scholarly journals A novel function for Cyclin A2: Control of cell invasion via RhoA signaling

2012 ◽  
Vol 196 (1) ◽  
pp. 147-162 ◽  
Author(s):  
Nikola Arsic ◽  
Nawal Bendris ◽  
Marion Peter ◽  
Christina Begon-Pescia ◽  
Cosette Rebouissou ◽  
...  
Keyword(s):  
Colon Adenocarcinoma ◽  
Embryonic Cells ◽  
Nucleotide Exchange ◽  
Rhoa Activity ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Primary Colon ◽  
Cyclin A2 ◽  
Rhoa Signaling

Cyclin A2 plays a key role in cell cycle regulation. It is essential in embryonic cells and in the hematopoietic lineage yet dispensable in fibroblasts. In this paper, we demonstrate that Cyclin A2–depleted cells display a cortical distribution of actin filaments and increased migration. These defects are rescued by restoration of wild-type Cyclin A2, which directly interacts with RhoA, or by a Cyclin A2 mutant unable to associate with Cdk. In vitro, Cyclin A2 potentiates the exchange activity of a RhoA-specific guanine nucleotide exchange factor. Consistent with this, Cyclin A2 depletion enhances migration of fibroblasts and invasiveness of transformed cells via down-regulation of RhoA activity. Moreover, Cyclin A2 expression is lower in metastases relative to primary colon adenocarcinoma in matched human tumors. All together, these data show that Cyclin A2 negatively controls cell motility by promoting RhoA activation, thus demonstrating a novel Cyclin A2 function in cytoskeletal rearrangements and cell migration.

scholarly journals Coordinated RhoA signaling at the leading edge and uropod is required for T cell transendothelial migration

2010 ◽  
Vol 190 (4) ◽  
pp. 553-563 ◽  
Author(s):  
Sarah J. Heasman ◽  
Leo M. Carlin ◽  
Susan Cox ◽  
Tony Ng ◽  
Anne J. Ridley
Keyword(s):  
T Cells ◽  
T Cell ◽  
Rho Gtpases ◽  
Leading Edge ◽  
Transendothelial Migration ◽  
Nucleotide Exchange ◽  
Rhoa Activity ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Rhoa Signaling

Transendothelial migration (TEM) is a tightly regulated process whereby leukocytes migrate from the vasculature into tissues. Rho guanosine triphosphatases (GTPases) are implicated in TEM, but the contributions of individual Rho family members are not known. In this study, we use an RNA interference screen to identify which Rho GTPases affect T cell TEM and demonstrate that RhoA is critical for this process. RhoA depletion leads to loss of migratory polarity; cells lack both leading edge and uropod structures and, instead, have stable narrow protrusions with delocalized protrusions and contractions. By imaging a RhoA activity biosensor in transmigrating T cells, we find that RhoA is locally and dynamically activated at the leading edge, where its activation precedes both extension and retraction events, and in the uropod, where it is associated with ROCK-mediated contraction. The Rho guanine nucleotide exchange factor (GEF) GEF-H1 contributes to uropod contraction but does not affect the leading edge. Our data indicate that RhoA activity is dynamically regulated at the front and back of T cells to coordinate TEM.

scholarly journals In Vitro Formation of Recycling Vesicles from Endosomes Requires Adaptor Protein-1/Clathrin and Is Regulated by Rab4 and the Connector Rabaptin-5

2004 ◽  
Vol 15 (11) ◽  
pp. 4990-5000 ◽  
Author(s):  
Adriana Pagano ◽  
Pascal Crottet ◽  
Cristina Prescianotto-Baschong ◽  
Martin Spiess
Keyword(s):  
Brefeldin A ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Vesicle Formation ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Vitro Assay ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and γ-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5.

