Characterization of the PP2A alpha gene mutation in okadaic acid-resistant variants of CHO-K1 cells.

Shima, Hiroshi; Tohda, Hiroko; Aonuma, Shizu; Nakayasu, Michie; DePaoli‐Roach, Anna A.; Sügimura, Takashi; Nagao, Minako

doi:10.1073/pnas.91.20.9267

Cited by 48 publications

(40 citation statements)

References 25 publications

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“…Ceramide also reportedly activates PP1 (47). Moreover OA penetrates cell membranes rapidly but accumulates slowly, making it difficult to control the intracellular concentration of the compound in vivo (61). Thus, at the elevated concentrations used in these and other studies, OA was incapable of distinguishing between PP2A and other protein phosphatases.…”

Section: Overexpression Of the Sv40 Small T Antigen Prevents The Inhimentioning

confidence: 89%

Regulation of Insulin Action by Ceramide

Stratford

Hoehn

Liu

et al. 2004

Journal of Biological Chemistry

351

118

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The sphingolipid ceramide negatively regulates insulin action by inhibiting Akt/protein kinase B (PKB), a serine/threonine kinase that is a central regulator of glucose uptake and anabolic metabolism. Despite considerable attention, the molecular mechanism accounting for this action of ceramide has remained both elusive and controversial. Herein we utilized deletion constructs encoding two different functional domains of Akt/PKB to identify which region of the enzyme conferred responsiveness to ceramide. Surprisingly the findings obtained with these separate domains reveal that ceramide blocks insulin stimulation of Akt/PKB by two independent mechanisms. First, using the isolated pleckstrin homology domain, we found that ceramide specifically blocks the translocation of Akt/PKB, but not its upstream activator phosphoinositide-dependent kinase-1, to the plasma membrane. Second, using a construct lacking this pleckstrin homology domain, which does not require translocation for activation, we found that ceramide stimulates the dephosphorylation of Akt/ PKB by protein phosphatase 2A. Collectively these findings identify at least two independent mechanisms by which excessive ceramide accumulation in peripheral tissues could contribute to the development of insulin resistance. Moreover the results obtained provide a unifying theory to account for the numerous dissenting reports investigating the actions of ceramide toward Akt/PKB.A strong correlation between intramyocellular lipid levels and the severity of insulin resistance has prompted investigators to hypothesize that insulin resistance results from the ectopic accumulation of fat in tissues other than adipose (1, 2). Specifically, many researchers have speculated that increased availability of lipids to peripheral tissues causes insulin resistance by promoting the accumulation of fat-derived metabolites capable of inhibiting insulin action (1, 2). Numerous recent studies support the hypothesis that the aberrant deposition of the sphingolipid ceramide in skeletal muscle and liver contributes to the development of insulin resistance resulting from lipid oversupply. First, insulin resistant rodents (3, 4) and humans (5, 6) have elevated ceramide levels in their peripheral tissues. Second, exercise training insulin-resistant rodents markedly improves their insulin sensitivity while substantially lowering intramuscular ceramide levels (7). Third, ceramide analogs or agents that induce endogenous ceramide accumulation inhibit insulin signaling and insulin-stimulated glycogen synthesis and glucose uptake (8 -14). Fourth, inhibitors of de novo ceramide synthesis prevent the antagonistic effects of saturated fatty acids on insulin signaling in cultured myotubes (9). The inhibitory effect of ceramides on insulin signaling result, at least partially, from their ability to block the phosphorylation and activation of Akt/protein kinase B, a serine/ threonine kinase that is a central mediator of many insulin effects (10, 15, 16). The studies described herein investigated the m...

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Section: Overexpression Of the Sv40 Small T Antigen Prevents The Inhimentioning

confidence: 89%

Regulation of Insulin Action by Ceramide

Stratford

Hoehn

Liu

et al. 2004

Journal of Biological Chemistry

351

118

View full text Add to dashboard Cite

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“…35,37 In one case, part of the OA resistance was probably due to mutations making PP2A slightly less sensitive to OA, but not to calyculin A. 38 The presently isolated OA-resistant cell clones (OAR1 and 2) were cross-resistant to OA, calyculin A and cantharidin, and the resistance was unaffected by verapamil. This argues against multidrug resistance up-regulation in OAR1 and 2.…”

Section: Discussionmentioning

confidence: 99%

“…Previous attempts to establish OA resistance through natural selection, when cells were exposed to low concentrations of OA for a period of several weeks to months, resulted in only a few cell clones. 35,38 Such a long treatment period predisposes to the accumulation of spontaneous mutations, gene duplications, and gene silencing. These potential problems were minimised with the present approach.…”

Section: Discussionmentioning

confidence: 99%

“…35 ± 37 In one case this was associated with a mutation in PP2A, making this enzyme sub-sensitive to OA. 38 So far, such cell lines have yielded limited information about events downstream of PP inhibition. One reason may be that the prolonged exposure to OA 35,37 may have favoured the induction of multidrug resistance and thereby prevented cells from developing protective alterations in their death signalling pathways.…”

Section: Introductionmentioning

confidence: 99%

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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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“…Known to be associated with FAs (25), the effect of PP2A on DLC1 activity was shown through OA-mediated inhibition (24). Because interactions with phosphatases are highly transient, a catalytically dead mutant of PP2A, PP2A C -CS, was generated on the basis of the conserved cysteine residue (30,31) to trap the PP2A-substrate complex. This mutant exhibited an acute binding to DLC1, peaking at 10 min after EGF stimulation, coinciding with maximal ERK activation ( Fig.…”

Section: Egf Stimulates Rhogap Activity and Phosphorylation Of Dlc1-mentioning

confidence: 99%

Epidermal Growth Factor Activates the Rho GTPase-activating Protein (GAP) Deleted in Liver Cancer 1 via Focal Adhesion Kinase and Protein Phosphatase 2A

Ravi

Kaushik

Ravichandran

et al. 2015

Journal of Biological Chemistry

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Background: DLC1 is a RhoGAP tumor suppressor that inactivates Rho GTPases, but its link to growth factor regulation remains unknown. Results: EGF activates the DLC1 phosphorylation-dephosphorylation cycle via MEK, FAK, and PP2A. Conclusion: EGF regulates the spatiotemporal activation of DLC1 to control cell migration. Significance: Signaling via the MEK/ERK-DLC1-FAK-PP2A quartet could integrate biochemical and mechanical cues to regulate cell dynamics.

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Characterization of the PP2A alpha gene mutation in okadaic acid-resistant variants of CHO-K1 cells.

Cited by 48 publications

References 25 publications

Regulation of Insulin Action by Ceramide

Regulation of Insulin Action by Ceramide

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

Epidermal Growth Factor Activates the Rho GTPase-activating Protein (GAP) Deleted in Liver Cancer 1 via Focal Adhesion Kinase and Protein Phosphatase 2A

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