Rac1 Protein Regulates Glycogen Phosphorylase Activation and Controls Interleukin (IL)-2-dependent T Cell Proliferation

Arrizabalaga, Onetsine; Lacerda, Hadriano M.; Zubiaga, Ana M.; Zugaza, José L.

doi:10.1074/jbc.m111.297804

Cited by 29 publications

(45 citation statements)

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“…Moreover, Rac 1 has also been found to participate in IL-2-induced actin cytoskeleton rearrangement in a murine T cell line (12). More recently, our group reported that Rac 1 binds and activates the glycogen phosphorylase muscle isoform (PYGM) 3 and thus established a novel metabolic pathway that participates actively in IL-2-stimulated cell proliferation in human T cells (13). Signals emanating from a large variety of membrane receptors: growth factor receptors (14,15), G protein-coupled receptors (16,17), and tyrosine kinases-linked receptors such as TCR (5,18), BCR (19,20), and IL2-R (13), actively regulate Rho GTPase effects.…”

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confidence: 99%

“…For transient transfections, cells were cultured in complete RPMI 1640 medium in the absence of IL-2 for 24 h. Thereafter, cells were washed, resuspended in 200 l of serum-free medium, and placed in an electroporation cuvette (0.4 mm, Sigma) containing 10 -20 g of different plasmids, or 15 ng of esiRNAs. Electroporation was carried out in a Gene Pulser Xcell Electroporator (Bio-Rad) at 260 V and 950 microfarads (13). The cuvette content was collected into 10 ml of complete RPMI 1640 medium and cultured in the absence of IL-2 for an additional 24 h.…”

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confidence: 99%

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Guanine Nucleotide Exchange Factor αPIX Leads to Activation of the Rac 1 GTPase/Glycogen Phosphorylase Pathway in Interleukin (IL)-2-stimulated T Cells

Llavero

Urzelai

Osinalde

et al. 2015

Journal of Biological Chemistry

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Background: Rac 1 GTPase mediates glycogen phosphorylase activation and controls IL-2-stimulated T cell proliferation. Results: PKC activates ␣PIX by serine phosphorylation and now this Rho-GEF activates Rac 1. Conclusion: IL-2-stimulated T cells migration and proliferation require the involvement of the PKC/␣PIX/Rac 1/PYGM pathway. Significance: This new signaling cascade may be a viable therapeutic target to block the inflammatory response mediated by T cells.

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confidence: 99%

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confidence: 99%

Guanine Nucleotide Exchange Factor αPIX Leads to Activation of the Rac 1 GTPase/Glycogen Phosphorylase Pathway in Interleukin (IL)-2-stimulated T Cells

Llavero

Urzelai

Osinalde

et al. 2015

Journal of Biological Chemistry

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“…In this way, they control a wide variety of cellular processes among which are the actin cytoskeleton reorganization, the formation of integrin complexes, cell adhesion, gene transcription, cell cycle progression, and cell proliferation [4,5,[32][33][34]. In fact, the control of CDKs by the activation of the growth factor-dependent Ras/MAP kinase pathway [35] , were maintained in the serum-free medium for 24 h and stained with PKH26.…”

Section: Discussionmentioning

confidence: 99%

“…This molecular structure and its potential to establish intermolecular interactions result in a complex and flexible platform for both recognition and processing information as well as the transduction of signals to the cell cytosol and the nucleus. B lymphocytes may be maintained in a quiescent state and stimulation of BCR leads to potent activation of signalling molecules, such as the small GTPases of the Ras superfamily [2][3][4][5] mediating the activation of intracellular signalling cascades [2][3][4][5][6][7][8][9]. Briefly, BCR stimulates the activity of both the Syk [10,11] and the Src family of protein tyrosine kinases [12] leading to the activation of the B cell signalosome, which includes the adapter protein BLNK (B cell linker-protein), Bruton's tyrosine kinase (BTK) and phospholipase Cc2 (PLCc2) [13,14].…”

