title: Type I phosphatidylinositol 4-phosphate 5-kinase homo- and heterodimerization determines its membrane localization and activity.
abstract: Type I phosphatidylinositol 4-phosphate 5-kinases (PIP5KIs; α, β, and γ) are a family of isoenzymes that produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] using phosphatidylinositol 4-phosphate as substrate. Their structural homology with the class II lipid kinases [type II phosphatidylinositol 5-phosphate 4-kinase (PIP4KII)] suggests that PIP5KI dimerizes, although this has not been formally demonstrated. Neither the hypothetical structural dimerization determinants nor the functional consequences of dimerization have been studied. Here, we used Förster resonance energy transfer, coprecipitation, and ELISA to show that PIP5KIβ forms homo- and heterodimers with PIP5KIγ_i2 in vitro and in live human cells. Dimerization appears to be a general phenomenon for PIP5KI isoenzymes because PIP5KIβ/PIP5KIα heterodimers were also detected by mass spectrometry. Dimerization was independent of actin cytoskeleton remodeling and was also observed using purified proteins. Mutagenesis studies of PIP5KIβ located the dimerization motif at the N terminus, in a region homologous to that implicated in PIP4KII dimerization. PIP5KIβ mutants whose dimerization was impaired showed a severe decrease in PI(4,5)P2 production and plasma membrane delocalization, although their association to lipid monolayers was unaltered. Our results identify dimerization as an integral feature of PIP5K proteins and a central determinant of their enzyme activity.
authors.affiliation: *Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas, Darwin 3, Campus de Cantoblanco, Madrid, Spain; Unidad de Biofísica Consejo Superior de Investigaciones Científicas, Universidad del País Vasco/Euskal Herriko Unibertsitatea, Campus de Leioa, Barrio Sarriena s/n, Leioa, Bizkaia, Spain; and Computational Biology and Bioinformatics Group, Instituto de Biomedicina de Sevilla-Hospital Universitario Virgen del Rocío-Consejo Superior de Investigaciones Científicas, Manuel Siurot s/n, Seville, Spain rlacalle@cnb.csic.es smanes@cnb.csic.es.
authors.forename: Rosa Ana
authors.initials: RA
authors.lastname: Lacalle
authors.affiliation: *Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas, Darwin 3, Campus de Cantoblanco, Madrid, Spain; Unidad de Biofísica Consejo Superior de Investigaciones Científicas, Universidad del País Vasco/Euskal Herriko Unibertsitatea, Campus de Leioa, Barrio Sarriena s/n, Leioa, Bizkaia, Spain; and Computational Biology and Bioinformatics Group, Instituto de Biomedicina de Sevilla-Hospital Universitario Virgen del Rocío-Consejo Superior de Investigaciones Científicas, Manuel Siurot s/n, Seville, Spain.
authors.forename: Juan C
authors.initials: JC
authors.lastname: de Karam
authors.affiliation: *Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas, Darwin 3, Campus de Cantoblanco, Madrid, Spain; Unidad de Biofísica Consejo Superior de Investigaciones Científicas, Universidad del País Vasco/Euskal Herriko Unibertsitatea, Campus de Leioa, Barrio Sarriena s/n, Leioa, Bizkaia, Spain; and Computational Biology and Bioinformatics Group, Instituto de Biomedicina de Sevilla-Hospital Universitario Virgen del Rocío-Consejo Superior de Investigaciones Científicas, Manuel Siurot s/n, Seville, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Martínez-Muñoz
authors.affiliation: *Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas, Darwin 3, Campus de Cantoblanco, Madrid, Spain; Unidad de Biofísica Consejo Superior de Investigaciones Científicas, Universidad del País Vasco/Euskal Herriko Unibertsitatea, Campus de Leioa, Barrio Sarriena s/n, Leioa, Bizkaia, Spain; and Computational Biology and Bioinformatics Group, Instituto de Biomedicina de Sevilla-Hospital Universitario Virgen del Rocío-Consejo Superior de Investigaciones Científicas, Manuel Siurot s/n, Seville, Spain.
authors.forename: Ibai
authors.initials: I
authors.lastname: Artetxe
authors.affiliation: *Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas, Darwin 3, Campus de Cantoblanco, Madrid, Spain; Unidad de Biofísica Consejo Superior de Investigaciones Científicas, Universidad del País Vasco/Euskal Herriko Unibertsitatea, Campus de Leioa, Barrio Sarriena s/n, Leioa, Bizkaia, Spain; and Computational Biology and Bioinformatics Group, Instituto de Biomedicina de Sevilla-Hospital Universitario Virgen del Rocío-Consejo Superior de Investigaciones Científicas, Manuel Siurot s/n, Seville, Spain.
