Integrin-linked kinase associated with integrin activation by Shigenori Honda, Hiroko Shirotani-Ikejima, Seiji Tadokoro, Yusuke Maeda, Taroh Kinoshita, Yoshiaki Tomiyama, and Toshiyuki Miyata Blood Volume 113(21):5304-5313 May 21, 2009 ©2009 by American Society of Hematology

Establishment of mutant cells expressing inactivated αIIbα6Bβ3. Establishment of mutant cells expressing inactivated αIIbα6Bβ3. (A) FACS sorting of mutant cells. CHO cells expressing constitutively activated αIIbα6Bβ3 (parental cells) were treated with a chemical mutagen EMS for 20 hours. After cell culture for 7 days, the cells were incubated with an activation-specific mAb against αIIbβ3, PAC-1 (conjugated with PE), and a β3-specific mAb Y2/51 (conjugated with fluorescein isothiocyanate). The cells that showed high levels of Y2/51 binding but did not bind PAC-1 were sorted out by a FACS. These cells were expanded and resorted, and then the mutant cells were cloned. (B) Characterization of mutant clones. Clones defective in PAC-1 binding were confirmed by flow cytometry (i), and the clones were examined for a phenotypic restoration of PAC-1 binding by DTT treatment (ii,iii). Nonspecific binding was shown by cells incubated with the secondary Ab alone (i). Mutant cells were treated with DTT or buffer and washed once, and then the cells were incubated with PAC-1 (ii,iii). (C) Flow cytometry showing expression of αIIbα6Bβ3 on parental or mutant cells. Cells were incubated with mAbs against αIIb (SZ22), β3 (Y2/51), and αIIbβ3 (HIP8). After washing, bound mAbs were detected with an AlexaFluor 488–conjugated secondary Ab. Nonspecific binding is shown by the cells stained with the secondary Ab alone (thin solid line). Results are a representative of 3 independent experiments. (D) Immunoblotting analysis of talin in the parental and mutant cells. Whole-cell lysates were electrophoresed, transferred to a polyvinylidene difluoride membrane, incubated with antitalin mAb or anti-GAPDH polyclonal Ab, and then detected by chemiluminescence. Shigenori Honda et al. Blood 2009;113:5304-5313 ©2009 by American Society of Hematology

cDNA cloning of a molecule that restores PAC-1 binding. cDNA cloning of a molecule that restores PAC-1 binding. The mutant cells transiently transfected with a cDNA library were analyzed in PAC-1 binding, and the cells that showed high levels of PAC-1 binding within a window were sorted. Plasmids were rescued from these sorted cells and amplified by E coli into which cDNA plasmids were transformed. After 3 rounds of cDNA transient transfection, cell sorting, and plasmid rescue, finally, 5 cDNA clones that exhibited significant restoration of PAC-1 binding in the mutant cells were isolated. Shigenori Honda et al. Blood 2009;113:5304-5313 ©2009 by American Society of Hematology

Sequence analysis of ILK mRNA and gene in mutant cells. Sequence analysis of ILK mRNA and gene in mutant cells. (A) Analysis of ILK mRNA sequence. ILK mRNA of parental cells and mutant cells was amplified by RT-PCR and the PCR products were directly sequenced. Two different nonsense mutations R317X and W383X were identified in the ILK kinase domain in the mutant cells in a heterozygous state. (B) Analysis of the Ilk gene sequence. The 895-bp region encompassing exon 9 through exon 12 in the Ilk gene was amplified by PCR. The PCR products were subcloned into pCR4-TOPO vector and sequenced. The vector inserts included one or the other mutation. Shigenori Honda et al. Blood 2009;113:5304-5313 ©2009 by American Society of Hematology

Analysis of ILK in mutant cells. Analysis of ILK in mutant cells. (A) Immunoblotting using anti-ILK Ab. Whole-cell lysates obtained from CHO cells, parental cells, mutant cells, and mutant cells transiently transfected with rat ILK cDNA were electrophoresed on SDS-PAGE and immunoblotted with rabbit anti-ILK polyclonal or rabbit anti-GAPDH polyclonal Ab. (B) Flow cytometry showing expression of activated β1 integrins. Cells were incubated with mAb specific for hamster β1 (7E2) or activated β1 (9EG7). Bound mAbs were detected with PE-conjugated secondary Ab. A thin solid line represents nonspecific binding to cells stained with the secondary Ab alone. Results are representative of 3 independent experiments. The mean fluorescence intensities of 7E2 and 9EG7 binding are indicated on the histograms. (C) Effects of ILK expression on β1 integrin activation. Mutant cells were transiently transfected with GFP-fused wild-type ILK (GFPILK-WT) cDNA or GFP cDNA. After 72 hours, 9EG7 binding to transfectants was analyzed by flow cytometry. 9EG7 binding was expressed as binding normalized to β1 expression (7E2 binding). Data are mean plus or minus SD of 3 independent experiments. (D) Specific binding of PAC-1 Fab fragment to parental cells and mutant cells. Nonspecific binding of PAC-1 Fab fragment to cells was measured in the presence of Ro44-9883, and specific PAC-1 Fab binding was estimated by subtracting the MFI of nonspecific binding from the MFI of PAC-1 Fab binding to the indicated cells. Data are mean plus or minus SD of 3 experiments, and the calculated values are displayed on the graph. (E) The activation index of ILK-transfected mutant cells. The activation index using PAC-1 Fab was calculated by the formula shown in “Flow cytometry.” A value of 100% represents maximal PAC-1 Fab binding to the DTT-treated cells. Data are mean plus or minus SD of 3 independent experiments, and the values are displayed on the graph. Shigenori Honda et al. Blood 2009;113:5304-5313 ©2009 by American Society of Hematology

