Inhibition of PI3K induces Rac Activation and Membrane Ruffling in Proto-Dbl Expressing Cells

Vanni, Cristina; Visco, Vincenzo; Mancini, Patrizia; Parodi, Alessia; Ottaviano, Catherine; Ognibene, Marzia; Manazza, Andrea D.; Retta, Saverio Francesco; Varesio, Luigi; Torrisi, Maria Rosaria; Eva, Alessandra

doi:10.4161/cc.5.22.3445

Cited by 5 publications

(6 citation statements)

References 45 publications

(58 reference statements)

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“…Rac1 activity assay was performed according to our previous study 24. Briefly, Rac1-GTP was pulled down with GST-PAK-PBD agarose, and its activity was examined by western blotting using an anti-Rac1 antibody 22, 24, 36. Western blotting were performed according to our previous papers 24, 37, 38.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Different roles of Rac1 in the acquisition and extinction of methamphetamine-associated contextual memory in the nucleus accumbens

Zhao¹,

Li²,

Liu³

et al. 2019

Theranostics

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Rationale: Repeated methamphetamine (METH) exposure induces long-term cognitive deficits and pathological drug-associated memory that can be disrupted by manipulating memory reconsolidation and extinction. The nucleus accumbens (NAc) is the key region of the brain reward system and predominantly consists of two subtypes of medium spiny neurons (MSNs) based on the expression of D1 or D2 dopamine receptors (D1-MSNs or D2-MSNs). Spine structural plasticity in the NAc is critical for the acquisition, reconsolidation and extinction of drug-associated memory. However, the molecular mechanisms underlying METH-associated memory and spine remodelling in each type of MSNs in the NAc remain unknown. Here, we explored whether Rac1 in the NAc mediates METH-associated contextual memory and spine remodelling.Methods: Pharmacological and genetic manipulations of Rac1 were used to investigate its role during the acquisition, reconsolidation and extinction of METH-associated contextual memory. Recombinant adeno-associated viruses expressing mCherry under the control of the dopamine D1 receptor gene promoter (Drd1-mCherry) or dopamine D2 receptor gene promoter (Drd2-mCherry) were used to specifically label D1-MSNs or D2-MSNs.Results: Using viral-mediated gene transfer, we demonstrated that decreased Rac1 activity was required for the acquisition of METH-associated contextual memory and the METH-induced increase in thin spine density, whereas increased Rac1 signalling was important for the extinction of METH-associated contextual memory and the related elimination of thin spines. Moreover, the increase of dendritic spines was both found in D1-MSNs and D2-MSNs during the acquisition process, but extinction training selectively decreased the spine density in D1-MSNs. Interestingly, Rac1 was responsible for METH-induced spine plasticity in D1-MSNs but not in D2-MSNs. Additionally, we found that microinjection of a Rac1 inhibitor or activator into the NAc was not sufficient to disrupt reconsolidation, and the pharmacological activation of Rac1 in the NAc facilitated the extinction of METH-associated contextual memory. Regarding cognitive memory, decreased Rac1 activity improved the METH-induced impairment in object recognition memory.Conclusion: Our findings indicate that Rac1 plays opposing roles in the acquisition and extinction of METH-associated contextual memory and reveal the cell-specific role of Rac1 in METH-associated spine remodelling, suggesting that Rac1 is a potential therapeutic target for reducing relapse in METH addiction and remediating METH-induced recognition memory impairment.

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Section: Methodsmentioning

confidence: 99%

“…Rac1, a member of the Rho GTPase family of small G proteins, is a crucial mediator of actin dynamics and plays a critical role in regulating spine morphological changes 20-22. Based on accumulating evidence, Rac1 contributes to addiction 23, 24 and diverse forms of learning and memory 5, 25, 26.…”

Section: Introductionmentioning

confidence: 99%

Different roles of Rac1 in the acquisition and extinction of methamphetamine-associated contextual memory in the nucleus accumbens

Zhao¹,

Li²,

Liu³

et al. 2019

Theranostics

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“…Small GTP-binding proteins Rac-1 and Cdc42 activate JNK and p38 (43)(44)(45) and activation of JNK directly correlates with Dbl transforming activity (36). On the other hand, we have shown that activation of p38 in Dbl-expressing cells correlates with inhibition of cell proliferation and induction of cell migration with localized activation of Rac (46). A potential balance of MAPK activities exists in which inhibition of p38 leads to an increase in cell proliferation while preventing cell migration and the activation of p38 leads to a decrease in cell proliferation while inducing cell migration (47).…”

Section: Discussionmentioning

confidence: 82%

“…A potential balance of MAPK activities exists in which inhibition of p38 leads to an increase in cell proliferation while preventing cell migration and the activation of p38 leads to a decrease in cell proliferation while inducing cell migration (47). Activation of Rac by onco-Dbl causes formation of membrane ruffles and cell migration (46) and Dbl transformed cells are characterized by abundant lamellipodia (30). Such morphology was evident in MEFs infected with a Dbl recombinant lentivirus, but disappeared when MEF knock-outs for the ezrin gene were used.…”

Section: Discussionmentioning

confidence: 99%

The Tumor Suppressor Hamartin Enhances Dbl Protein Transforming Activity through Interaction with Ezrin

