Type I phosphatidylinositol 4‐phosphate 5‐kinase homo‐ and heterodimerization determines its membrane localization and activity
| Autor: | Mario Mellado, Juan C. de Karam, Félix M. Goñi, Rosa Ana Lacalle, Ibai Artetxe, Jesús Sot, Ana M. Rojas, Laura Martínez-Muñoz, Rosa M. Peregil, Santos Mañes |
|---|---|
| Přispěvatelé: | Fundación 'la Caixa', Ministerio de Ciencia e Innovación (España), Comunidad de Madrid |
| Rok vydání: | 2015 |
| Předmět: |
Phosphatidylinositol 4
5-Diphosphate Phosphatidylinositol 4-phosphate Immunoblotting Enzyme-Linked Immunosorbent Assay HL-60 Cells Biology Biochemistry Isozyme Substrate Specificity PI(4 5)P2 chemistry.chemical_compound Phosphatidylinositol Phosphates Fluorescence Resonance Energy Transfer Genetics Pi Humans Phosphatidylinositol Molecular Biology Lipid kinase Microscopy Confocal Kinase Cell Membrane Mutagenesis In vitro Isoenzymes Phosphotransferases (Alcohol Group Acceptor) HEK293 Cells Förster resonance energy transfer PIP5K chemistry Mutation Protein Multimerization Dimerization Biotechnology |
| Zdroj: | Digital.CSIC. Repositorio Institucional del CSIC instname |
| ISSN: | 1530-6860 0892-6638 |
| Popis: | 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.—Lacalle, R. A., de Karam, J. C., Martínez-Muñoz, L., Artetxe, I., Peregil, R. M., Sot, J., Rojas, A. M., Goñi, F. M., Mellado, M., Mañes, S. Type I phosphatidylinositol 4-phosphate 5-kinase homo- and heterodimerization determines its membrane localization and activity. J.C.d.K. is a Ph.D. fellow of the La Caixa Foundation International Fellowship Programme. This work was supported in part by the Spanish Ministry of Science and Innovation (SAF2011-24453) and the Comunidad de Madrid (IMMUNOTHERCAN; S2010/BMD-2326). |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Plný text