The Leukemia-Associated Rho Guanine Nucleotide Exchange Factor (LARG) Is Essential for Survival and Proliferation of AML1-ETO Associated AML Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 923-923
Author(s):  
Shuhong Shen ◽  
Yi Zheng ◽  
James C. Mulloy
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Fusion Gene ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Rhoa Signaling

Abstract Abstract 923 AML1-ETO (AE), the product of translocation 8;21, is the most frequently observed fusion gene in acute myeloid leukemia (AML). Although AE is not sufficient to induce leukemia by itself, it endows a significant survival and growth advantage to hematopoietic progenitors. In a search of various AML microarray databases, we found that the Leukemia-Associated Rho Guanine nucleotide exchange factor (LARG) gene expression is consistently upregulated in samples from AE-positive AML patients compared to other types of AML. This observation was confirmed in an independent cohort of AML samples by qPCR and Western Blot analysis, and in a pre-leukemia cell model generated by AE expression in CD34+ human cord blood (CB) cells compared with CB cells transduced with the MLL-AF9 (MA9) fusion gene or control retroviral vector. ChIP-PCR assays further indicate that AE directly binds to the LARG regulatory region through cis-elements containing AML1 (Runx1) binding sites. LARG, a RhoA GEF, can activate the RhoA pathway which is important for interaction between cells and their environment. We found that the AE cell lines were more adhesive to fibronectin than the MA9 cell lines. When we used lentiviral vectors expressing shRNA to suppress LARG expression in AE+ pre-leukemic cells, the cells showed a significant decrease in adhesion to fibronectin. In addition, knockdown of LARG also significantly interfered with the growth of AE cells in that the shRNA transduced cells displayed a competitive disadvantage relative to non-transduced or control-transduced cells in a proliferation assay. Cell cycle analysis revealed that LARG knockdown induced cell cycle exit, while Annexin V staining showed that LARG suppression promoted apoptosis of AE cells. Because LARG is a well-known guanine nucleotide exchange factor (GEF) for RhoA GTPase, we examined the downstream signaling events of the LARG/Rho axis, including phosphorylation of MLC and AURORA, targets of Rho-associated protein kinase (ROCK). pMLC and pAURORA were significantly down-regulated upon knockdown of LARG. Phosphorylation of Stat3, another protein downstream of RhoA signaling important for the proliferation and survival of myeloid cells, was also decreased upon LARG knockdown. These findings suggest that LARG is a transcriptional target of the AML1-ETO fusion protein and the LARG-RhoA signaling pathway plays an essential role in the proliferation and survival of AE cells. The LARG/RhoA pathway may serve as a new therapeutic target in t(8;21) AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Novel Functions of Ect2 in Polar Lamellipodia Formation and Polarity Maintenance during “Contractile Ring-Independent” Cytokinesis in Adherent Cells

2008 ◽  
Vol 19 (1) ◽  
pp. 8-16 ◽  
Author(s):  
Masamitsu Kanada ◽  
Akira Nagasaki ◽  
Taro Q.P. Uyeda
Keyword(s):  
Mammalian Cells ◽  
Polar Regions ◽  
Nucleotide Exchange ◽  
Contractile Ring ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Ht1080 Cells ◽  
Lamellipodia Formation

Some mammalian cells are able to divide via both the classic contractile ring-dependent method (cytokinesis A) and a contractile ring-independent, adhesion-dependent method (cytokinesis B). Cytokinesis A is triggered by RhoA, which, in HeLa cells, is activated by the guanine nucleotide-exchange factor Ect2 localized at the central spindle and equatorial cortex. Here, we show that in HT1080 cells undergoing cytokinesis A, Ect2 does not localize in the equatorial cortex, though RhoA accumulates there. Moreover, Ect2 depletion resulted in only modest multinucleation of HT1080 cells, enabling us to establish cell lines in which Ect2 was constitutively depleted. Thus, RhoA is activated via an Ect2-independent pathway during cytokinesis A in HT1080 cells. During cytokinesis B, Ect2-depleted cells showed narrower accumulation of RhoA at the equatorial cortex, accompanied by compromised pole-to-equator polarity, formation of ectopic lamellipodia in regions where RhoA normally would be distributed, and delayed formation of polar lamellipodia. Furthermore, C3 exoenzyme inhibited equatorial RhoA activation and polar lamellipodia formation. Conversely, expression of dominant active Ect2 in interphase HT1080 cells enhanced RhoA activity and suppressed lamellipodia formation. These results suggest that equatorial Ect2 locally suppresses lamellipodia formation via RhoA activation, which indirectly contributes to restricting lamellipodia formation to polar regions during cytokinesis B.

