Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1
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{"title"=>"Liposome reconstitution and modulation of recombinant prenylated human Rac1 by GEFs, GDI1 and Pak1", "type"=>"journal", "authors"=>[{"first_name"=>"Si Cai", "last_name"=>"Zhang", "scopus_author_id"=>"55537305400"}, {"first_name"=>"Lothar", "last_name"=>"Gremer", "scopus_author_id"=>"6602975391"}, {"first_name"=>"Henrike", "last_name"=>"Heise", "scopus_author_id"=>"56240627000"}, {"first_name"=>"Petra", "last_name"=>"Janning", "scopus_author_id"=>"6506621935"}, {"first_name"=>"Aliaksei", "last_name"=>"Shymanets", "scopus_author_id"=>"34168454900"}, {"first_name"=>"Ion C.", "last_name"=>"Cirstea", "scopus_author_id"=>"14044613800"}, {"first_name"=>"Eberhard", "last_name"=>"Krause", "scopus_author_id"=>"7102585503"}, {"first_name"=>"Bernd", "last_name"=>"Nürnberg", "scopus_author_id"=>"7006609721"}, {"first_name"=>"Mohammad Reza", "last_name"=>"Ahmadian", "scopus_author_id"=>"7005332220"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"25014207", "scopus"=>"2-s2.0-84904245088", "doi"=>"10.1371/journal.pone.0102425", "sgr"=>"84904245088", "pui"=>"373524411"}, "id"=>"0c71008b-a028-31df-acc0-098e430eadef", "abstract"=>"Small Rho GTPases are well known to regulate a variety of cellular processes by acting as molecular switches. The regulatory function of Rho GTPases is critically dependent on their posttranslational modification at the carboxyl terminus by isoprenylation and association with proper cellular membranes. Despite numerous studies, the mechanisms of recycling and functional integration of Rho GTPases at the biological membranes are largely unclear. In this study, prenylated human Rac1, a prominent member of the Rho family, was purified in large amount from baculovirus-infected Spodoptera frugiperda insect cells using a systematic detergent screening. In contrast to non-prenylated human Rac1 purified from Escherichia coli, prenylated Rac1 from insect cells was able to associate with synthetic liposomes and to bind Rho-specific guanine nucleotide dissociation inhibitor 1 (GDI1). Subsequent liposome reconstitution experiments revealed that GDI1 efficiently extracts Rac1 from liposomes preferentially in the inactive GDP-bound state. The extraction was prevented when Rac1 was activated to its GTP-bound state by Rac-specific guanine nucleotide exchange factors (GEFs), such as Vav2, Dbl, Tiam1, P-Rex1 and TrioN, and bound by the downstream effector Pak1. We found that dissociation of Rac1-GDP from its complex with GDI1 strongly correlated with two distinct activities of especially Dbl and Tiam1, including liposome association and the GDP/GTP exchange. Taken together, our results provided first detailed insights into the advantages of the in vitro liposome-based reconstitution system to study both the integration of the signal transducing protein complexes and the mechanisms of regulation and signaling of small GTPases at biological membranes.", "link"=>"http://www.mendeley.com/research/liposome-reconstitution-modulation-recombinant-prenylated-human-rac1-gefs-gdi1-pak1", "reader_count"=>13, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Researcher"=>4, "Student > Ph. D. Student"=>2, "Student > Bachelor"=>3, "Lecturer > Senior Lecturer"=>2}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Researcher"=>4, "Student > Ph. D. Student"=>2, "Student > Bachelor"=>3, "Lecturer > Senior Lecturer"=>2}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Biochemistry, Genetics and Molecular Biology"=>6, "Agricultural and Biological Sciences"=>5, "Computer Science"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>5}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>6}}, "reader_count_by_country"=>{"Canada"=>1, "United