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Nucleophosmin1 is a negative regulator of the small GTPase Rac1.

Zoughlami Y, van Stalborgh AM, van Hennik PB, Hordijk PL - PLoS ONE (2013)

Bottom Line: We have previously identified several effector proteins of Rac1 that also act as Rac1 regulatory proteins, including caveolin-1 and PACSIN2.Conversely, we found that expression of NPM1 limits Rac1 GTP loading and cell spreading.In conclusion, this study identifies NPM1 as a novel, negative regulator of Rac1.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

ABSTRACT
The Rac1 GTPase is a critical regulator of cytoskeletal dynamics and controls many biological processes, such as cell migration, cell-cell contacts, cellular growth and cell division. These complex processes are controlled by Rac1 signaling through effector proteins. We have previously identified several effector proteins of Rac1 that also act as Rac1 regulatory proteins, including caveolin-1 and PACSIN2. Here, we report that Rac1 interacts through its C-terminus with nucleophosmin1 (NPM1), a multifunctional nucleo-cytoplasmic shuttling protein with oncogenic properties. We show that Rac1 controls NPM1 subcellular localization. In cells expressing active Rac1, NPM1 translocates from the nucleus to the cytoplasm. In addition, Rac1 regulates the localization of the phosphorylated pool of NPM1 as this pool translocated from the nucleus to the cytosol in cells expressing activated Rac1. Conversely, we found that expression of NPM1 limits Rac1 GTP loading and cell spreading. In conclusion, this study identifies NPM1 as a novel, negative regulator of Rac1.

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Phospho-NPM1 shows dispersed localization throughout the nucleus.HeLa cells were grown on glass cover slips and transfected with GFP-NPM1. After 24 hours, cells were fixed and stained with a phospho-specific rabbit antibody against NPM1 followed by a goat anti-Rabbit IgG Alexa568, the nuclear dye DAPI and the F-actin binding toxin Phalloidin fluorescently labeled with Alexa633 and analyzed by confocal laser scanning microscopy. Higher magnification images of the boxed areas are included. Scale bars, 20 µm.
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pone-0068477-g005: Phospho-NPM1 shows dispersed localization throughout the nucleus.HeLa cells were grown on glass cover slips and transfected with GFP-NPM1. After 24 hours, cells were fixed and stained with a phospho-specific rabbit antibody against NPM1 followed by a goat anti-Rabbit IgG Alexa568, the nuclear dye DAPI and the F-actin binding toxin Phalloidin fluorescently labeled with Alexa633 and analyzed by confocal laser scanning microscopy. Higher magnification images of the boxed areas are included. Scale bars, 20 µm.

Mentions: Because NPM1 function is regulated by phosphorylation and because the Rac1 C-terminus associates to phosphorylated NPM1 (pNPM1; Figure 1C), we questioned whether Rac1 activity alters the distribution of pNPM1. First, we used a phospho-specific NPM1 antibody to document the localization of pNPM1. This staining revealed that the phosphorylated fraction of endogenous NPM1 localized markedly different from the total NPM1 pool. Phospho-NPM1 was diffusely dispersed throughout the nucleus and also localized to small dotted structures inside the nucleoplasm (Figure 5).


Nucleophosmin1 is a negative regulator of the small GTPase Rac1.

Zoughlami Y, van Stalborgh AM, van Hennik PB, Hordijk PL - PLoS ONE (2013)

Phospho-NPM1 shows dispersed localization throughout the nucleus.HeLa cells were grown on glass cover slips and transfected with GFP-NPM1. After 24 hours, cells were fixed and stained with a phospho-specific rabbit antibody against NPM1 followed by a goat anti-Rabbit IgG Alexa568, the nuclear dye DAPI and the F-actin binding toxin Phalloidin fluorescently labeled with Alexa633 and analyzed by confocal laser scanning microscopy. Higher magnification images of the boxed areas are included. Scale bars, 20 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3713031&req=5

pone-0068477-g005: Phospho-NPM1 shows dispersed localization throughout the nucleus.HeLa cells were grown on glass cover slips and transfected with GFP-NPM1. After 24 hours, cells were fixed and stained with a phospho-specific rabbit antibody against NPM1 followed by a goat anti-Rabbit IgG Alexa568, the nuclear dye DAPI and the F-actin binding toxin Phalloidin fluorescently labeled with Alexa633 and analyzed by confocal laser scanning microscopy. Higher magnification images of the boxed areas are included. Scale bars, 20 µm.
Mentions: Because NPM1 function is regulated by phosphorylation and because the Rac1 C-terminus associates to phosphorylated NPM1 (pNPM1; Figure 1C), we questioned whether Rac1 activity alters the distribution of pNPM1. First, we used a phospho-specific NPM1 antibody to document the localization of pNPM1. This staining revealed that the phosphorylated fraction of endogenous NPM1 localized markedly different from the total NPM1 pool. Phospho-NPM1 was diffusely dispersed throughout the nucleus and also localized to small dotted structures inside the nucleoplasm (Figure 5).

Bottom Line: We have previously identified several effector proteins of Rac1 that also act as Rac1 regulatory proteins, including caveolin-1 and PACSIN2.Conversely, we found that expression of NPM1 limits Rac1 GTP loading and cell spreading.In conclusion, this study identifies NPM1 as a novel, negative regulator of Rac1.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

ABSTRACT
The Rac1 GTPase is a critical regulator of cytoskeletal dynamics and controls many biological processes, such as cell migration, cell-cell contacts, cellular growth and cell division. These complex processes are controlled by Rac1 signaling through effector proteins. We have previously identified several effector proteins of Rac1 that also act as Rac1 regulatory proteins, including caveolin-1 and PACSIN2. Here, we report that Rac1 interacts through its C-terminus with nucleophosmin1 (NPM1), a multifunctional nucleo-cytoplasmic shuttling protein with oncogenic properties. We show that Rac1 controls NPM1 subcellular localization. In cells expressing active Rac1, NPM1 translocates from the nucleus to the cytoplasm. In addition, Rac1 regulates the localization of the phosphorylated pool of NPM1 as this pool translocated from the nucleus to the cytosol in cells expressing activated Rac1. Conversely, we found that expression of NPM1 limits Rac1 GTP loading and cell spreading. In conclusion, this study identifies NPM1 as a novel, negative regulator of Rac1.

Show MeSH