Nucleophosmin1 Is a Negative Regulator of the Small GTPase Rac1

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

Rac1 interacts through its C-terminus with NPM1.

<p>(A) Schematic representation of the Rho-like GTPase C-terminal peptides fused to a protein transduction domain as used in this study. (B) Pull-down (PD) experiments were performed using lysates from HeLa cells (upper panel) or Jurkat T-cells (lower panel) with a control peptide, wild-type and mutant Rac1 C-terminal peptides, Rac2, RhoA and RhoG C-terminal peptides. Association of endogenous NPM1 was detected by immunoblotting (IB) with an NPM1 specific monoclonal antibody (representative example out of three independent experiments is shown). (C) Pull-down (PD) experiment was performed using lysates from HeLa cells with a control peptide, wild-type and mutant Rac1 C-terminal peptides. Association of phosphorylated NPM1 (pNPM1) was detected by immunoblotting (IB) with a phospho-specific NPM1 antibody. (representative example out of two independent experiments is shown). (D) Pull-down (PD) experiment using full-length Rac1 and Rac1 lacking the C-terminus (ΔC) both fused to GST or GST alone was performed with lysates from HeLa cells exogenously expressing GFP-NPM1. Association of NPM1 was detected by immunoblotting (IB) with a GFP specific monoclonal antibody. The ponceau staining shows the presence of the different GST constructs. ED: effector domain of Rac1, HV: hypervariable domain of Rac1, PTD: protein transduction domain, Rac1 PPP→AAA, Rac1 RKR→AAA: Rac1 C-terminal peptide mutants where the three prolines, or RKR sequence were replaced by alanine residues, respectively, TCL: total cell lysates, PD: pull-down, IB: immunoblotting.</p

Rac1 activity drives NPM1 out of nucleoli.

<p>HeLa cells were grown on glass cover slips and (co)-transfected with GFP-NPM1 (A) or mCherry Rac1Q61L and GFP-NPM1 (B). After 24 hours, cells were fixed and stained with the nuclear dye DAPI and the F-actin binding toxin Phalloidin fluorescently-labeled with Alxa633. Samples were analyzed by confocal laser scanning microscopy. Higher magnification images of the boxed areas are included. Scale bars, 20 µm.</p

Phospho-NPM1 shows dispersed localization throughout the nucleus.

<p>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.</p

Rac1 activity alters phospho-NPM1 distribution.

<p>HeLa cells were grown on glass cover slips and transfected with either mCherry Rac1Q61L (A) or mCherry Rac1V12G (B). After 24 hours, cells were fixed and stained with a phospho-specific rabbit antibody against NPM1 followed by a goat anti-Rabbit IgG Alexa488, the nuclear dye DAPI and the F-actin binding toxin Phalloidin fluorescently labeled with Alexa 633 and analyzed by confocal laser scanning microscopy. Higher magnification images of the boxed areas are included. (C) Hela cells were transfected with an empty vector, myc-tagged Rac1 WT or two different myc-tagged constitutively active Rac1 mutants; Rac1V12G and Rac1Q61L and after 24 hours lysates were made and endogenous NPM1 as well as phospho-NPM1 (pNPM1) were detected by immunoblotting (IB) with a NPM1 specific or a NPM1 phospho-specific antibody. EV: empty vector. Scale bars, 20 µm.</p

NPM1 is a negative regulator of Rac1.

<p>(A) Rac1 GTP loading was measured by biotinylated Pak-CRIB peptide-based pull-down (PD) with lysates of GFP control-transfected HeLa cells or HeLa cells transfected with GFP-NPM1. Rac1, Rac1 GTP and GFP-NPM1 were detected by immunoblotting with Rac1 and GFP specific monoclonal antibodies respectively. The bar graph shows the relative levels of Rac1 GTP levels compared to that in control as determined by quantification of Western blots (representative example out of three independent experiments is shown). TCL: total cell lysates, PD: pull-down. (B) Cell spreading on fibronectin coated ECIS-electrodes was determined for mock-transfected HeLa cells or HeLa cells expressing GFP-tagged NPM1. The results are depicted as normalized mean resistance of three independent experiments. (n = 3) *p<0.05, **p<0.01. (C) Rac1 GTP loading was measured by biotinylated Pak-CRIB peptide-based pull-down (PD) with lysates of GFP control-transfected HeLa cells or HeLa cells transfected with GFP-NPM1 in the absence or presence of the GEF protein Tiam-C-1199 tagged with HA. Rac1 and Rac1 GTP were detected by immunoblotting with Rac1 specific monoclonal antibody. GFP-NPM1 and Tiam-C-1199-HA were detected by immunoblotting with GFP and HA specific monoclonal antibodies, respectively.</p

Active Rac1 polybasic mutant has decreased capacity to relocalize NPM1.

<p>HeLa cells were grown on glass cover slips and co-transfected with mCherry Rac1Q61L-RKR and GFP-NPM1. After 24 hours, cells were fixed and stained with the nuclear dye DAPI and the F-actin binding toxin Phalloidin fluorescently-labeled with Alexa633. Samples were analyzed by confocal laser scanning microscopy. Higher magnification images of the boxed areas are included. Scale bars, 20 µm. Bar graph represents the percentage of cells (mean±SD) expressing Rac1Q61L-RKR with either NPM1 exclusively in the nucleus or NPM1 in both the nuclear and cytoplasmic compartment (nucleo-cytoplasmic) Fifty cells expressing Rac1Q61L-RKR in two independent experiments were analyzed.</p

Active RhoA does not affect NPM1 localization.

<p>HeLa cells were grown on glass cover slips and co-transfected with HA-tagged active RhoA mutant, RhoAV14, and GFP-NPM1. After 24 hours, cells were fixed and stained with the nuclear dye DAPI and the F-actin binding toxin Phalloidin fluorescently labeled with Alxa633. RhoA was detected by a monoclonal anti-HA antibody followed by an anti-mouse Alexa568 antibody. Samples were analyzed by confocal laser scanning microscopy. Higher magnification images of the boxed areas are included. Scale bars, 20 µm.</p