Vimentin phosphorylation and assembly are regulated by the small GTPase Rab7a

Cogli, Laura; Progida, Cinzia; Bramato, Roberta; Bucci, Cecilia

doi:10.1016/j.bbamcr.2013.02.024

Cited by 57 publications

(89 citation statements)

References 71 publications

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“…Co-immunoprecipitation of endogenous proteins in HeLa cells was performed using a crosslink immunprecipitation kit (Pierce, Rockford, IL), following the manufacturer's instructions and as described previously (Cogli et al, 2013a).…”

Section: Co-immunoprecipitation Pull-down and Direct Interaction Expmentioning

confidence: 99%

The Rab-interacting lysosomal protein (RILP) regulates vacuolar ATPase acting on the V1G1 subunit

Luca

Cogli

Progida

et al. 2014

Journal of Cell Science

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There was an error published in J. Cell Sci. 127, 2697-2708.In Fig. 2, the V1C1 western blot was inadvertently duplicated in panels A and B. The V1C1 western blot has been replaced with the correct image in panel B in the figure shown below. There are no changes to the figure legend, which is accurate. This error does not affect the conclusions of the study.The authors apologise to the readers for any confusion that this error might have caused. or cells expressing HA-RILP were subjected to western blot analysis using primary antibodies against V1G1, HA, V1C1, V0D1 and tubulin. (B) Cells treated with control siRNA (scr), RILP-specific siRNA (RILPi) or V1G1-specific siRNA (V1G1i) were subjected to western blot analysis using primary antibodies against V1G1, RILP, V1C1, V0D1 and tubulin. (C) Lysates of control HeLa cells or cells expressing HA-RILP, HA-RILPΔC2 or HA-RILPC33 were subjected to western blot analysis using anti-V1G1, anti-HA and anti-tubulin antibodies. (D) Cells treated with control siRNA or RILP-specific siRNA and transfected with HA-RILP, as indicated, were subjected to western blot analysis using anti-V1G1, anti-RILP and anti-tubulin antibodies. (E-G) Real-time PCR was performed on control HeLa cells, or cells expressing HA-RILP (RILP1 and RILP2) or silenced for RILP (RILPi1 and RILPi2), and the amount of RILP (E,F) or of V1G1 (G) was quantified relative to the control GAPDH RNA transcript. (H) Control cells or cells expressing HA-RILP, as indicated, were either untreated or treated with the proteasomal inhibitor MG132. Lysates were then subjected to western blot analysis with antibodies against V1G1, HA and tubulin. (I) Control cells or cells transfected with HA-V1G1 and RILP, as indicated, were either untreated or treated with MG132. Lysates were subjected to western blot analysis using antibodies against RILP, V1G1 and tubulin, and to immunoprecipitation using an anti-HA antibody. Immunoprecipitates were subjected to western blot analysis using an anti-ubiquitin antibody. BSTRACTRab-interacting lysosomal protein (RILP) is a downstream effector of the Rab7 GTPase. GTP-bound Rab7 recruits RILP to endosomal membranes and, together, they control late endocytic traffic, phagosome and autophagosome maturation and are responsible for signaling receptor degradation. We have identified, using different approaches, the V1G1 (officially known as ATP6V1G1) subunit of the vacuolar ATPase (V-ATPase) as a RILP-interacting protein. V1G1 is a component of the peripheral stalk and is fundamental for correct V-ATPase assembly. We show here that RILP regulates the recruitment of V1G1 to late endosomal and lysosomal membranes but also controls V1G1 stability. Indeed, we demonstrate that V1G1 can be ubiquitylated and that RILP is responsible for proteasomal degradation of V1G1. Furthermore, we demonstrate that alterations in V1G1 expression levels impair VATPase activity. Thus, our data demonstrate for the first time that RILP regulates the activity of the V-ATPase through its interaction with V1G1. Given the impor...

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Section: Co-immunoprecipitation Pull-down and Direct Interaction Expmentioning

confidence: 99%

The Rab-interacting lysosomal protein (RILP) regulates vacuolar ATPase acting on the V1G1 subunit

Luca

Cogli

Progida

et al. 2014

Journal of Cell Science

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“…We demonstrated that the interaction is direct and that Rab7a regulates vimentin and peripherin filament assembly [17,18]. Overexpression of Rab7a wt or expression of the constitutively active Rab7a Q67L mutant protein increased vimentin phosphorylation and caused redistribution of vimentin in the soluble fraction, while, coherently, Rab7a-depletion determined a decreased phosphorylation of vimentin and increased the amount of filamentous vimentin [17,18].…”

Section: Introductionmentioning

confidence: 95%

“…In fact, vimentin regulates Rac1 activation while activation of Rac1 induces vimentin phosphorylation and disassembly [51][52][53][54]. Furthermore, we have previously shown that Rab7a interacts with vimentin and that depletion of Rab7a determines a lower phosphorylated state of the head domain of vimentin and a higher abundance of vimentin in the insoluble, filamentous fraction [17]. As both Rab7a and Rac1 regulate vimentin assembly and since it has previously been shown that vimentin filaments are oriented towards the leading edge during migration [55], we tested whether vimentin filament orientation was impaired in Rab7a-depleted cells in a wound healing assay.…”

