Characterization of natural toxins with inhibitory activity against serine/threonine protein phosphatases

Honkanan, Richard E.; Codispoti, Burt A.; Tse, Kathy; Boynton, Alton L.

doi:10.1016/0041-0101(94)90086-8

Cited by 199 publications

(118 citation statements)

References 29 publications

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“…Cultures treated with 1 or 10 nM at days 7, 9, or 11 did not show a dose dependent difference, thus the results in figure 5c are shown only for 10 nM. When Microcystin-LR, which is reported to act like okadaic acid [41,42], was added on day 7, although there was a slight decrease in the rate of mineral accretion, the yield at day 21 was not altered. When added on day 9 the yield was decreased, while the rate of mineral accretion was comparable to that in the controls.…”

Section: Phosphatase Inhibitor Studiesmentioning

confidence: 89%

Modulation of extracellular matrix protein phosphorylation alters mineralization in differentiating chick limb-bud mesenchymal cell micromass cultures

Boskey

Doty

Kudryashov

et al. 2008

Bone

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Protein phosphorylation and dephosphorylation are important regulators of cellular and extracellular events. The purpose of this study was to define how these events regulate cartilage matrix calcification in a cell culture system that mimics endochondral ossification. The presence of casein kinase II (CK2), an enzyme known to phosphorylate matrix proteins, was confirmed by immunohistochemistry. The importance of phosphoprotein phosphorylation and dephosphorylation was examined by comparing effects of inhibiting CK2 or phosphoprotein phosphatases on mineral accretion relative to untreated mineralizing controls. Specific inhibitors were added to differentiating chick limb bud mesenchymal cell micromass cultures during the development of a mineralized matrix at the times of cell differentiation, proliferation, formation of the mineralized matrix, or proliferation of the mineral crystals. The mineralizing media for these cultures contained 4mM inorganic phosphate and no organic-phosphate esters; control cultures had 1mM inorganic phosphate. Mineralization was monitored based on 45 Ca uptake and infrared characterization of the mineral; cell viability was assessed by three independent methods. Treatments that caused cell toxicity were excluded from the analysis.Inhibition of CK2 activity with apigenin or CK2 inhibitor II reduced the rate of mineral deposition, but did not block mineral accretion. Effects were greatest during the time of mineralized matrix formation. Inhibition of phosphoprotein phosphatase activities with okadaic acid, calyculin A, and microcystin LR, at early time points also markedly inhibited mineral accretion. Inhibition after mineralization had commenced increased the mineral yield. Levamisole, an alkaline phosphatase inhibitor, had no effect on mineral accretion in this system, suggesting the involvement of other phosphatases. Adding additional inorganic phosphate to the inhibited cultures after mineralization had started, but not earlier, reversed the inhibition indicating the phosphatases were, in part, providing a source of inorganic phosphate.To characterize the roles of specific phosphoproteins blocking studies were performed. Blocking with anti-osteopontin antibody confirmed osteopontin's previously reported role as a mineralization inhibitor. Blocking antibodies to bone sialoprotein added from day 9 or on days 9 and 11 retarded mineralization, supporting its role as a mineralization nucleator. Antibodies to osteonectin slightly stimulated early mineralization, but had no effect after the time that initial mineral deposition occurs. Taken together, the results of this study demonstrate the importance of the phosphorylation state of extracellular matrix proteins in regulating mineralization in this culture system.

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Section: Phosphatase Inhibitor Studiesmentioning

confidence: 89%

Modulation of extracellular matrix protein phosphorylation alters mineralization in differentiating chick limb-bud mesenchymal cell micromass cultures

Boskey

Doty

Kudryashov

et al. 2008

Bone

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“…To assess the potential involvement of PP2A in the dephosphorylation of CXCR2, we used okadaic acid (OA), a potent cell-permeant inhibitor of PP1 and PP2A (28,29). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Phosphorylation-independent Association of CXCR2 with the Protein Phosphatase 2A Core Enzyme