States"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1589155"], "description"=>"<p>(<b>A</b>) Pak1 interferes with GDI1 binding to GppNHp-bound Rac1<sup>Ic</sup> and potentiates Rac1<sup>Ic</sup>-GppNHp association with the liposomes. An excess amount of 20-fold of Pak1 was used in liposome sedimentation assay. (<b>B</b>) Competitive inhibition of the GDI1-mediated Rac1<sup>Ic</sup>-GppNHp extraction from the liposomes by Pak1. (<b>C</b>) Tiam1-mediated Rac1<sup>Ic</sup> activation on the liposomes. Liposome-bound Rac1<sup>Ic</sup>-GDP was incubated with Pak1 in the presence or absence of Tiam1-DHPH and an excess of GppNHp. Rac1<sup>Ic</sup> activation on liposome was evaluated by detecting Pak1 in the pellet, which is bound to Tiam1-activated Rac1<sup>Ic</sup>. (<b>D</b>) Tiam1-mediated Rac1<sup>Ic</sup> activation on the liposomes in the presence of GDI1. (<b>E</b>) Partial displacement from the GDI1 complex, activation by Tiam1 and association of Rac1<sup>Ic</sup> with liposomes and Pak1. CBB, coomassie brilliant blue; <i>Ec</i>, <i>E. coli</i>; Ic, insect cells; P, liposome pellet; S, supernatant.</p>", "links"=>[], "tags"=>["Biochemistry", "biomechanics", "biophysics", "molecular biology", "binding", "activated", "counteracting", "extraction", "liposomes"], "article_id"=>1100544, "categories"=>["Biological Sciences"], "users"=>["Si-Cai Zhang", "Lothar Gremer", "Henrike Heise", "Petra Janning", "Aliaksei Shymanets", "Ion C. Cirstea", "Eberhard Krause", "Bernd Nürnberg", "Mohammad Reza Ahmadian"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0102425.g003", "stats"=>{"downloads"=>0, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Pak1_binding_to_activated_Rac1_Ic_counteracting_its_extraction_from_the_liposomes_by_GDI1_/1100544", "title"=>"Pak1 binding to activated Rac1<sup>Ic</sup> counteracting its extraction from the liposomes by GDI1.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-07-11 02:43:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/1589147"], "description"=>"<p>(<b>A</b>) Schematic workflow for the isolation of insect cell membrane fraction, detergent extraction and pull-down assay using GST-GDI1. (<b>B</b>) Effects of eighteen various detergents on Rac1 extraction from the membrane fraction of insect cells (upper panel) and inspection of Rac1 prenylation <i>via</i> pull-down with GST-GDI1 (lower panel). Membrane fractions mixed with two different concentrations (0.5% and 1%) of the respective detergents (Table S1 in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102425#pone.0102425.s001\" target=\"_blank\">File S1</a>) were incubated at room temperature for 30 min, separated in supernatants (S1) and pellets (P1) by centrifugation and immunoblotted using anti-Rac1 antibody. The Supernatants S1 were used in pull-down assays (PD) by using GST-GDI1, which selectively binds to the intact, nucleotide-bound Rac1. Resulted pellets (P2, corresponding to the GSH beads) and supernatant (S2) were visualized by anti-Rac1 antibody in immunoblots. Underlined detergents, especially CHAPS, showed the best properties in the extraction of GDP-bound Rac1 from the insect cell membranes.</p>", "links"=>[], "tags"=>["Biochemistry", "biomechanics", "biophysics", "molecular biology", "optimal", "extraction", "gdp-bound", "rac1"], "article_id"=>1100541, "categories"=>["Biological Sciences"], "users"=>["Si-Cai Zhang", "Lothar Gremer", "Henrike Heise", "Petra Janning", "Aliaksei Shymanets", "Ion C. Cirstea", "Eberhard Krause", "Bernd Nürnberg", "Mohammad Reza Ahmadian"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0102425.g001", "stats"=>{"downloads"=>3, "page_views"=>54, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Detergent_screening_for_optimal_extraction_of_GDP_bound_Rac1_from_the_insect_cell_membrane_/1100541", "title"=>"Detergent screening for optimal extraction of GDP-bound Rac1 from the insect cell membrane.