Activation of Erk1/Erk2 and transiently increased p53 levels together may account for p21 expression associated with phorbol ester-induced transient growth inhibition in HepG2 cells

Sipeki, Szabolcs; Bander, Erzsébet; Ways, D. Kirk; Farago, Anna F.

doi:10.1016/s0898-6568(01)00229-7

Cited by 13 publications

(4 citation statements)

References 22 publications

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“…It has been reported that TPA causes growth inhibition, migration, and G1 arrest in HepG2 cells, the parent line of C3A (15)(16)(17). Similar findings apply to C3A cells.…”

Section: Effect Of G1 Arrest On Ptdins Synthesissupporting

confidence: 69%

Regulation of de novo phosphatidylinositol synthesis

Nuwayhid

Vega

Walden

et al. 2006

Journal of Lipid Research

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Mechanisms that function to regulate the rate of de novo phosphatidylinositol (PtdIns) synthesis in mammalian cells have not been elucidated. In this study, we characterize the effect of phorbol ester treatment on de novo PtdIns synthesis in C3A human hepatoma cells. Incubation of cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA) initially (1-6 h) results in a decrease in precursor incorporation into PtdIns; however, at later times (18-24 h), a marked increase is observed. TPA-induced glucose uptake from the medium is not required for observation of the stimulation of PtdIns synthesis, because the effect is apparent in glucose-free medium. Inhibition of the activation of arachidonic acid substantially blocks the synthesis of PtdIns but has no effect on the synthesis of phosphatidylcholine (PtdCho). Increasing the concentration of cellular phosphatidic acid by blocking its conversion to diacylglycerol, on the other hand, enhances the synthesis of PtdIns and inhibits the synthesis of PtdCho. The TPA-induced stimulation of PtdIns synthesis is not the result of the concomitant TPA-induced G1 arrest, because G1 arrest induced by mevastatin has no effect on PtdIns synthesis. Inhibition of protein kinase C activity blocks the stimulatory action of TPA on de novo synthesis of PtdIns but has no effect on TPA-induced inhibition.Potential sites of enzymatic regulation are discussed.-Nuwayhid, S. J., M. Vega, P. D. Walden, and M. E. Monaco. Phosphatidylinositol (PtdIns) is an essential acidic phospholipid found in the membranes of mammalian cells (1) as well as on the chromatin (2). A single molecular species composed of stearate (R9) and arachidonate (R99) accounts for z80% of the total PtdIns. There are two pathways for the synthesis of PtdIns: the de novo pathway and the salvage pathway, which converge at the level of phosphatidic acid (PtdOH). The de novo pathway uses dihydroxyacetone phosphate derived from glucose to make glycerol phosphate, which is subsequently acylated at the sn-1 position with stearate and then at the sn-2 position with arachidonate to yield PtdOH. In the salvage pathway, diacylglycerol (DAG), derived from phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ), is converted to PtdOH through the action of DAG kinase. In the first instance, there is a net increase in PtdIns, whereas in the second, there is not. The central element of both pathways is the production of PtdOH. For the salvage pathway, it appears that the conversion of DAG to PtdOH by DAG kinase is stimulated as a result of agonist-induced polyphosphoinositide turnover. Whether this results from an increase in DAG substrate and/or an increase in DAG kinase enzyme activity is unclear. This laboratory has previously demonstrated that the two pathways can be differentiated in situ on the basis of the inhibition by triacsin C, a specific inhibitor of arachidonate-specific long-chain fatty acyl-CoA synthetase (ACSL4) in situ (3, 4).Although regulation of the salvage pathway via the activation ...

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“…It has been reported that TPA causes growth inhibition, migration, and G1 arrest in HepG2 cells, the parent line of C3A (15)(16)(17). Similar findings apply to C3A cells.…”

Section: Effect Of G1 Arrest On Ptdins Synthesissupporting

confidence: 69%

Regulation of de novo phosphatidylinositol synthesis

Nuwayhid

Vega

Walden

et al. 2006

Journal of Lipid Research

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“…Many studies have shown that p53 interacts with MAPK [4,26–29]. In mammalian cells, MAPKs are a family of serine/threonine protein kinases that consist of three distinct components, namely, ERKs, JNKs, and p38 kinases.…”

Section: Discussionmentioning

confidence: 99%

Mutant human tumor suppressor p53 modulates the activation of mitogen‐activated protein kinase and nuclear factor‐κB, but not c‐Jun N‐terminal kinase and activated protein‐1