Fan

Yang

Sai

et al. 2001

Journal of Biological Chemistry

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Protein phosphatase 2A (PP2A) is postulated to be involved in the dephosphorylation of G protein-coupled receptors. In the present study, we demonstrate that the carboxyl terminus of CXCR2 physically interacts with the PP2A core enzyme, a dimer formed by PP2Ac and PR65, but not with the PP2Ac monomer, suggesting direct interaction of the receptor with PR65. The integrity of a sequence motif in the C terminus of CXCR2, KFRHGL, which is conserved in all CC and CXC chemokine receptors, is required for the receptor binding to the PP2A core enzyme. CXCR2 co-immunoprecipitates with the PP2A core enzyme in HEK293 cells and in human neutrophils. Overexpression of dominant negative dynamin 1 (dynamin 1 K44A) in CXCR2-expressing cells blocks the receptor association with the PP2A core enzyme, and an internalization-deficient mutant form of CXCR2 (I323A,L324A) also exhibits impaired association with the PP2A core enzyme, suggesting that the receptor internalization is required for the receptor binding to PP2A. A phosphorylation-deficient mutant of CXCR2 (331T), which has previously been shown to undergo internalization in HEK293 cells, binds to an almost equal amount of the PP2A core enzyme in comparison with the wild-type CXCR2, suggesting that the interaction of the receptor with PP2A is phosphorylation-independent. The dephosphorylation of CXCR2 is reversed by treatment of the cells with okadaic acid. Moreover, pretreatment of the cells with okadaic acid increases basal phosphorylation of CXCR2 and attenuates CXCR2-mediated calcium mobilization and chemotaxis. Taken together, these data indicate that PP2A is involved in the dephosphorylation of CXCR2. We postulate that this interaction results from direct binding of the regulatory subunit A (PR65) of PP2A to the carboxyl terminus of CXCR2 after receptor sequestration and internalization.Chemokines comprise a family of about 50 low molecular weight proteins that mediate inflammatory responses, chemotaxis, immune cell development, and leukocyte homing. These have been classified into C, CC, CXC, and CX3C chemokines, based on the presence and the position of conserved cysteine amino acid residues (1-3). The biological functions of chemokines are mediated through interaction with their cognate receptors, which are members of the G protein-coupled receptor (GPCR) 1 superfamily. Like other members of the GPCR superfamily, the functional status of many chemokine receptors is determined largely by the phosphorylation state (4 -6). Agonist treatment enhances phosphorylation of the receptors by protein kinases, presumably G protein-coupled receptor kinases and protein kinase C, which results in desensitization of the receptors (4,7,8). This phenomenon is common to many hormonal and neurotransmitter signaling systems (9), but the underlying mechanisms are still only partially understood, especially in the case of chemokine receptors. Based on work on several chemokine receptors, the phosphorylated receptor is then internalized via clathrin-coated pits into early endosomes (6, 10 -...

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“…CalA differs also from OA in being more potent for PP1, while CA is less potent than OA for PP2A and PP1. 21,54,55 OA had a molar potency for cell death induction in C3H/10T1/2 Cl 8 cells ( Figure 4A) that was between that of CalA ( Figure 4B) and CA ( Figure 4C). OAR1 and OAR2 were cross-resistant to OA, CalA and CA ( Figure 4).…”

Section: Optimisation Of the Selection Systemmentioning

confidence: 98%

“…94 It was also assayed for capacity to bind [ 125 I] microcystin-YR (present at 5 nM and about 6000 d.p.m.). PP2A, PP1, PP4, and PP5 have high-affinity interaction with microcystins, 19,21,94 and should be detected by the assay. The incubation was routinely carried out over night at 28C, and [ 125 I] microcystin-YR bound to PP was determined by scintillation counting of the macromoleculeassociated [ 125 I] microcystin-YR, as described previously.…”

Section: Determination Of Apoptosismentioning

confidence: 99%

Establishment of okadaic acid resistant cell clones using a cDNA expression library

2001

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The mechanism whereby the universal apoptogen and serine/ threonine phosphatase inhibitor okadaic acid (OA) kills cells, is still unclear. To create a novel tool for probing of OA action, fibroblasts were selectedfor OA-resistance after infection with a retroviral Jurkat T-cell cDNA expression library. Twenty-one cloneswereselected. Twoof these(OAR1,OAR2)werestudied in detail. OAR1 and 2 had each a retrovirally introduced short cDNA, corresponding to a human gene (oar1 and oar2, respectively) with unknown function. Reintroduction of oar1 or oar2 cDNA into wild-type cells reproduced the OA-resistant phenotype. OAR1 and 2 were cross-resistant to other phosphatase inhibitors (calyculin A, cantharidin), but not to staurosporineor microinjectedCytochromec,thus,indicating a disturbance in a limited number of death pathways, upstream or independent of apaf-1/caspases-3/9. The action of OA involved caspase-dependent and caspase-independent components. Both components were less efficient in OAR1 and 2, than in wild-type cells. Subtle differences existed between OAinduced phosphoprotein patterns in wild-type cells, OAR1, and OAR2, indicating that a narrow selection of protein phosphorylation events had been targeted. We propose that the clones have defects in a hitherto non-elucidated signal pathway linking OA-induced protein phosphorylation to initiation of a death execution pathway provided with a caspase-dependent amplification loop. The novel OAresistant cell clones will be used to elucidate the significance for apoptosis of oar1 and 2, their link to altered protein phosphorylation, and the potential link of the latter to initiation of apoptosis. Cell Death and Differentiation (2001) 8, 754 ± 766.

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Characterization of natural toxins with inhibitory activity against serine/threonine protein phosphatases

Cited by 199 publications

References 29 publications

Modulation of extracellular matrix protein phosphorylation alters mineralization in differentiating chick limb-bud mesenchymal cell micromass cultures

Modulation of extracellular matrix protein phosphorylation alters mineralization in differentiating chick limb-bud mesenchymal cell micromass cultures

Phosphorylation-independent Association of CXCR2 with the Protein Phosphatase 2A Core Enzyme

Establishment of okadaic acid resistant cell clones using a cDNA expression library

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