Journal: The Journal of Biological Chemistry

Article Title: Type I gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling

doi: 10.1016/j.jbc.2025.110573

Figure Lengend Snippet: PIPKIγi5 interacts with YAP1. A , HEK-293 cells coexpressing HA-PIPKIγi5 and FLAG-YAP1 were subjected to HA antibody immunoprecipitation and subsequently immunoblotted with the indicated antibodies. B , CAL27 cells were immunoprecipitated utilizing YAP1 antibody, then followed by immunoblotting with the indicated antibodies. C , recombinant FLAG-YAP1 and HA-PIPKIγi5 proteins (purified via Halo-tag system) were subjected into in vitro pull-down assays with Anti-FLAG Magnetic Beads. D , the diagram depicts the domain architecture of PIPKIγi1, i2, and i5. Developed with BioRender.com . E , the Myc-tagged PIPKIγi1, PIPKIγi2, PIPKIγi5, or PIPKIγi5 kinase dead mutants (D316A; referred to as PIPKIγi5KD) were coexpressed with FLAG-YAP1 in HEK-293 cells, followed by immunoprecipitation from cell lysates using anti-Myc antibody. F , quantification of YAP1 interaction with PIPKIγi1, PIPKIγi2, PIPKIγi5, or PIPKIγi5KD. G , the interaction between purified HA-PIPKIγi5 and FLAG-YAP1 was assessed using an in vitro solid-phase binding assay, with or without the presence of PI3,5P 2 or PI4,5P 2 as indicated. H , quantification of the interaction between PIPKIγi5 and YAP1 in the solid-phase binding assay. The values presented in the graphs indicate the mean ± SD from three independent experiments. Statistical significance was determined using one-way ANOVA and Tukey’s HSD ( F and H ) (∗∗∗ p < 0.0005; ∗∗∗∗ p < 0.0001). HEK-293, human embryonic kidney 293 cell line; HSD, honestly significant difference test; PIPKIγi5, type I gamma phosphatidylinositol phosphate kinase i5; YAP1, Yes-associated protein 1.

Article Snippet: YAP1 human recombinant protein (catalog no.: TP325864) was purchased from OriGene.

Techniques: Immunoprecipitation, Western Blot, Recombinant, Purification, In Vitro, Magnetic Beads, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Type I gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling

doi: 10.1016/j.jbc.2025.110573

Figure Lengend Snippet: Identification of the binding regions responsible for the interaction between PIPKIγi5 and YAP1. A , a diagrammatic representation of the sequence of PIPKIγi5 C-terminal truncation mutants. Schematic diagram was created using BioRender.com . B , FLAG-YAP1 was coexpressed with HA-tagged wildtype PIPKIγi5 or a variety of PIPKIγi5 C-terminal truncation mutants, HA antibody was used for immunoprecipitation from cell lysates. C , levels of YAP1 interaction with wildtype PIPKIγi5 or PIPKIγi5 C-terminal truncation mutants were quantified. D , schematic representation of the domains of YAP1 and its truncation mutants. Schematic diagram was created using BioRender.com . E , MYC-PIPKIγi5 was coexpressed with HA-YAP1-N (1-263) or HA-YAP1-C (264-504), and MYC antibody was used for immunoprecipitation from cell lysates. The values presented in the graphs indicate the mean ± SD derived from three independent experiments. One-way ANOVA and Tukey’s HSD ( C ) (∗∗∗∗ p < 0.0001, and ns, nonsignificant). C, C terminus; HSD, honestly significant difference test; KD, kinase domain; N, N terminus; PDZ, PDZ-binding motif; PIPKIγi5, type I gamma phosphatidylinositol phosphate kinase i5; P-rich, proline-rich; SH3, SH3 domain; TAD, transcription activation domain; TEAD, transcriptional enhanced associate domain; WW, tryptophan–tryptophan domain; YAP1, Yes-associated protein 1.

Article Snippet: YAP1 human recombinant protein (catalog no.: TP325864) was purchased from OriGene.

Techniques: Binding Assay, Sequencing, Immunoprecipitation, Derivative Assay, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Type I gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling

doi: 10.1016/j.jbc.2025.110573

Figure Lengend Snippet: Loss of PIPKIγi5 enhances YAP1 target gene expression. Control siRNA or PIPKIγi5 siRNA-1 were transfected into CAL27 cells in low cell density ( e.g. , sparsity) ( A ) or high cell density ( e.g. , confluence) ( B ) conditions. The PIPKIγi5 protein levels were examined by Western blot in sparsity ( C ) and confluence cells ( D ). The mRNA levels of indicated YAP1 target genes were examined by real-time PCR ( E ). The values shown on graphs represent the mean ± SD from three independent experiments. One-way ANOVA and Tukey’s HSD ( E ) (∗ p < 0.05; ∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001). HSD, honestly significant difference test; PIPKIγi5, type I gamma phosphatidylinositol phosphate kinase i5; YAP1, Yes-associated protein 1.