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Peregil
authors.affiliation: *Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas, Darwin 3, Campus de Cantoblanco, Madrid, Spain; Unidad de Biofísica Consejo Superior de Investigaciones Científicas, Universidad del País Vasco/Euskal Herriko Unibertsitatea, Campus de Leioa, Barrio Sarriena s/n, Leioa, Bizkaia, Spain; and Computational Biology and Bioinformatics Group, Instituto de Biomedicina de Sevilla-Hospital Universitario Virgen del Rocío-Consejo Superior de Investigaciones Científicas, Manuel Siurot s/n, Seville, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Sot
authors.affiliation: *Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas, Darwin 3, Campus de Cantoblanco, Madrid, Spain; Unidad de Biofísica Consejo Superior de Investigaciones Científicas, Universidad del País Vasco/Euskal Herriko Unibertsitatea, Campus de Leioa, Barrio Sarriena s/n, Leioa, Bizkaia, Spain; and Computational Biology and Bioinformatics Group, Instituto de Biomedicina de Sevilla-Hospital Universitario Virgen del Rocío-Consejo Superior de Investigaciones Científicas, Manuel Siurot s/n, Seville, Spain.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Rojas
authors.affiliation: *Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas, Darwin 3, Campus de Cantoblanco, Madrid, Spain; Unidad de Biofísica Consejo Superior de Investigaciones Científicas, Universidad del País Vasco/Euskal Herriko Unibertsitatea, Campus de Leioa, Barrio Sarriena s/n, Leioa, Bizkaia, Spain; and Computational Biology and Bioinformatics Group, Instituto de Biomedicina de Sevilla-Hospital Universitario Virgen del Rocío-Consejo Superior de Investigaciones Científicas, Manuel Siurot s/n, Seville, Spain.
authors.forename: Félix M
authors.initials: FM
authors.lastname: Goñi
authors.affiliation: *Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas, Darwin 3, Campus de Cantoblanco, Madrid, Spain; Unidad de Biofísica Consejo Superior de Investigaciones Científicas, Universidad del País Vasco/Euskal Herriko Unibertsitatea, Campus de Leioa, Barrio Sarriena s/n, Leioa, Bizkaia, Spain; and Computational Biology and Bioinformatics Group, Instituto de Biomedicina de Sevilla-Hospital Universitario Virgen del Rocío-Consejo Superior de Investigaciones Científicas, Manuel Siurot s/n, Seville, Spain.
authors.forename: Mario
authors.initials: M
authors.lastname: Mellado
authors.affiliation: *Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas, Darwin 3, Campus de Cantoblanco, Madrid, Spain; Unidad de Biofísica Consejo Superior de Investigaciones Científicas, Universidad del País Vasco/Euskal Herriko Unibertsitatea, Campus de Leioa, Barrio Sarriena s/n, Leioa, Bizkaia, Spain; and Computational Biology and Bioinformatics Group, Instituto de Biomedicina de Sevilla-Hospital Universitario Virgen del Rocío-Consejo Superior de Investigaciones Científicas, Manuel Siurot s/n, Seville, Spain rlacalle@cnb.csic.es smanes@cnb.csic.es.
authors.forename: Santos
authors.initials: S
authors.lastname: Mañes
mesh_terms: D002462:Cell Membrane / Q000201:enzymology*; D004797:Enzyme-Linked Immunosorbent Assay; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D018922:HL-60 Cells; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; C037178:phosphatidylinositol 4-phosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase
keywords: PI(4,5)P2; PIP5K; dimerization; lipid kinase