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-07-11 02:43:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/1589154"], "description"=>"<p>(<b>A</b>) GST-GDI1 pull-down of Rac1<sup>Ic</sup> but not of Rac1<i><sup>Ec</sup></i>. Input is the total mixture of beads and proteins, and output is the pull-down (PD). (<b>B</b>) Liposome binding of Rac1<sup>Ic</sup> but not of Rac1<i><sup>Ec</sup></i>. In the liposome sedimentation assay, Rac1<sup>Ic</sup> efficiently binds to liposomes in the absence of GDI1 and independent of whether it was loaded with GDP or GppNHp, a non-hydrolysable GTP analog. Rac1<i><sup>Ec</sup></i> failed to bind to liposomes under the same conditions. (<b>C</b>) Preferential binding Rac1<sup>Ic</sup> to GDI1 than to liposomes. GDI1 binds to both GDP-bound and GppNHp-bound Rac1<sup>Ic</sup> proteins and prevents their association with the liposomes. (<b>D</b>, <b>E</b>) GDI1 efficiently extracted GDP-bound Rac1<sup>Ic</sup> from the liposomes and to a lower extend also Rac1<sup>Ic</sup>-GppNHp. Same amount of GDP-bound and GppNHp-bound forms of Rac1<sup>Ic</sup> associated with the liposomes were prepared before incubation with 5-fold molar excess of GDI1 and sedimentation at 20,000x<i>g</i> (<b>D</b>). Using increasing molar excess of GDI1 (2-, 5-, 10-, 15- and 20-fold) showed that higher concentrations of GDI1 are required to extract Rac1<sup>Ic</sup>-GppNHp from the liposomes to supernatants in comparison to Rac1<sup>Ic</sup>-GDP (<b>E</b>). CBB, coomassie brilliant blue; <i>Ec</i>, <i>E. coli</i>; Ic, insect cells; P, liposome pellet; S, supernatant.</p>", "links"=>[], "tags"=>["Biochemistry", "biomechanics", "biophysics", "molecular biology", "extraction", "liposomes"], "article_id"=>1100542, "categories"=>["Biological Sciences"], "users"=>["Si-Cai Zhang", "Lothar Gremer", "Henrike Heise", "Petra Janning", "Aliaksei Shymanets", "Ion C. Cirstea", "Eberhard Krause", "Bernd Nürnberg", "Mohammad Reza Ahmadian"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0102425.g002", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Nucleotide_independent_extraction_of_Rac1_Ic_from_the_liposomes_by_GDI1_/1100542", "title"=>"Nucleotide-independent extraction of Rac1<sup>Ic</sup> from the liposomes by GDI1.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-07-11 02:43:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/1589157"], "description"=>"<p>(<b>A</b>) Differential binding of Vav2, Dbl, TrioN, Tiam1 and P-Rex1 to various liposomes. Lipo<sup>+PIP2</sup> is composed of PE, PC, PS, SM and PIP2. In Lipo<sup>+PIP3</sup>, PIP2 is replaced by PIP3. (<b>B</b>) Efficient Rac1 activation by Dbl and Vav2 on Lipo<sup>+PIP2</sup>. (<b>C</b>) Displacement from the GDI1 complex, activation by GEFs and association of Rac1<sup>Ic</sup> with liposomes and Pak1. (<b>D</b>) Tiam1 but not Rac1<sup>Ic</sup> binding to Folch III. Rac1GDP, Pak1, Tiam1, GppNHp and different liposomes were preincubated, afterwards the liposome sedimentation assay was conducted. Rac1, PAKα and Tiam1 from the liposome pellet were detected by GST antibody. (<b>E</b>) Rac1<sup>Ic</sup> repulsion by PC but not PS. Liposomes (PS and PE or PC and PE) at increasing amounts of PS and PC were sedimented after incubation with Rac1<sup>Ic</sup>. (F) Thin layer chromatography of Folch I and Folch III liposomes. Relative lipid content of Folch I and Folch III liposomes was analyzed by thin layer chromatography. PE, PS and PC as well as Folch III containing PS and PS were used as controls. CBB, coomassie brilliant blue; <i>Ec</i>, <i>E. coli</i>; Ic, insect cells; P, liposome pellet; S, supernatant.