The interaction of SV40 small tumor antigen with protein phosphatase 2A stimulates the map kinase pathway and induces cell proliferation

ABSTRACT Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase that carries out multiple functions. Although numerous observations suggest that PP2A plays a major role in downregulation of the mitogen-activated protein (MAP) kinase pathway, the precise mechanisms are unknown. To clarify the role of PP2A in growth factor (insulin, epidermal growth factor [EGF], and insulin-like growth factor 1 [IGF-1]) stimulation of the Ras/MAP kinase pathway, simian virus 40 small t antigen was expressed in Rat-1 fibroblasts which overexpress insulin receptors. Small t antigen is known to specifically inhibit PP2A by binding to the A PP2A regulatory subunit, interfering with the ability of PP2A to bind to its cellular substrates. Overexpressed small t protein was coimmunoprecipitated with PP2A and inhibited cellular PP2A activity but did not inhibit protein phosphatase 1 (PP1) activity. Insulin, IGF-1, and EGF stimulation also inhibited PP2A activity. Growth factor-stimulated Ras, Raf-1, MAP kinase, and mitogen-activated extracellular-signal-regulated kinase kinase (MEK) activities were elevated in small-t-antigen-expressing cells. Furthermore, Shc tyrosine phosphorylation and its association with Grb2 were also elevated in small-t-antigen-expressing cells. Expression levels of Shc, Ras, MEK, or MAP kinase and phosphorylation of insulin, EGF, and IGF-1 receptors were not altered. Interestingly, we found that PP2A associated with Shc in the basal state and dissociated in response to insulin and EGF and that this dissociation was inhibited by 65% in small-t-antigen-expressing cells. In addition, we found that PP2A associates with the phosphotyrosine-binding domain (PTB domain) of Shc and that phosphorylation of tyrosine 317 of Shc was required for PP2A-Shc dissociation. We conclude (i) that PP2A negatively regulates the Ras/MAP kinase pathway by binding to Shc, inhibiting tyrosine phosphorylation; (ii) that the Shc-PP2A association is mediated by the Shc PTB domain but the interaction is independent of phosphotyrosine binding, indicating a new molecular function for the PTB domain; (iii) that growth factor stimulation, or small-t-antigen expression, causes dissociation of the PP2A-Shc complex, facilitating Shc phosphorylation and downstream activations of the Ras/MAP kinase pathway; and (iv) that this defines a new mechanism of small-t-antigen action to promote mitogenesis.

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Constitutive Cell Proliferation Regulating Inhibitor of Protein Phosphatase 2A (CIP2A) Mediates Drug Resistance to Erlotinib in an EGFR Activating Mutated NSCLC Cell Line

Cells ◽

10.3390/cells10040716 ◽

2021 ◽

Vol 10 (4) ◽

pp. 716

Author(s):

Hisham Saafan ◽

Ahmad Alahdab ◽

Robin Michelet ◽

Linus Gohlke ◽

Janine Ziemann ◽

...

Keyword(s):