scholarly journals PDZRhoGEF and myosin II localize RhoA activity to the back of polarizing neutrophil-like cells

2007 ◽  
Vol 179 (6) ◽  
pp. 1141-1148 ◽  
Author(s):  
Kit Wong ◽  
Alexandra Van Keymeulen ◽  
Henry R. Bourne
Keyword(s):  
Rho Gtpase ◽  
Myosin Ii ◽  
Nucleotide Exchange ◽  
Dependent Protein Kinase ◽  
Rhoa Activity ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Long Tails ◽  
Dependent Protein ◽  
Atpase Inhibition

Chemoattractants such as formyl-Met-Leu-Phe (fMLP) induce neutrophils to polarize by triggering divergent pathways that promote formation of a protrusive front and contracting back and sides. RhoA, a Rho GTPase, stimulates assembly of actomyosin contractile complexes at the sides and back. We show here, in differentiated HL60 cells, that PDZRhoGEF (PRG), a guanine nucleotide exchange factor (GEF) for RhoA, mediates RhoA-dependent responses and determines their spatial distribution. As with RNAi knock-down of PRG, a GEF-deleted PRG mutant blocks fMLP-dependent RhoA activation and causes neutrophils to exhibit multiple fronts and long tails. Similarly, inhibition of RhoA, a Rho-dependent protein kinase (ROCK), or myosin II produces the same morphologies. PRG inhibition reduces or mislocalizes monophosphorylated myosin light chains in fMLP-stimulated cells, and myosin II ATPase inhibition reciprocally disrupts normal localization of PRG. We propose a cooperative reinforcing mechanism at the back of cells, in which PRG, RhoA, ROCK, myosin II, and actomyosin spatially cooperate to consolidate attractant-induced contractility and ensure robust cell polarity.

scholarly journals Gef1p, a New Guanine Nucleotide Exchange Factor for Cdc42p, Regulates Polarity inSchizosaccharomyces pombe

2003 ◽  
Vol 14 (1) ◽  
pp. 313-323 ◽  
Author(s):  
Pedro M. Coll ◽  
Yadira Trillo ◽  
Amagoia Ametzazurra ◽  
Pilar Perez
Keyword(s):  
Cell Morphology ◽  
Rho Gtpases ◽  
Genetic Interaction ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Schizosaccharomyces pombe cdc42+regulates cell morphology and polarization of the actin cytoskeleton. Scd1p/Ral1p is the only described guanine nucleotide exchange factor (GEF) for Cdc42p in S. pombe. We have identified a new GEF, named Gef1p, specifically regulating Cdc42p. Gef1p binds to inactive Cdc42p but not to other Rho GTPases in two-hybrid assays. Overexpression of gef1+increases specifically the GTP-bound Cdc42p, and Gef1p is capable of stimulating guanine nucleotide exchange of Cdc42p in vitro. Overexpression ofgef1+causes changes in cell morphology similar to those caused by overexpression of the constitutively active cdc42G12V allele. Gef1p localizes to the septum. gef1+deletion is viable but causes a mild cell elongation and defects in bipolar growth and septum formation, suggesting a role for Gef1p in the control of cell polarity and cytokinesis. The double mutant gef1Δ scd1Δ is not viable, indicating that they share an essential function as Cdc42p activators. However, both deletion and overexpression of either gef1+orscd1+causes different morphological phenotypes, which suggest different functions. Genetic evidence revealed a link between Gef1p and the signaling pathway of Shk1/Orb2p and Orb6p. In contrast, no genetic interaction between Gef1p and Shk2p-Mkh1p pathway was observed.

scholarly journals Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1

2003 ◽  
Vol 160 (1) ◽  
pp. 17-23 ◽  
Author(s):  
Metello Innocenti ◽  
Emanuela Frittoli ◽  
Isabella Ponzanelli ◽  
John R. Falck ◽  
Saskia M. Brachmann ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Physical Interaction ◽  
Nucleotide Exchange ◽  
Downstream Target ◽  
Signaling Complex ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Phosphoinositide 3 Kinase

Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8–Abi1–Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Here, we show that Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8–Abi1–Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.