</p>", "links"=>[], "tags"=>["Biochemistry", "biomechanics", "biophysics", "molecular biology", "activation", "liposomes", "dbl", "proteins"], "article_id"=>1100546, "categories"=>["Biological Sciences"], "users"=>["Si-Cai Zhang", "Lothar Gremer", "Henrike Heise", "Petra Janning", "Aliaksei Shymanets", "Ion C. Cirstea", "Eberhard Krause", "Bernd Nürnberg", "Mohammad Reza Ahmadian"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0102425.g004", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Rac1_Ic_activation_on_liposomes_by_different_Dbl_family_proteins_and_association_with_Pak1_/1100546", "title"=>"Rac1<sup>Ic</sup> activation on liposomes by different Dbl family proteins and association with Pak1.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-07-11 02:43:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/1589162"], "description"=>"<div><p>Small Rho GTPases are well known to regulate a variety of cellular processes by acting as molecular switches. The regulatory function of Rho GTPases is critically dependent on their posttranslational modification at the carboxyl terminus by isoprenylation and association with proper cellular membranes. Despite numerous studies, the mechanisms of recycling and functional integration of Rho GTPases at the biological membranes are largely unclear. In this study, prenylated human Rac1, a prominent member of the Rho family, was purified in large amount from baculovirus-infected <i>Spodoptera frugiperda</i> insect cells using a systematic detergent screening. In contrast to non-prenylated human Rac1 purified from <i>Escherichia coli</i>, prenylated Rac1 from insect cells was able to associate with synthetic liposomes and to bind Rho-specific guanine nucleotide dissociation inhibitor 1 (GDI1). Subsequent liposome reconstitution experiments revealed that GDI1 efficiently extracts Rac1 from liposomes preferentially in the inactive GDP-bound state. The extraction was prevented when Rac1 was activated to its GTP-bound state by Rac-specific guanine nucleotide exchange factors (GEFs), such as Vav2, Dbl, Tiam1, P-Rex1 and TrioN, and bound by the downstream effector Pak1. We found that dissociation of Rac1-GDP from its complex with GDI1 strongly correlated with two distinct activities of especially Dbl and Tiam1, including liposome association and the GDP/GTP exchange. Taken together, our results provided first detailed insights into the advantages of the <i>in vitro</i> liposome-based reconstitution system to study both the integration of the signal transducing protein complexes and the mechanisms of regulation and signaling of small GTPases at biological membranes.</p></div>", "links"=>[], "tags"=>["Biochemistry", "biomechanics", "biophysics", "molecular biology", "reconstitution", "modulation", "recombinant", "prenylated", "rac1", "gdi1"], "article_id"=>1100551, "categories"=>["Biological Sciences"], "users"=>["Si-Cai Zhang", "Lothar Gremer", "Henrike Heise", "Petra Janning", "Aliaksei Shymanets", "Ion C. Cirstea", "Eberhard Krause", "Bernd Nürnberg", "Mohammad Reza Ahmadian"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0102425", "stats"=>{"downloads"=>26, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Liposome_Reconstitution_and_Modulation_of_Recombinant_Prenylated_Human_Rac1_by_GEFs_GDI1_and_Pak1_/1100551", "title"=>"Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-07-11 02:43:46"}

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Relative Metric

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