The Integrin-Linked Kinase-PINCH-Parvin Complex Supports Integrin αIIbβ3 Activation
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{"title"=>"The integrin-linked kinase-PINCH-parvin complex supports integrin αIIbβ3 activation", "type"=>"journal", "authors"=>[{"first_name"=>"Shigenori", "last_name"=>"Honda", "scopus_author_id"=>"7402362335"}, {"first_name"=>"Hiroko", "last_name"=>"Shirotani-Ikejima", "scopus_author_id"=>"7801452506"}, {"first_name"=>"Seiji", "last_name"=>"Tadokoro", "scopus_author_id"=>"13605646000"}, {"first_name"=>"Yoshiaki", "last_name"=>"Tomiyama", "scopus_author_id"=>"7102307978"}, {"first_name"=>"Toshiyuki", "last_name"=>"Miyata", "scopus_author_id"=>"56381961600"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"84893238618", "issn"=>"19326203", "scopus"=>"2-s2.0-84893238618", "pmid"=>"24376884", "pui"=>"372285527", "doi"=>"10.1371/journal.pone.0085498"}, "id"=>"548bb012-2bca-3f6b-af7b-ebd0a2dccda6", "abstract"=>"Integrin-linked kinase (ILK) is an important signaling regulator that assembles into the heteroternary complex with adaptor proteins PINCH and parvin (termed the IPP complex). We recently reported that ILK is important for integrin activation in a Chinese hamster ovary (CHO) cell system. We previously established parental CHO cells expressing a constitutively active chimeric integrin (αIIbα6Bβ3) and mutant CHO cells expressing inactive αIIbα6Bβ3 due to ILK deficiency. In this study, we further investigated the underlying mechanisms for ILK-dependent integrin activation. ILK-deficient mutant cells had trace levels of PINCH and α-parvin, and transfection of ILK cDNA into the mutant cells increased not only ILK but also PINCH and α-parvin, resulting in the restoration of αIIbα6Bβ3 activation. In the parental cells expressing active αIIbα6Bβ3, ILK, PINCH, and α-parvin were co-immunoprecipitated, indicating the formation of the IPP complex. Moreover, short interfering RNA (siRNA) experiments targeting PINCH-1 or both α- and β-parvin mRNA in the parent cells impaired the αIIbα6Bβ3 activation as well as the expression of the other components of the IPP complex. In addition, ILK mutants possessing defects in either PINCH or parvin binding failed to restore αIIbα6Bβ3 activation in the mutant cells. Kindlin-2 siRNA in the parental cells impaired αIIbα6Bβ3 activation without disturbing the expression of ILK. For CHO cells stably expressing wild-type αIIbβ3 that is an inactive form, overexpression of a talin head domain (THD) induced αIIbβ3 activation and the THD-induced αIIbβ3 activation was impaired by ILK siRNA through a significant reduction in the expression of the IPP complex. In contrast, overexpression of all IPP components in the αIIbβ3-expressing CHO cells further augmented THD-induced αIIbβ3 activation, whereas they did not induce αIIbβ3 activation without THD. These data suggest that the IPP complex rather than ILK plays an important role and supports integrin activation probably through stabilization of the active conformation.", "link"=>"http://www.mendeley.com/research/integrinlinked-kinasepinchparvin-complex-supports-integrin-%CE%B1iib%CE%B23-activation", "reader_count"=>12, "reader_count_by_academic_status"=>{"Researcher"=>3, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>1, "Student > Postgraduate"=>1, "Student > Master"=>1, "Student > Bachelor"=>4, "Professor"=>1}, "reader_count_by_user_role"=>{"Researcher"=>3, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>1, "Student > Postgraduate"=>1, "Student > Master"=>1, "Student > Bachelor"=>4, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>3, "Medicine and Dentistry"=>2, "Agricultural and Biological Sciences"=>3, "Business, Management and Accounting"=>1, "Sports and Recreations"=>1, "Chemistry"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Chemistry"=>{"Chemistry"=>1}, "Sports and Recreations"=>{"Sports and Recreations"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>3}, "Business, Management and Accounting"=>{"Business, Management and Accounting"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>3}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"United Kingdom"=>1, "Germany"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1327100"], "description"=>"<p>αIIbα6Bβ3-active parental cells were transiently transfected with PINCH siRNAs (p157 and p755) (A), α-parvin siRNAs (pa503 and pa761) (C), β-parvin siRNAs (pb900 and pb1011) (C), kindlin-2 siRNAs (k770 and k1733) (E), negative control siRNAs, and scrambled siRNAs. Cell lysates were electrophoresed on SDS-PAGE gels, and the separated proteins were immunoblotted with the indicated Abs. GAPDH and β-actin are shown as internal loading controls. The activation indexes of transfected cells (B, D, F) were calculated using the formula shown in Materials and Methods. A value of 100% implies the maximum PAC-1 binding to the cells treated with dithiothreitol (DTT). Data represent means ± standard deviation (SD) of three (B, F) or four (D) independent experiments. ** indicates <i>P</i> < 0.01.