Cell Proliferation ◽

Drug Resistance ◽

Cell Line ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Nsclc Cell ◽

Egfr Mutations ◽

Nsclc Cell Line ◽

Acquired Drug Resistance ◽

Phosphatase 2A

Exploring mechanisms of drug resistance to targeted small molecule drugs is critical for an extended clinical benefit in the treatment of non-small cell lung cancer (NSCLC) patients carrying activating epidermal growth factor receptor (EGFR) mutations. Here, we identified constitutive cell proliferation regulating inhibitor of protein phosphatase 2A (CIP2A) in the HCC4006rErlo0.5 NSCLC cell line adapted to erlotinib as a model of acquired drug resistance. Constitutive CIP2A resulted in a constitutive activation of Akt signaling. The proteasome inhibitor bortezomib was able to reduce CIP2A levels, which resulted in an activation of protein phosphatase 2A and deactivation of Akt. Combination experiments with erlotinib and bortezomib revealed a lack of interaction between the two drugs. However, the effect size of bortezomib was higher in HCC4006rErlo0.5, compared to the erlotinib-sensitive HCC4006 cells, as indicated by an increase in Emax (0.911 (95%CI 0.867–0.954) vs. 0.585 (95%CI 0.568–0.622), respectively) and decrease in EC50 (52.4 µM (95%CI 46.1–58.8 µM) vs. 73.0 µM (95%CI 60.4–111 µM), respectively) in the concentration–effect model, an earlier onset of cell death induction, and a reduced colony surviving fraction (0.38 ± 0.18 vs. 0.95 ± 0.25, respectively, n = 3, p < 0.05). Therefore, modulation of CIP2A with bortezomib could be an interesting approach to overcome drug resistance to erlotinib treatment in NSCLC.

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Extracellular Zinc Triggers ERK-dependent Activation of Na+/H+Exchange in Colonocytes Mediated by the Zinc-sensing Receptor

Journal of Biological Chemistry ◽

10.1074/jbc.m406581200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 51804-51816 ◽

Cited By ~ 73

Author(s):

Hagit Azriel-Tamir ◽

Haleli Sharir ◽

Betty Schwartz ◽

Michal Hershfinkel

Keyword(s):

Cell Proliferation ◽

Map Kinase ◽

Ph Homeostasis ◽

Ht29 Cells ◽

Intracellular Release ◽

Map Kinase Pathway ◽

Zinc Sensing ◽

Multiple Signaling ◽

Kinase Pathway

Extracellular zinc promotes cell proliferation and its deficiency leads to impairment of this process, which is particularly important in epithelial cells. We have recently characterized a zinc-sensing receptor (ZnR) linking extracellular zinc to intracellular release of calcium. In the present study, we addressed the role of extracellular zinc, acting via the ZnR, in regulating the MAP kinase pathway and Na+/H+exchange in colonocytes. We demonstrate that Ca2+release, mediated by the ZnR, induces phosphorylation of ERK1/2, which is highly metal-specific, mediated by physiological concentrations of extracellular Zn2+but not by Cd2+, Fe2+, Ni2+, or Mn2+. Desensitization of the ZnR by Zn2+, is followed by ∼90% inhibition of the Zn2+-dependent ERK1/2 phosphorylation, indicating that the ZnR is a principal link between extracellular Zn2+and ERK1/2 activation. Application of both the IP3pathway and PI 3-kinase antagonists largely inhibited Zn2+-dependent ERK1/2 phosphorylation. The physiological significance of the Zn2+-dependent activation of ERK1/2 was addressed by monitoring Na+/H+exchanger activity in HT29 cells and in native colon epithelium. Preincubation of the cells with zinc was followed by robust activation of Na+/H+exchange, which was eliminated by cariporide (0.5 μm); indicating that zinc enhances the activity of NHE1. Activation of NHE1 by zinc was totally blocked by the ERK1/2 inhibitor, U0126. Prolonged acidification, in contrast, stimulates NHE1 by a distinct pathway that is not affected by extracellular Zn2+or inhibitors of the MAP kinase pathway. Desensitization of ZnR activity eliminates the Zn2+-dependent, but not the prolonged acidification-dependent activation of NHE1, indicating that Zn2+-dependent activation of H+extrusion is specifically mediated by the ZnR. Our results support a role for extracellular zinc, acting through the ZnR, in regulating multiple signaling pathways that affect pH homeostasis in colonocytes. Furthermore activation of both, ERK and NHE1, by extracellular zinc may provide the mechanism linking zinc to enhanced cell proliferation.