Effects of mutant ILKs lacking kinase activity in mutant cells. Effects of mutant ILKs lacking kinase activity in mutant cells. (A) Immunoblotting showing protein expression of GFPILK-WT and kinase-dead ILK mutants (GFPILK-K220M and GFPILK-S343A). Whole-cell lysates were electrophoresed on SDS-PAGE and immunoblotted with rabbit anti-GFP polyclonal, rabbit anti-ILK polyclonal, or rabbit anti-GAPDH polyclonal Ab. (B) The activation indexes of transfectants. The activation index was determined by the formula shown in “Flow cytometry.” A value of 100% represents maximal PAC-1 binding in the cells treated with DTT. Data are mean plus or minus SD of 3 independent experiments. Shigenori Honda et al. Blood 2009;113:5304-5313 ©2009 by American Society of Hematology

Effects of ILK siRNA on activated αIIbα6Bβ3 in parental cells. Effects of ILK siRNA on activated αIIbα6Bβ3 in parental cells. (A) Flow cytometric analysis of PAC-1 binding to siRNA transfectants. Parental cells were transiently transfected with scrambled siRNAs, ILK-specific siRNAs (Ilk467 and Ilk1255), or talin-specific siRNA (Tln5465) at a final concentration of 100 nM. After 72 hours, PAC-1 binding to transfectants was analyzed by flow cytometry in either DTT-treated or nontreated condition. The thin line histogram represents cells incubated with a PE-conjugated secondary Ab alone. Results are representative of 3 independent experiments. (B) The activation index of transfectants. The activation index was determined by the formula shown in “Flow cytometry.” Data are mean plus or minus SD of 3 experiments. (C) Immunoblotting of ILK and talin showing knockdown effects of siRNA transfection. Whole-cell lysates from the indicated transfectants were electrophoresed on SDS-PAGE and immunoblotted with rabbit anti-ILK polyclonal Ab, mouse antitalin mAb 8D4, and rabbit anti-GAPDH polyclonal Ab. (D) Adhesion of siRNA transfected cells or ILK-transfected cells to immobilized fibrinogen. (i) Parental cells were transiently transfected with scrambled Ilk1255 or Ilk1255 at 100 nM. After 72 hours, cell adhesion to microtiter wells coated with the indicated concentrations of fibrinogen was tested in triplicate. (ii) Mutant cells were transfected with an empty plasmid (2 μg) or a plasmid encoding rat ILK (2 μg), and cell adhesion was tested. Results are representative of 3 independent experiments. Data are mean plus or minus SD of triplicate measurements of optical density at 405 nm. Shigenori Honda et al. Blood 2009;113:5304-5313 ©2009 by American Society of Hematology

Effects of ILK overexpression on impaired PAC-1 binding in talin knocked-down parental cells. Effects of ILK overexpression on impaired PAC-1 binding in talin knocked-down parental cells. (A) Dot plot analysis of PAC-1 binding to cotransfectants of empty plasmid, talin-F3, or ILK with shRNA. GFP was used as a transfection marker. An empty plasmid (2 μg), a plasmid encoding a talin-F3 domain (2 μg), or rat ILK (2 μg) was cotransfected with plasmids encoding EGFP (0.5 μg) and shRNA (4 μg) to parental cells. Cells were stained with PAC-1 for flow cytometry. (B) Quantitative estimates of PAC-1 binding to the indicated transfectants. PAC-1 binding was measured in cells that showed high levels of GFP fluorescence (boxed regions in panel A) using flow cytometry. Nonspecific PAC-1 binding was measured in the presence of Ro44-9883, and specific PAC-1 binding was determined by subtracting the MFI of nonspecific binding from the MFI of PAC-1 binding to the indicated transfectants. Data are mean ± SD of 2 experiments. Shigenori Honda et al. Blood 2009;113:5304-5313 ©2009 by American Society of Hematology