Nuclear localization of enhanced green fluorescent protein homomultimers

Seibel, Nicole Maria; Eljouni, Jihane; Nalaskowski, Marcus M.; Hampe, Wolfgang

doi:10.1016/j.ab.2007.05.025

Cited by 172 publications

(144 citation statements)

References 24 publications

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“…Moreover, TP was induced by ERK-dependent cytoplasmic hnRNP K accumulation and forced expression of phosphomimetic S284/353D mutant hnRNP K, but not by forced expression of dominant-negative S284/353A mutant hnRNP K. Using EGFP-tagged hnRNP K and its mutant proteins to determine the cellular location, a smaller proportion of cytoplasmic hnRNP K ( Figure 5) as compared to the endogenous protein (Figure 4c) was observed under our experimental conditions. This might be due to the nature of EGFP, which is prone to localize to the nucleus resulting in the overall reduction of cytoplasmic proportion of EGFP-tagged hnRNP K (Seibel et al, 2007). Taken together, our findings indicate that cytoplasmic accumulation of hnRNP K is controlled by ERK signaling in NPC cells, which is critical for the induction of TP expression.…”

Section: Discussionmentioning

confidence: 68%

Thymidine phosphorylase mRNA stability and protein levels are increased through ERK-mediated cytoplasmic accumulation of hnRNP K in nasopharyngeal carcinoma cells

Liu

et al. 2009

Oncogene

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The cytoplasmic level of heterogeneous nuclear ribonucleoprotein K (hnRNP K) is significantly correlated with the elevated expression of thymidine phosphorylase (TP), and high levels of both proteins are predictive of a poor prognosis in nasopharyngeal carcinoma (NPC). We herein show that TP is highly induced by serum deprivation in NPC cells, and that this is due to an increase in the halflife of the TP mRNA, as shown by nuclear run-on and actinomycin D assays. We further show that the CU-rich element of the TP mRNA directly interacts with hnRNP K, as demonstrated by immunoprecipitation RT-PCR assays, and the nucleus-to-cytoplasm translocation of hnRNP K. Blockade of hnRNP K expression reduces TP expression, suggesting that hnRNP K acts in the upregulation of TP. Mechanistically, both MEK inhibitor and the hnRNP K ERK-phosphoacceptor-site mutant decrease cytoplasmic accumulation of hnRNP K, suggesting that ERK-dependent phosphorylation is critical for TP induction. Furthermore, we found that hnRNP Kmediated TP induction allows NPC cells to resist hypoxia-induced apoptosis. Our results collectively establish the regulation and role of ERK-mediated cytoplasmic accumulation of hnRNP K as an upstream modulator of TP, suggesting that hnRNP K may be an attractive candidate as a future therapeutic target for cancer.

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Section: Discussionmentioning

confidence: 68%

Thymidine phosphorylase mRNA stability and protein levels are increased through ERK-mediated cytoplasmic accumulation of hnRNP K in nasopharyngeal carcinoma cells

Liu

et al. 2009

Oncogene

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“…Furthermore, none of these studies appear to have compared the activity and/or function of these constructs to the endogenous protein (Asano et al, 2008;Calleja et al, 2003;Carpten et al, 2007;Cenni et al, 2003;Currie et al, 1999;Du et al, 2014;Feng et al, 2014;Gonzalez and McGraw, 2009;Huang et al, 2011;Imazaki et al, 2010;Kontos et al, 1998;Lasserre et al, 2008;Parikh et al, 2012;Rodríguez-Escudero et al, 2005;Servant et al, 2000;Terashima et al, 2005;Watton and Downward, 1999;Zhang et al, 2009). While GFP remains the most commonly used fluorescent protein, there are a growing number of publications that report fusion protein dysfunction (Goto et al, 2003;Huang and Shusta, 2006;Kalatskaya et al, 2006;Liu et al, 1999;Yantsevich et al, 2009) and mislocalisation (Skube et al, 2010;Zhu et al, 2013), which one group has attributed to the affinity of eGFP for the nucleus (Seibel et al, 2007). Cellular stress responses have also been reported in cultured cells stably expressing GFP (Zhang et al, 2003), and eGFP expression has also been reported to increase production of superoxide and hydrogen peroxide (Ganini et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

An improved Akt reporter reveals intra- and inter-cellular heterogeneity and oscillations in signal transduction

Norris

Yang

Krycer

et al. 2017

Journal of Cell Science

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Akt is a key node in a range of signal transduction cascades and play a critical role in diseases such as cancer and diabetes. Fluorescentlytagged Akt reporters have been used to discern Akt localisation, yet it has not been clear how well these tools recapitulate the behaviour of endogenous Akt proteins. Here, we observed that fusion of eGFP to Akt2 impaired both its insulin-stimulated plasma membrane recruitment and its phosphorylation. Endogenous-like responses were restored by replacing eGFP with TagRFP-T. The improved response magnitude and sensitivity afforded by TagRFP-T-Akt2 over eGFP-Akt2 enabled monitoring of signalling outcomes in single cells at physiological doses of insulin with subcellular resolution and revealed two previously unreported features of Akt biology. In 3T3-L1 adipocytes, stimulation with insulin resulted in recruitment of Akt2 to the plasma membrane in a polarised fashion. Additionally, we observed oscillations in plasma membrane localised Akt2 in the presence of insulin with a consistent periodicity of 2 min. Our studies highlight the importance of fluorophore choice when generating reporter constructs and shed light on new Akt signalling responses that may encode complex signalling information.This article has an associated First Person interview with the first author of the paper.

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“…S1). Small proteins tagged with GFP are known to readily enter the nucleus without the requirement for a nuclear localization signal (38); apparently this is the case for AfrLEA2-GFP, given that bioinformatic algorithms failed to detect a nuclear localization signal (25).…”

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confidence: 99%

Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation

Chakraborty

Borcar

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Expression of late embryogenesis abundant (LEA) proteins is highly correlated with desiccation tolerance in anhydrobiotic animals, selected land plants, and bacteria. Genes encoding two LEA proteins, one localized to the cytoplasm/nucleus (AfrLEA2) and one targeted to mitochondria (AfrLEA3m), were stably transfected into human HepG2 cells. A trehalose transporter was used for intracellular loading of this disaccharide. Cells were rapidly and uniformly desiccated to low water content (<0.12 g H 2 O/g dry weight) with a recently developed spin-drying technique. Immediately on rehydration, control cells without LEA proteins or trehalose exhibited 0% membrane integrity, compared with 98% in cells loaded with trehalose and expressing AfrLEA2 or AfrLEA3m; surprisingly, AfrLEA3m without trehalose conferred 94% protection. Cell proliferation across 7 d showed an 18-fold increase for cells dried with AfrLEA3m and trehalose, compared with 27-fold for nondried controls. LEA proteins dramatically enhance desiccation tolerance in mammalian cells and offer the opportunity for engineering biostability in the dried state.water stress | biopreservation | intrinsically disordered proteins | osmolyte | Artemia franciscana

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Nuclear localization of enhanced green fluorescent protein homomultimers

Cited by 172 publications

References 24 publications

Thymidine phosphorylase mRNA stability and protein levels are increased through ERK-mediated cytoplasmic accumulation of hnRNP K in nasopharyngeal carcinoma cells

Thymidine phosphorylase mRNA stability and protein levels are increased through ERK-mediated cytoplasmic accumulation of hnRNP K in nasopharyngeal carcinoma cells

An improved Akt reporter reveals intra- and inter-cellular heterogeneity and oscillations in signal transduction

Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation

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