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NK Cell Proliferation and Cytokine Production Depends on the mTOR- and Calcineurin-Pathway but not p38 MAP-Kinase Pathway

Transplantation Journal ◽

10.1097/00007890-201211271-02223 ◽

2012 ◽

Vol 94 (10S) ◽

pp. 1119

Author(s):

C. Neudörfl ◽

C. S. Falk

Keyword(s):

Cell Proliferation ◽

Map Kinase ◽

Cytokine Production ◽

Nk Cell ◽

P38 Map Kinase ◽

Map Kinase Pathway ◽

Kinase Pathway ◽

Calcineurin Pathway

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Protein Phosphatase 2A Suppresses MAP Kinase Signalling and Ectopic Protein Expression

Cellular Signalling ◽

10.1016/s0898-6568(99)00033-9 ◽

1999 ◽

Vol 11 (8) ◽

pp. 575-580 ◽

Cited By ~ 36

Author(s):

Heekyoung Chung ◽

David L. Brautigan

Keyword(s):

Protein Expression ◽

Map Kinase ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Phosphatase 2A

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Association of protein phosphatase 2A with polyoma virus medium tumor antigen.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.87.7.2521 ◽

1990 ◽

Vol 87 (7) ◽

pp. 2521-2525 ◽

Cited By ~ 105

Author(s):

G. Walter ◽

R. Ruediger ◽

C. Slaughter ◽

M. Mumby

Keyword(s):

Tumor Antigen ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Polyoma Virus ◽

Phosphatase 2A

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Protein Phosphatase 2A Negatively Regulates Insulin's Metabolic Signaling Pathway by Inhibiting Akt (Protein Kinase B) Activity in 3T3-L1 Adipocytes

Molecular and Cellular Biology ◽

10.1128/mcb.24.19.8778-8789.2004 ◽

2004 ◽

Vol 24 (19) ◽

pp. 8778-8789 ◽

Cited By ~ 157

Author(s):

Satoshi Ugi ◽

Takeshi Imamura ◽

Hiroshi Maegawa ◽

Katsuya Egawa ◽

Takeshi Yoshizaki ◽

...

Keyword(s):

Protein Kinase ◽

Signaling Pathway ◽

Glucose Transport ◽

Protein Phosphatase ◽

T Antigen ◽

Metabolic Signaling ◽

Phosphatase 2A ◽

Map Kinase Pathway ◽

Small T Antigen ◽

Kinase Pathway

ABSTRACT Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase which has multiple functions, including inhibition of the mitogen-activated protein (MAP) kinase pathway. Simian virus 40 small t antigen specifically inhibits PP2A function by binding to the PP2A regulatory subunit, interfering with the ability of PP2A to associate with its cellular substrates. We have reported that the expression of small t antigen inhibits PP2A association with Shc, leading to augmentation of insulin and epidermal growth factor-induced Shc phosphorylation with enhanced activation of the Ras/MAP kinase pathway. However, the potential involvement of PP2A in insulin's metabolic signaling pathway is presently unknown. To assess this, we overexpressed small t antigen in 3T3-L1 adipocytes by adenovirus-mediated gene transfer and found that the phosphorylation of Akt and its downstream target, glycogen synthase kinase 3β, were enhanced both in the absence and in the presence of insulin. Furthermore, protein kinase C λ (PKC λ) activity was also augmented in small-t-antigen-expressing 3T3-L1 adipocytes. Consistent with this result, both basal and insulin-stimulated glucose uptake were enhanced in these cells. In support of this result, when inhibitory anti-PP2A antibody was microinjected into 3T3-L1 adipocytes, we found a twofold increase in GLUT4 translocation in the absence of insulin. The small-t-antigen-induced increase in Akt and PKC λ activities was not inhibited by wortmannin, while the ability of small t antigen to enhance glucose transport was inhibited by dominant negative Akt (DN-Akt) expression and Akt small interfering RNA (siRNA) but not by DN-PKC λ expression or PKC λ siRNA. We conclude that PP2A is a negative regulator of insulin's metabolic signaling pathway by promoting dephosphorylation and inactivation of Akt and PKC λ and that most of the effects of PP2A to inhibit glucose transport are mediated through Akt.

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