvarial osteoblastic cells by treatment with osteotropic factors such as 1a,25-dihydroxyvitamin D 3 , prostaglandin E 2 , interleukin-1 (IL-1) or parathyroid hormone.2,3) Osteoblasts or stromal cells are the target cells for these factors in bone. Recently, an essential factor provided by osteoblasts or stromal cells has been identified and named osteoclast differentiation factor (ODF)/osteoprotegerin ligand (OPGL)/tumor necrosis factor-related activation-induced cytokine (TRANCE)/receptor activator of nuclear factor-kB (NF-kB) ligand (RANKL). 4,5) It has been shown that RANKL induces OCL formation in cultures of bone marrow cells in the presence of M-CSF without requiring osteoblasts or stromal cells.6) RAW cells are also known to differentiate into osteoclasts in the presence of RANKL. ; rats fed onions increased their bone mass. 8) Onion also inhibits bone resorption stimulated in ovariectomized rat, 9,10) and an extract from onion is known to prevent tibial cortical and cancellous bone loss induced by a combination of low protein intake and diet-mediated mild hyperparathyroidism in rats.11) Rutin (quercetin-3-O-glucose rhamnose) ( Fig. 1) has recently been reported to inhibit ovariectomy-stimulated bone resorption in rats.12) These facts taken together suggest that rutin is one of the principal components of onions that effectively facilitate bone resorption, and that its inhibitory effect on bone loss could in part be responsible for its effects on increasing bone mass.Quercetin (Fig. 1) is the major representative of the flavonoid subclass of flavonols commonly found in fruits and vegetables, 13,14) and is also abundant in onion extracts (200-600 mg quercetin/kg onion) and primarily in the form of glycoside (rutin).14) Dietary glycosides like rutin are thought to be converted into aglycone (like quercetin) in the large intestine by the glycosidase activity of intestinal bacteria.15) While these facts suggest that quercetin inhibits bone resorption in animals, its target cells for bone resorption and its mode of action has not been fully elucidated. We investigated the effects of quercetin on the differentiation and activation of osteoclasts, and on bone resorption in cultures. We show here that quercetin inhibits pOC formation induced by sRANKL Although quercetin has suppressed bone resorption in several animal studies, its target cells and the mechanism of its action related to bone resorption has not been fully elucidated. We investigated the effect of quercetin on the differentiation and activation of osteoclasts. We used cocultures of mouse spleen cells and ST2 cells, and cultures of osteoclast progenitor cells {M-CSF-dependent (MD) cells from mouse bone marrow and murine monocytic RAW 264 (RAW) cells}. Quercetin dose-dependently inhibite...

Sanguinarine, a plant alkaloid, was identified as a potent and specific protein phosphatase (PP) 2C inhibitor. It inhibited PP2C competitively with respect to alpha-casein (Ki=0.68 microM) and showed selectivity for PP2C as compared with PP1, PP2A, and PP2B in vitro. In vivo, sanguinarine showed cytotoxicity toward human promyelocytic leukemia cell line HL60, with an IC(50) value of 0.37 microM, and induced apoptosis through a caspase-3/7-dependent mechanism involving the phosphorylation of p38, a PP2Calpha substrate. The apoptosis activity induced by sanguinarine was partially inhibited by a p38 inhibitor, SB203580, and was involved in the phospho-p38 protein in HL60 cells.