Section: Rab7a Regulates Vimentin Filaments Orientation During Cell Mmentioning

confidence: 99%

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Rab7a regulates cell migration through Rac1 and vimentin

Margiotta

Progida

Bakke

et al. 2017

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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Rab7a, a small GTPase of the Rab family, is localized to late endosomes and controls late endocytic trafficking. The discovery of several Rab7a interacting proteins revealed that Rab7a function is closely connected to cytoskeletal elements. Indeed, Rab7a recruits on vesicles RILP and FYCO that are responsible for the movement of Rab7a-positive vesicles and/or organelles on microtubule tracks, but also directly interacts with Rac1, a fundamental regulator of actin cytoskeleton, and with peripherin and vimentin, two intermediate filament proteins. Considering all these interactions and, in particular, the fact that Rac1 and vimentin are key factors for cellular motility, we investigated a possible role of Rab7a in cell migration. We show here that Rab7a is needed for cell migration as Rab7a depletion causes slower migration of NCI H1299 cells affecting cell velocity and directness.Rab7a depletion negatively affects adhesion and spreading onto fibronectin substrates, altering β1-integrin activation, localization and intracellular trafficking, and myosin X localization. In fact, Rab7a-depleted cells show 40% less filopodia and active integrin accumulates at the leading edge of migrating cells. Furthermore, Rab7a depletion decreases the amount of active Rac1 but not its abundance and reduces the number of cells with vimentin filaments facing the wound, indicating that Rab7a has a role in the orientation of vimentin filaments during migration. In conclusion, our results demonstrate a key role of Rab7a in the regulation of different aspects of cell migration.

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“…There is accumulating evidence that the organization of intermediate filaments is regulated by phosphorylation. Changes in the phosphorylation of the intermediate filaments have been suggested as a potential mechanism to modulate their assembly and distribution (Steinert et al, 1993;Sin et al, 1998;Valgeirsdóttir et al, 1998;Satelli and Li, 2011;Cogli et al, 2013). Using immunofluorescence analyses, we found that enoxaparin 1 gefitinib treatment redistributed p-vimentin to the perinuclear area.…”

Section: Sensitization Of Lung Cancer Cells By Enoxaparin To Gefitinibmentioning

confidence: 79%

Enoxaparin Sensitizes Human Non–Small-Cell Lung Carcinomas to Gefitinib by Inhibiting DOCK1 Expression, Vimentin Phosphorylation, and Akt Activation

Pan

Duan

et al. 2014

Mol Pharmacol

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Gefitinib is widely used for the treatment of lung cancer in patients with sensitizing epidermal growth factor receptor mutations, but patients tend to develop resistance after an average of 10 months. Low molecular weight heparins, such as enoxaparin, potently inhibit experimental metastasis. This study aimed to determine the potential of combined enoxaparin and gefitinib (enoxaparin 1 gefitinib) treatment to inhibit tumor resistance to gefitinib both in vitro and in vivo. A549 and H1975 cell migration was analyzed in wound closure and Transwell assays. Akt and extracellular signalrelated kinase 1/2 signaling pathways were identified, and a proteomics analysis was conducted using SDS-PAGE/liquid chromatography-tandem mass spectrometry analysis. Molecular interaction networks were visualized using the Cytoscape bioinformatics platform. Protein expression of dedicator of cytokinesis 1 (DOCK1) and cytoskeleton intermediate filament vimentin were identified using an enzyme-linked immunosorbent assay, Western blot, and small interfering RNA transfection of A549 cells. In xenograft A549-luc-C8 tumors in nude mice, enoxaparin 1 gefitinib inhibited tumor growth and reduced lung colony formation compared with gefitinib alone. Furthermore, the combination had stronger inhibitory effects on cell migration than either agent used individually. Additional enoxaparin administration resulted in better effective inhibition of Akt activity compared with gefitinib alone. Proteomics and network analysis implicated DOCK1 as the key node molecule. Western blot verified the effective inhibition of the expression of DOCK1 and vimentin phosphorylation by enoxaparin 1 gefitinib compared with gefitinib alone. DOCK1 knockdown confirmed its role in cell migration, Akt expression, and vimentin phosphorylation. Our data indicate that enoxaparin sensitizes gefitinib antitumor and antimigration activity in lung cancer by suppressing DOCK1 expression, Akt activity, and vimentin phosphorylation.

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Vimentin phosphorylation and assembly are regulated by the small GTPase Rab7a

Cited by 57 publications

References 71 publications

The Rab-interacting lysosomal protein (RILP) regulates vacuolar ATPase acting on the V1G1 subunit

The Rab-interacting lysosomal protein (RILP) regulates vacuolar ATPase acting on the V1G1 subunit

Rab7a regulates cell migration through Rac1 and vimentin

Enoxaparin Sensitizes Human Non–Small-Cell Lung Carcinomas to Gefitinib by Inhibiting DOCK1 Expression, Vimentin Phosphorylation, and Akt Activation

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