</p>", "links"=>[], "tags"=>["kindlin-2"], "article_id"=>885505, "categories"=>["Biological Sciences"], "users"=>["Shigenori Honda", "Hiroko Shirotani-Ikejima", "Seiji Tadokoro", "Yoshiaki Tomiyama", "Toshiyuki Miyata"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085498.g003", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Knockdown_effects_of_PINCH_parvins_and_kindlin_2_in_945_IIb_945_6B_946_3_active_parental_cells_/885505", "title"=>"Knockdown effects of PINCH, parvins, and kindlin-2 in αIIbα6Bβ3-active parental cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-23 03:46:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/1327105"], "description"=>"<p>The activation indexes of transfected cells (A, C, E). ILK-deficient mutant cells were transiently transfected with GFP cDNA, GFP-fused wild-type ILK (GFPILK-WT) cDNA, GFP-fused ILK mutant with defective PINCH binding (GFPILK-H99D/F109A/W110A) cDNA (A, E), or GFP-fused ILK mutant with defective parvin binding (GFPILK-M402A/K403A) cDNA (C, E). After transfection, the binding of either PAC-1 (A, C) or fibrinogen (E) to the cells was analyzed by flow cytometry. The activation index was determined by the formula shown in Materials and Methods. A value of 100% represents the maximal binding of PAC-1 or fibrinogen to the cells treated with dithiothreitol. Data represent means ± SD of three independent experiments. ** indicates <i>P</i> < 0.01. Immunoblotting showing protein expression of GFP (B, D), GFP-fused wild-type ILK (GFPILK-WT) (B, D), GFP-fused ILK mutant with defective PINCH binding (GFPILK-H99D/F109A/W110A) (B), and GFP-fused ILK mutant with defective parvin binding (GFPILK-M402A/K403A) (D) in ILK-deficient mutant cells. Cell lysates were electrophoresed and immunoblotted with indicated Abs. </p>", "links"=>[], "tags"=>["ilk", "mutants", "defects", "pinch", "parvin"], "article_id"=>885507, "categories"=>["Biological Sciences"], "users"=>["Shigenori Honda", "Hiroko Shirotani-Ikejima", "Seiji Tadokoro", "Yoshiaki Tomiyama", "Toshiyuki Miyata"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085498.g004", "stats"=>{"downloads"=>0, "page_views"=>33, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effects_of_ILK_mutants_with_defects_in_either_PINCH_or_parvin_binding_/885507", "title"=>"Effects of ILK mutants with defects in either PINCH or parvin binding.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-23 03:46:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/1327108"], "description"=>"<p>(A) Dot plot detecting PAC-1 binding to cotransfected cells. Inactive αIIbβ3-expressing CHO cells were transiently cotransfected with GFP cDNA plus scrambled ILK siRNA (scramble Ilk1255), with THD-GFP cDNA plus scrambled ILK siRNA (scramble Ilk1255), or with THD-GFP cDNA plus ILK siRNA (Ilk1255). Highly transfected cells (cells in gated regions) were analyzed for PAC-1 binding or HIP8 (an αIIbβ3-specific mAb) binding. (B) The activation indexes of transfected cells. The activation index was determined by the formula shown in Materials and Methods. A value of 100% implies the median fluorescence intensity of HIP8 binding to the cells in gated regions. Data represent means ± SD of three independent experiments. ** indicates <i>P</i> < 0.01. (C) Immunoblotting to evaluate expression levels of IPP and THD-GFP. Cell lysates were electrophoresed and immunoblotted with indicated Abs. </p>", "links"=>[], "tags"=>["ilk", "thd-mediated"], "article_id"=>885509, "categories"=>["Biological Sciences"], "users"=>["Shigenori Honda", "Hiroko Shirotani-Ikejima", "Seiji Tadokoro", "Yoshiaki Tomiyama", "Toshiyuki Miyata"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085498.g005", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Knockdown_effects_of_ILK_on_THD_mediated_945_IIb_946_3_activation_/885509", "title"=>"Knockdown effects of ILK on THD-mediated αIIbβ3 activation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-23 03:46:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/1327112"], "description"=>"<p>(A) Dot plot detecting PAC-1 binding to transfected cells. Inactive αIIbβ3-expressing CHO cells were transiently transfected with GFP cDNA, with THD-GFP cDNA, with THD-GFP cDNA plus IPP (ILK, PINCH, and α-parvin) cDNAs, with GFP cDNA plus IPP cDNAs, or with THD-GFP cDNA plus kindling-2 cDNA. Highly transfected cells in the gated regions were analyzed for PAC-1 binding or HIP8 (an αIIbβ3-specific mAb) binding. (B) The activation indexes of transfected cells. The index was determined by the formula shown