Article Snippet: YAP1 human recombinant protein (catalog no.: TP325864) was purchased from OriGene.

Techniques: Targeted Gene Expression, Control, Transfection, Western Blot, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Type I gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling

doi: 10.1016/j.jbc.2025.110573

Figure Lengend Snippet: Effects of PIPKIγi5 on YAP1 expression and phosphorylation. CAL27 cells were transfected with either control, PIPKIγi5 siRNA-1, or PIPKIγi2 siRNA, and the specified protein levels were analyzed using Western blot ( A ). Quantification of total YAP1, YAP1 phosphorylation (S127 and S397), LATS1 and LATS2 in CAL27 cells ( B ). UM-SCC-1 cells were transfected with either control, PIPKIγi5 siRNA-1, or PIPKIγi2 siRNA, and the specified protein levels were analyzed using Western blot ( C ). Quantification of total YAP1, YAP1 phosphorylation (S127 and S397), LATS1 and LATS2 in UM-SCC-1 cells ( D ). The values shown on graphs represent the mean ± SD from three independent experiments. One-way ANOVA and Tukey’s HSD ( B , D ) (∗ p < 0.05, and ns, nonsignificant). HSD, honestly significant difference test; LATS1/2, large tumor suppressor kinase 1 and 2; PIPKIγi5, type I gamma phosphatidylinositol phosphate kinase i5; YAP1, Yes-associated protein 1.

Article Snippet: YAP1 human recombinant protein (catalog no.: TP325864) was purchased from OriGene.

Techniques: Expressing, Phospho-proteomics, Transfection, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Type I gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling

doi: 10.1016/j.jbc.2025.110573

Figure Lengend Snippet: PIPKIγi5 promotes YAP1 interaction with 14-3-3. A , FLAG-YAP1 were coexpressed with Myc-PIPKIγi5 or Myc-PIPKIγi5 KD in HEK-293 cells, and the cells were subjected to FLAG antibody immunoprecipitation and subsequently immunoblotted with the specified antibodies. B , quantification of YAP1 interaction with 14-3-3. C , HA-PIPKIγi5 was coexpressed with or without FLAG-YAP1 in HEK-293 cells, and then cells were subjected to HA antibody immunoprecipitation and subsequently immunoblotted with the specified antibodies. D , quantification of PIPKIγi5 interaction with 14-3-3. E , FLAG-YAP1 was expressed in control or PIPKIγi5-knockdown CAL27 cells and subjected to FLAG antibody immunoprecipitation and subsequently immunoblotted with the specified antibodies. F , quantification of YAP1 interaction with 14-3-3 in control or PIPKIγi5-knockdown cells. The values shown on graphs represent the mean ± SD from three independent experiments. One-way ANOVA and Tukey’s HSD ( B ) (∗ p < 0.05; ∗∗∗ p < 0.0005). Unpaired two-tailed Student’s t test ( D , F ) (∗ p < 0.05; ∗∗ p < 0.001). HEK-293, human embryonic kidney 293 cell line; HSD, honestly significant difference test; KD, kinase domain; PIPKIγi5, type I gamma phosphatidylinositol phosphate kinase i5; YAP1, Yes-associated protein 1.

Article Snippet: YAP1 human recombinant protein (catalog no.: TP325864) was purchased from OriGene.

Techniques: Immunoprecipitation, Control, Knockdown, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Type I gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling

doi: 10.1016/j.jbc.2025.110573

Figure Lengend Snippet: Depletion of PIPKIγi5 increases the nuclear translocation of YAP1. CAL27 cells were transfected with either control siRNA or PIPKIγi5 siRNA-1. A , control or PIPKIγi5-knockdown CAL27 cells were stained with YAP1 antibody. Nuclei were stained with DAPI (scale bars represent 10 μm). High magnifications of the respective framed regions were shown on the right (scale bars represent 5 μm). B , quantification of YAP1 nuclear IF staining. Error bars indicate mean ± SD. (n = 60 cells from three independent experiments). Control or PIPKIγi5-knockdown CAL27 cells were treated with or without the MST1/2 inhibitor XMU-MP-1, and then the cytoplasmic and nuclear fractions were isolated using nuclear extraction kits. Cytoplasmic constituents were subjected to immunoblotting using indicated antibodies ( C ). Nuclear components were subjected to immunoblotting using specified antibodies ( D ). The levels of nuclear YAP1 in control or PIPKIγi5-knockdown CAL27 cells were quantified ( E ). The PIPKIγi5 expression levels, MST1/2 expression levels, and MST1/2 phosphorylation (MST1 (Thr183)/MST2 (Thr180)) levels were examined by Western blot ( F ). The values shown on graphs represent the mean ± SD from three independent experiments. Unpaired two-tailed Student’s t test ( B ) (∗∗∗ p < 0.0005). One-way ANOVA and Tukey’s HSD ( E ) (∗∗ p < 0.001; ∗∗∗ p < 0.0005, ∗∗∗∗ p < 0.0001). DAPI, 4′,6-diamidino-2-phenylindole; HSD, honestly significant difference test; IF, immunofluorescence; MST1/2, mammalian STE20-like protein kinase 1/2; PIPKIγi5, type I gamma phosphatidylinositol phosphate kinase i5; YAP1, Yes-associated protein 1