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confidence: 99%

Rac1/p21‐activated kinase pathway controls retinoblastoma protein phosphorylation and E2F transcription factor activation in B lymphocytes

et al. 2016

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Small GTPases of the Ras superfamily are capable of activating E2F-dependent transcription leading to cell proliferation, but the molecular mechanisms are poorly understood. In this study, using immortalized chicken DT40 B cell lines to investigate the role of the Vav/Rac signalling cascade on B cell proliferation, it is shown that the proliferative response triggered by B cell receptor activation is dramatically reduced in the absence of Vav3 expression. Analysis of this proliferative defect shows that in the absence of Vav3 expression, retinoblastoma protein (RB) phosphorylation and the subsequent E2F activation do not take place. By combining pharmacological and genetic approaches, phosphatidylinositol-3-kinase and phospholipase Cc2 (PLCc2) were identified as the key regulatory signalling molecules upstream of the Vav3/Rac pathway leading to RB phosphorylation and E2F transcription factor activation. Additionally, vav3À/À and plcc2 À/À DT40 B cells were not able to activate the RB-E2F complex wild-type phenotype when these genetically modified cells were transfected with constitutively active forms of RhoA or Cdc42. However, when these knockout cells were transfected with different constitutively active versions of PLCc, Vav or Rac1, not only activation of the RB-E2F complex wild-type phenotype was recovered but also the cellular proliferation. Furthermore, by evaluating the effect of two known effector mutants of Rac1 (Rac1 Q61L/F37A and Rac1), the RB-E2F complex activation dependency on p21-activated kinase (PAK) and protein kinase Ce (PKCe) activities was established, being independent of both actin cytoskeleton reorganization and Ras activity. These results suggest that PAK1 and PKCe may be potential therapeutic targets to stop uncontrolled B cell proliferation mediated by the Vav/Rac pathway.Abbreviations BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N 0 ,N 0 -tetraacetic acid tetrakis(acetoxymethyl ester); BCR, B cell receptor; CDK, cyclindependent kinase; DAG, diacylglycerol; GEF, guanine nucleotide exchange factor; PAK, p21-activated kinase; PI3K, phosphatidylinositol-3-kinase; PKC, protein kinase C; PKD, protein kinase D; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate; RasGRP, RAS guanyl releasing protein; RB, retinoblastoma protein.

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“…There were also a number of studies by other groups using Taqman based gene expression analysis with RNA extracted with TRIzol reagent [156][157][158][159][160][161][162] …”

Section: Taqman Gene Expression Assay Using Rna Extracted With Trizolmentioning

confidence: 99%

Technique and method development for intervertebral disc (IVD) research

Lee¹,

李芷恩²

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Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. The direct and indirect cost of managing back pain posts heavy socioeconomic burden to the society. Improved technologies and techniques together with well documented experiments will benefit the research field by saving effort in doing the optimization in every laboratory and avoiding experiment failure due to incomplete understanding of the procedures. These can accelerate scientific discovery, reduce the sacrifice of animals and enable a more effective use of funding.Nucleus pulposus (NP) is the central part of IVD. Differences in matrix compositions in human NP clinical samples demand different cell isolation protocols for optimal results but there is no clear guide about this to date. Sub-optimal protocols may result in low cell yield, limited reliability of results or even failure of experiments. We experimented different isolation protocols to study different parameters involved and suggested some rules for cell isolation in three main applications: RNA extraction for phenotyping, cell isolation for cell culture, and characterization by flow cytometry.In addition, instead of extracting RNA from isolated cells, extraction of RNA from tissues directly may avoid the change of RNA levels during the cell isolation process.However, extraction of RNA directly from human and large animal IVD tissue is technically challenging due to its tough nature, low cell-to-matrix ratio and high proteoglycan content. Thus we developed a method for RNA extraction from bovine disc tissues by integrating the use of cryosectioning, additional phase separation and high salt precipitation into conventional guanidinium thiocyanate based method. With this method, RNA could be extracted from the NP tissue directly but the concentration was low. A shift toward 270 nm was observed in its UV spectrum which was due to phenol contamination. This caused an overestimation of RNA concentration. Hence we developed a computational method based on UV spectra for correcting the In short, different methods related to IVD research were developed and optimized. With improved methods together with a better understanding of the underlying rationale, researchers can save time and cost in their experiments and reduce experiment failure rate. This will help to accelerate researches. New methods also enable studies which were not feasible in the past.(484 words)

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Rac1 Protein Regulates Glycogen Phosphorylase Activation and Controls Interleukin (IL)-2-dependent T Cell Proliferation

Cited by 29 publications

References 71 publications

Guanine Nucleotide Exchange Factor αPIX Leads to Activation of the Rac 1 GTPase/Glycogen Phosphorylase Pathway in Interleukin (IL)-2-stimulated T Cells

Guanine Nucleotide Exchange Factor αPIX Leads to Activation of the Rac 1 GTPase/Glycogen Phosphorylase Pathway in Interleukin (IL)-2-stimulated T Cells

Rac1/p21‐activated kinase pathway controls retinoblastoma protein phosphorylation and E2F transcription factor activation in B lymphocytes

Technique and method development for intervertebral disc (IVD) research

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