in Materials and Methods. A value of 100% implies the median fluorescence intensity of HIP8 binding to the cells in gated regions. Data represent means ± SD of three independent experiments. * and ** indicate <i>P</i> < 0.05 and <i>P</i> < 0.01, respectively. (C) Immunoblotting to evaluate expression levels of IPP, THD-GFP, and kindlin-2. Cell lysates obtained from transfected cells were electrophoresed and immunoblotted with indicated Abs. </p>", "links"=>[], "tags"=>["ipp", "overexpression", "thd-mediated", "integrin", "activation", "inactive", "cho"], "article_id"=>885513, "categories"=>["Biological Sciences"], "users"=>["Shigenori Honda", "Hiroko Shirotani-Ikejima", "Seiji Tadokoro", "Yoshiaki Tomiyama", "Toshiyuki Miyata"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085498.g006", "stats"=>{"downloads"=>1, "page_views"=>25, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effects_of_IPP_overexpression_on_THD_mediated_integrin_activation_in_inactive_945_IIb_946_3_expressing_CHO_cells_/885513", "title"=>"Effects of IPP overexpression on THD-mediated integrin activation in inactive αIIbβ3-expressing CHO cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-23 03:46:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/1327096"], "description"=>"<p>(A) Immunoblotting for ILK, PINCH, α-parvin, talin, and kindlin-2. Cell lysates obtained from parental cells with constitutively active αIIbα6Bβ3, ILK-deficient mutant cells with inactive αIIbα6Bβ3, and mutant cells transiently transfected with rat ILK cDNA were electrophoresed on SDS-PAGE gels and immunoblotted with indicated Abs. GAPDH shows an internal loading control. (B) Flow cytometry analysis showing PAC-1 (an activation-specific mAb for αIIbβ3) binding to mutant cells transiently transfected with either ILK plasmid or empty plasmid. Bound PAC-1 was detected with a PE-conjugated secondary mAb. </p>", "links"=>[], "tags"=>["ilk-deficient", "mutant", "cells", "expressing", "inactive"], "article_id"=>885501, "categories"=>["Biological Sciences"], "users"=>["Shigenori Honda", "Hiroko Shirotani-Ikejima", "Seiji Tadokoro", "Yoshiaki Tomiyama", "Toshiyuki Miyata"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085498.g001", "stats"=>{"downloads"=>1, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Characterization_of_ILK_deficient_mutant_cells_expressing_inactive_945_IIb_945_6B_946_3_/885501", "title"=>"Characterization of ILK-deficient mutant cells expressing inactive αIIbα6Bβ3.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-23 03:46:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/1327098"], "description"=>"<p>Cell lysates obtained from αIIbα6Bβ3-active parental cells were immunoprecipitated with Abs against PINCH (A), α-parvin (B, C), and ILK (D). The co-precipitates were detected by Abs for α-parvin (A), ILK (B), and PINCH (C, D). IgG means immunoprecipitation (IP) using non-immune control IgG. IB stands for immunoblotting. Arrows indicate the predicted sizes of the indicated proteins. Arrowheads (D) indicate the antibody heavy chains used in the IP. Different mobilities between those of the two IgG antibodies are probably caused by differences in the amino acid compositions of them. </p>", "links"=>[], "tags"=>["ipp", "proteins"], "article_id"=>885503, "categories"=>["Biological Sciences"], "users"=>["Shigenori Honda", "Hiroko Shirotani-Ikejima", "Seiji Tadokoro", "Yoshiaki Tomiyama", "Toshiyuki Miyata"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085498.g002", "stats"=>{"downloads"=>0, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Detection_of_IPP_complex_proteins_in_945_IIb_945_6B_946_3_active_parental_cells_/885503", "title"=>"Detection of IPP complex proteins in αIIbα6Bβ3-active parental cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-23 03:46:51"}

PMC Usage Stats | Further Information

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Relative Metric

{"start_date"=>"2013-01-01T00:00:00Z", "end_date"=>"2013-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences/Biochemistry", "average_usage"=>[266, 468, 593, 703, 804, 903, 993, 1084, 1171, 1256, 1339, 1422, 1492]}, {"subject_area"=>"/Biology and life sciences/Genetics", "average_usage"=>[284, 491, 620, 738, 843, 945, 1043, 1137, 1225, 1315, 1400, 1479, 1555]}, {"subject_area"=>"/Biology and life sciences/Molecular biology", "average_usage"=>[272, 466, 589, 702, 806, 903, 995, 1086, 1176, 1258, 1347, 1422, 1493]}, {"subject_area"=>"/Medicine and health sciences", "average_usage"=>[264, 460, 584, 692, 794, 887, 978, 1067, 1154, 1241, 1328, 1408, 1474]}, {"subject_area"=>"/Medicine and health sciences/Hematology", "average_usage"=>[238, 424, 545, 650, 749, 839, 926, 1006, 1090, 1167, 1243, 1316, 1380]}]}
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