Article Snippet: YAP1 human recombinant protein (catalog no.: TP325864) was purchased from OriGene.

Techniques: Translocation Assay, Transfection, Control, Knockdown, Staining, Isolation, Extraction, Western Blot, Expressing, Phospho-proteomics, Two Tailed Test, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Type I gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling

doi: 10.1016/j.jbc.2025.110573

Figure Lengend Snippet: The kinase activity of PIPKIγi5 is required for the regulation of YAP1. A , the interaction between purified GST-14-3-3 and FLAG-YAP1 was assessed using an in vitro solid-phase binding assay, with or without the presence of PI3,5P 2 or PI4,5P 2 as specified. B , quantification of the interaction between 14-3-3 and YAP1 in the solid-phase binding assay. Myc-PIPKIγi5, Myc-PIPKIγi5KD, or Myc-PIPKIγi1 were individually expressed in either control or PIPKIγi5-knockdown CAL27 cells, and then PIPKIγi5 expression was identified using Western blot analysis ( C ), the expression of YAP1 target genes, ANKRD1 and CTGF, was assessed using real-time PCR ( D ). The values shown on graphs represent the mean ± SD from three independent experiments. One-way ANOVA and Tukey’s HSD ( B , D ) (∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; and ns, nonsignificant). HSD, honestly significant difference test; PI3,5P 2, phosphatidylinositol-3,5-bisphosphate; PI4,5P 2 , phosphatidylinositol-4,5-bisphosphate; PIPKIγi5, type I gamma phosphatidylinositol phosphate kinase i5; YAP1, Yes-associated protein 1.

Article Snippet: YAP1 human recombinant protein (catalog no.: TP325864) was purchased from OriGene.

Techniques: Activity Assay, Purification, In Vitro, Binding Assay, Control, Knockdown, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Type I gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling

doi: 10.1016/j.jbc.2025.110573

Figure Lengend Snippet: Model for PIPKIγi5 regulation of Hippo/YAP1 signaling pathway. PIPKIγi5 interacts with YAP1, inhibiting its nuclear translocation and thereby suppressing YAP1-mediated gene transcription. By facilitating the interaction between YAP1 and 14-3-3 protein, PIPKIγi5 sequesters YAP1 in the cytosol. Therefore, reduction of PIPKIγi5 enhances YAP1 signaling. Schematic diagram was created using BioRender.com . PIPKIγi5, type I gamma phosphatidylinositol phosphate kinase i5; YAP1, Yes-associated protein 1.

Article Snippet: YAP1 human recombinant protein (catalog no.: TP325864) was purchased from OriGene.

Techniques: Translocation Assay

Journal: Nature Communications

Article Title: SKP2- and OTUD1-regulated non-proteolytic ubiquitination of YAP promotes YAP nuclear localization and activity