The guanine-nucleotide-exchange factor P-Rex1 is activated by protein phosphatase 1α

2012 ◽  
Vol 443 (1) ◽  
pp. 173-183 ◽  
Author(s):  
Mark A. Barber ◽  
Annick Hendrickx ◽  
Monique Beullens ◽  
Hugo Ceulemans ◽  
David Oxley ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Guanine Nucleotide Exchange Factor ◽  
Site Directed Mutagenesis ◽  
Similar Degree ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

P-Rex1 is a GEF (guanine-nucleotide-exchange factor) for the small G-protein Rac that is activated by PIP3 (phosphatidylinositol 3,4,5-trisphosphate) and Gβγ subunits and inhibited by PKA (protein kinase A). In the present study we show that PP1α (protein phosphatase 1α) binds P-Rex1 through an RVxF-type docking motif. PP1α activates P-Rex1 directly in vitro, both independently of and additively to PIP3 and Gβγ. PP1α also substantially activates P-Rex1 in vivo, both in basal and PDGF (platelet-derived growth factor)- or LPA (lysophosphatidic acid)-stimulated cells. The phosphatase activity of PP1α is required for P-Rex1 activation. PP1β, a close homologue of PP1α, is also able to activate P-Rex1, but less effectively. PP1α stimulates P-Rex1-mediated Rac-dependent changes in endothelial cell morphology. MS analysis of wild-type P-Rex1 and a PP1α-binding-deficient mutant revealed that endogenous PP1α dephosphorylates P-Rex1 on at least three residues, Ser834, Ser1001 and Ser1165. Site-directed mutagenesis of Ser1165 to alanine caused activation of P-Rex1 to a similar degree as did PP1α, confirming Ser1165 as a dephosphorylation site important in regulating P-Rex1 Rac-GEF activity. In summary, we have identified a novel mechanism for direct activation of P-Rex1 through PP1α-dependent dephosphorylation.

scholarly journals PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility

2008 ◽  
Vol 180 (1) ◽  
pp. 187-203 ◽  
Author(s):  
Yangmi Lim ◽  
Ssang-Taek Lim ◽  
Alok Tomar ◽  
Margaret Gardel ◽  
Joie A. Bernard-Trifilo ◽  
...  
Keyword(s):  
Cell Motility ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Cellular Contact

Integrin binding to matrix proteins such as fibronectin (FN) leads to formation of focal adhesion (FA) cellular contact sites that regulate migration. RhoA GTPases facilitate FA formation, yet FA-associated RhoA-specific guanine nucleotide exchange factors (GEFs) remain unknown. Here, we show that proline-rich kinase-2 (Pyk2) levels increase upon loss of focal adhesion kinase (FAK) in mouse embryonic fibroblasts (MEFs). Additionally, we demonstrate that Pyk2 facilitates deregulated RhoA activation, elevated FA formation, and enhanced cell proliferation by promoting p190RhoGEF expression. In normal MEFs, p190RhoGEF knockdown inhibits FN-associated RhoA activation, FA formation, and cell migration. Knockdown of p190RhoGEF-related GEFH1 does not affect FA formation in FAK−/− or normal MEFs. p190RhoGEF overexpression enhances RhoA activation and FA formation in MEFs dependent on FAK binding and associated with p190RhoGEF FA recruitment and tyrosine phosphorylation. These studies elucidate a compensatory function for Pyk2 upon FAK loss and identify the FAK–p190RhoGEF complex as an important integrin-proximal regulator of FA formation during FN-stimulated cell motility.

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