Ognibene

Vanni

Segalerba

et al. 2011

Journal of Biological Chemistry

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The Rho guanine nucleotide exchange factor (GEF) Dbl binds to the N-terminal region of ezrin, a member of the ERM (ezrin, radixin, moesin) proteins known to function as linkers between the plasma membrane and the actin cytoskeleton. Here we have characterized the interaction between ezrin and Dbl. We show that binding of Dbl with ezrin involves positively charged amino acids within the region of the pleckstrin homology (PH) domain comprised between ␤1 and ␤2 sheets. In addition, we show that Dbl forms a complex with the tuberous sclerosis-1 (TSC-1) gene product hamartin and with ezrin. We demonstrate that hamartin and ezrin are both required for activation of Dbl. In fact, the knock-down of ezrin and hamartin, as well as the expression of a mutant hamartin, unable to bind ezrin, inhibit Dbl transforming and exchange activity. These results suggest that Dbl is regulated by hamartin through association with ezrin.ERMs (ezrin, radixin, moesin) 2 are ubiquitously expressed proteins that serve as membrane-cytoskeleton linkers and control diverse actin-based cellular functions including cell adhesion, cell motility, and protein localization, and participate in signaling pathways (1). ERM proteins share a conserved structure containing two domains: the C-terminal domain, that binds to actin, and the N-terminal FERM domain, which binds to protein targets at the plasma membrane and with several signaling proteins (2-14). In the inactive conformation the C terminus binds to the N terminus, thus preventing both interaction with membrane targets and actin. Activation of ERM proteins causes the release of this intramolecular binding and subsequent association of ERMs with the cytoskeleton and membrane proteins.Two distinct signals have been proposed as effectors of ERM protein activation: the phosphorylation, by Rho-dependent kinase ROCK, of a conserved C-terminal threonine residue, localized in a consensus C-terminal region of ERM proteins (15)(16)(17)(18)(19)(20), and the binding of phosphatidylinositol-4,5-biphosphate (21-23), produced by the activity of phopshatidylinositol 4-phosphate 5-kinase (24, 25), which in turn is stimulated by activated ROCK (26). Thus Rho, upon activation by guanine nucleotide exchange factors, may act as an upstream activator of ERMs.On the other hand, in vitro and in vivo studies have indicated that ERM proteins can act as upstream regulators of Rho GTPases by binding to the Rho GDP dissociation inhibitor (RhoGDI). This association is thought to displace RhoGDI from Rho GTPases, allowing them to be activated by their specific guanine nucleotide exchange factors (2). In this regard, a functional dependence of Rho GEFs on ezrin has been shown (27) and the association of ERM proteins with Rho GEF Dbl has been demonstrated (28 -30). Moreover, association of ezrin with a novel GEF that activates the small GTPase RhoG has been reported (8). Therefore, ERM proteins may act as upstream activators of Rho GTPases not only through their association with Rho GDI but also through their interaction with R...

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“…51 Briefly, glutathione-coupled Sepharose 4B beads bound to recombinant GST-PAK CRIB domain fusion protein were incubated with cell extracts at 4 C for 45 min, eluted in Laemmli buffer and analyzed for the presence of Rac1 and Cdc42 by Western blot.…”

Section: In Vivo Rho Gtpases Activation Assaymentioning

confidence: 99%

Dbl oncogene expression in MCF-10 A epithelial cells disrupts mammary acinar architecture, induces EMT and angiogenic factor secretion

et al. 2015

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The proteins of the Dbl family are guanine nucleotide exchange factors (GEFs) of Rho GTPases and are known to be involved in cell growth regulation. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders, neoplastic transformation, and tumor metastasis. We have previously demonstrated that expression of Dbl oncogene in lens epithelial cells modulates genes encoding proteins involved in epithelialmesenchymal-transition (EMT) and induces angiogenesis in the lens. Our present study was undertaken to investigate the role of Dbl oncogene in epithelial cells transformation, providing new insights into carcinoma progression.To assess how Dbl oncogene can modulate EMT, cell migration, morphogenesis, and expression of pro-apoptotic and angiogenic factors we utilized bi-and 3-dimensional cultures of MCF-10 A cells. We show that upon Dbl expression MCF-10 A cells undergo EMT. In addition, we found that Dbl overexpression sustains Cdc42 and Rac activation inducing morphological alterations, characterized by the presence of lamellipodia and conferring a high migratory capacity to the cells. Moreover, Dbl expressing MCF-10 A cells form altered 3D structures and can induce angiogenesis by producing proangiogenic factors such as CCL2. These results support a role for Dbl oncogene in epithelial cell differentiation and transformation and suggest the relevance of GEF deregulation in tumor onset and progression.

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Inhibition of PI3K induces Rac Activation and Membrane Ruffling in Proto-Dbl Expressing Cells

Cited by 5 publications

References 45 publications

Different roles of Rac1 in the acquisition and extinction of methamphetamine-associated contextual memory in the nucleus accumbens

Different roles of Rac1 in the acquisition and extinction of methamphetamine-associated contextual memory in the nucleus accumbens

The Tumor Suppressor Hamartin Enhances Dbl Protein Transforming Activity through Interaction with Ezrin

Dbl oncogene expression in MCF-10 A epithelial cells disrupts mammary acinar architecture, induces EMT and angiogenic factor secretion

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