title: The activation loop of PIP5K functions as a membrane sensor essential for lipid substrate processing.
abstract: Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), a representative member of the phosphatidylinositol phosphate kinase (PIPK) family, is a major enzyme that biosynthesizes the signaling molecule PI(4,5)P2(phosphatidylinositol 4,5-bisphosphate) in eukaryotic cells. The stringent specificity toward lipid substrates and the high sensitivity to the membrane environment strongly suggest a membrane-sensing mechanism, but the underlying structural basis is still largely unknown. We present a nuclear magnetic resonance (NMR) study on a peptide commensurate with a PIP5K's activation loop, which has been reported to be a determinant of lipid substrate specificity and subcellular localization of PIP5K. Although the activation loop is severely disordered in the crystal structure of PIP5K, the NMR experiments showed that the largely unstructured peptide folded into an amphipathic helix upon its association with the 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellar surface. Systematic mutagenesis and functional assays further demonstrated the crucial roles of the amphipathic helix and its hydrophobic surface in kinase activity and membrane-sensing function, supporting a working model in which the activation loop is a critical structural module conferring a membrane-sensing mechanism on PIP5K. The activation loop, surprisingly functioning as a membrane sensor, represents a new paradigm of kinase regulation by the activation loop through protein-membrane interaction, which also lays a foundation on the regulation of PIP5K (and other PIPKs) by membrane lipids for future studies.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Aizhuo
authors.initials: A
authors.lastname: Liu
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Dexin
authors.initials: D
authors.lastname: Sui
authors.affiliation: Department of Pharmacology, Yale School of Medicine, New Haven, CT 06511, USA.
authors.forename: Dianqing
authors.initials: D
authors.lastname: Wu
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.; Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Jian
authors.initials: J
authors.lastname: Hu
mesh_terms: D000818:Animals; D006801:Humans; D008823:Micelles; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry*; D010742:Phospholipid Ethers / Q000737:chemistry*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary
chemical_list: C052298:1,2-dihexadecyl-sn-glycero-3-phosphocholine; D008823:Micelles; D019269:Phosphatidylinositol 4,5-Diphosphate; D010742:Phospholipid Ethers; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase
keywords: NMR; Phosphatidylinositol 4-phosphate 5-kinase; activation loop; amphipathic helix; membrane sensing; phosphatidylinositol phosphate kinase