Gulati

Yang

Harter

et al. 2005

Molecular Carcinogenesis

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The roles of the mitogen-activated kinase protein (MAPK) pathway, nuclear factor-kappa B (NF-kappaB), and activator protein-1 (AP-1) in cellular responses to growth factors and mitogen are well established. However, the manner by which these proliferative pathways are affected by the tumor suppressor protein p53 is not fully understood. We report here the results of an investigation of the status of p53 on two human melanoma cell lines with wild-type p53 (SK-Mel-186) or mutant p53 (SK-Mel-110). The basal levels of the activated extracellular-signal regulated kinases 1 and 2 (ERK1/2) were high in cells with wild-type p53, but low in cells with mutant p53. The 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of ERK1/2 through the phosphorylation of threonine and tyrosine at 202 and 204, respectively, was demonstrated in both cell lines, however, in a discrete manner. TPA-induced activation of ERK1/2 was sustained in wild-type p53 cells, while only a transient activation was seen in mutant p53 cells. Inhibition of MAPK kinase (MEK), an upstream kinase, by U0126, blocked TPA-induced activation of ERK1/2 in wild-type p53 cells and in mutant p53 cells. Treatment of wild-type p53 (SK-Mel 186) cells with small interfering RNA (siRNA) of p53 displayed a transient induction of activation of ERK1/2 following TPA treatment, indicating that p53 has a role in the regulation of the activation of ERK1/2. NF-kappaB activity decreased significantly in cells with wild-type p53, while enhanced NF-kappaB activity was evident in cells with mutant p53. The expression of either wild-type or mutant p53 had a similar effect on TPA-induced Jun N-terminal kinase (JNK) activation, indicating specificity for the ERK pathway. Similarly, AP-1 binding activity showed a transient variation in both cell lines after TPA treatment but with different kinetics. These observations suggest that both wild-type and mutant p53 can modulate the activation pathways for ERK1/2, and NF-kappaB distinctively, while modulating the pathways of JNK and AP-1 similarly. These differences may influence cellular processes such as proliferation, differentiation, and apoptosis.

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“…For in vitro phosphorylation, the beads were resuspended in 100 l of protein kinase C reaction buffer (5 mM MgCl 2 , 1 mM dithiothreitol, 2 mM CaCl 2 , 100 M ATP, 1ϫ PKC micelle, 2.5 ng of purified rat brain protein kinase C, and 20 mM TrisHCl, pH 7.5), and incubated for 1 h at 32°C. PKC micelle (10ϫ) was prepared by dissolving 470 g of dried L-␣-phosphatidylserine in 10 l of 10% Triton X-100, 40 l of 20 mM Tris-HCl, pH 7.5, and 0.8 l of 1 mM phorbol 12-myristate 13-acetate (Sipeki et al, 2002). After washing with ice-cold lysis buffer, bound protein was eluted by adding SDS sample buffer and boiling for 3 min, and subjected to immunoblotting.…”

Section: Methodsmentioning

confidence: 99%

Bidirectional Activity-Dependent Regulation of Neuronal Ion Channel Phosphorylation

Misonou¹,

Menegola²,

Mohapatra³

et al. 2006

J. Neurosci.

103

131

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Activity-dependent dephosphorylation of neuronal Kv2.1 channels yields hyperpolarizing shifts in their voltage-dependent activation and homoeostatic suppression of neuronal excitability. We recently identified 16 phosphorylation sites that modulate Kv2.1 function. Here, we show that in mammalian neurons, compared with other regulated sites, such as serine (S)563, phosphorylation at S603 is supersensitive to calcineurin-mediated dephosphorylation in response to kainate-induced seizures in vivo, and brief glutamate stimulation of cultured hippocampal neurons. In vitro calcineurin digestion shows that supersensitivity of S603 dephosphorylation is an inherent property of Kv2.1. Conversely, suppression of neuronal activity by anesthetic in vivo causes hyperphosphorylation at S603 but not S563. Distinct regulation of individual phosphorylation sites allows for graded and bidirectional homeostatic regulation of Kv2.1 function. S603 phosphorylation represents a sensitive bidirectional biosensor of neuronal activity.

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Activation of Erk1/Erk2 and transiently increased p53 levels together may account for p21 expression associated with phorbol ester-induced transient growth inhibition in HepG2 cells

Cited by 13 publications

References 22 publications

Regulation of de novo phosphatidylinositol synthesis

Regulation of de novo phosphatidylinositol synthesis

Mutant human tumor suppressor p53 modulates the activation of mitogen‐activated protein kinase and nuclear factor‐κB, but not c‐Jun N‐terminal kinase and activated protein‐1

Bidirectional Activity-Dependent Regulation of Neuronal Ion Channel Phosphorylation

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