doi: 10.1038/s41467-018-04620-y

Figure Lengend Snippet: SKP2 interacts with YAP and induces its K63-linked polyubiquitination. a The HEK293T SFB-YAP stable cell line was transfected with MYC-tagged E3 ligases, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against FLAG and MYC. b The HEK293T SFB-YAP stable cell line was co-transfected with the K63-specific mutant of HA-ubiquitin and the indicated E3 ligase, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. c Co-immunoprecipitation of endogenous SKP2 with endogenous YAP. d In vitro binding of bacterially purified MBP-SKP2 to in vitro translated and purified GST-YAP. e In vitro binding of bacterially purified MBP-SKP2 to SFB-YAP purified from the HEK293T SFB-YAP stable cell line. MBP-SKP2 or MBP protein was incubated with SFB-YAP protein with or without CIP treatment at 37 °C for 1 h, followed by immunoblotting with antibodies against SKP2, p-YAP (S127), and FLAG. f The HEK293T SFB-YAP stable cell line was transfected with HA-ubiquitin (wild-type, K48R or K63R) and MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. g The HEK293T SFB-YAP stable cell line was transfected with HA-ubiquitin (wild-type, K48-specific or K63-specific) and MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. h Total K63-linkage specific ubiquitinated proteins in control or SKP2 knockout HEK293A cells (generated by CRISPR-Cas9) were immunoprecipitated by a K63-linked polyubiquitin-specific antibody, followed by immunoblotting with antibodies against YAP and ubiquitin. i siRNA targeting CUL1 or SKP1 was transfected into the HEK293T SFB-YAP stable cell line. Forty-eight hours after siRNA transfection, cells were transfected with HA-ubiquitin and MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. j The HEK293T UBCH5c shRNA stable cell line was transfected with SFB-YAP and HA-ubiquitin with or without MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. k Purified GST-YAP protein was incubated with ATP, E1, UBCH5c, and the K63-specific mutant of His-ubiquitin with or without the SCF SKP2 complex, followed by immunoprecipitation with a YAP-specific antibody and immunoblotting with antibodies against ubiquitin and YAP

Article Snippet: For pulldown of SFB-tagged proteins, cell extracts were incubated with S-protein beads (Millipore, 69704) at 4 °C for 2 h. For in vitro binding assays, purified GST-YAP (Novus Biologicals, H00010413-P01) was incubated with purified MBP-SKP2, and purified His-YAP (MyBioSource, MBS717875) was incubated with purified GST-OTUD1, followed by pulldown with Glutathione Sepharose beads (GE healthcare, 17-0756-01).

Techniques: Stable Transfection, Transfection, Immunoprecipitation, Western Blot, Mutagenesis, In Vitro, Binding Assay, Purification, Incubation, Knock-Out, Generated, CRISPR, shRNA

Journal: Nature Communications

Article Title: SKP2- and OTUD1-regulated non-proteolytic ubiquitination of YAP promotes YAP nuclear localization and activity

doi: 10.1038/s41467-018-04620-y

Figure Lengend Snippet: OTUD1 interacts with YAP and antagonizes its K63-linked ubiquitination. a SFB-tagged DUBs were co-transfected with MYC-YAP into HEK293T cells, followed by pulldown with S-protein beads and immunoblotting with antibodies against FLAG and MYC. b Seven SFB-DUBs were co-transfected with MYC-YAP and HA-ubiquitin into HEK293T cells, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. c The HEK293T SFB-YAP stable cell line was transfected with four YAP-interacting DUBs along with an 8× GTIIC luciferase reporter and a TK-Renilla luciferase reporter. Reporter activity was measured 48 h after transfection. Error bars are s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001. n = 3 biological replicates. d Co-immunoprecipitation of endogenous OTUD1 with endogenous YAP. e In vitro binding of purified GST-OTUD1 to purified His-YAP. f HEK293T cells were co-transfected with SFB-OTUD1 (wild-type, C320S or H431R), HA-ubiquitin and MYC-YAP, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. g Ubiquitinated SFB-YAP was purified with S-protein beads and incubated with His-OTUD1 (wild-type or C320S) purified from bacteria. After the in vitro deubiquitination reaction, the bound proteins were eluted by boiling in Laemmli sample buffer and immunoblotted with antibodies against HA, FLAG, and OTUD1. h HEK293T cells were co-transfected with SFB-OTUD1, HA-ubiquitin (wild-type, K48R or K63R) and MYC-YAP, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. i siRNA targeting OTUD1 was transfected into the HEK293T SFB-YAP stable cell line. Forty-eight hours later, cells were transfected with HA-ubiquitin (wild-type, K48R or K63R), followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. j Total K63-linkage specific ubiquitinated proteins in control or OTUD1 knockout HEK293A cells (generated by CRISPR-Cas9) were immunoprecipitated by a K63-linked polyubiquitin-specific antibody, followed by immunoblotting with antibodies against YAP and ubiquitin

Article Snippet: For pulldown of SFB-tagged proteins, cell extracts were incubated with S-protein beads (Millipore, 69704) at 4 °C for 2 h. For in vitro binding assays, purified GST-YAP (Novus Biologicals, H00010413-P01) was incubated with purified MBP-SKP2, and purified His-YAP (MyBioSource, MBS717875) was incubated with purified GST-OTUD1, followed by pulldown with Glutathione Sepharose beads (GE healthcare, 17-0756-01).

Techniques: Transfection, Western Blot, Immunoprecipitation, Stable Transfection, Luciferase, Activity Assay, Two Tailed Test, In Vitro, Binding Assay, Purification, Incubation, Bacteria, Knock-Out, Generated, CRISPR