Purification of an insect juvenile hormone receptor complex enables insights into its post-translational phosphorylation
| Autor: | Thomas Nebl, Joel P. Mackay, Jason Low, William J. McKinstry, Jan Shaw, Bin Ren, George O. Lovrecz, Lenka Bittova, Lindsay G. Sparrow, Ronald J. Hill, Marek Jindra, Louis Lu, Tram Phan |
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| Rok vydání: | 2021 |
| Předmět: |
Receptor complex
MFBS1 MET–FISC binding site 1 NCBI National Center for Biotechnology Information Ligand Binding Protein JH juvenile hormone Biochemistry EcR ecdysone receptor AHR aryl hydrocarbon receptor IMAC immobilized metal affinity chromatography Aedes protein purification Protein purification Sf9 Cells AaTAI Aedes aegypti taiman Protein phosphorylation MET methoprene-tolerant SFM serum-free media SEC–MALLS size-exclusion chromatography in combination with multiangle laser-light scattering Phosphorylation HEK293 human embryonic kidney 293 cell line FA formic acid Tribolium Ha Helicoverpa armigera TAI Taiman ESI electrospray ionization JHRE juvenile hormone response element Chemistry Kr-h1 Krüppel-homolog 1 GCE germ cell–expressed JHR juvenile hormone receptor ET early trypsin Ligand (biochemistry) ligand-binding protein Juvenile Hormones bHLH–PAS basic helix–loop–helix/Per–Arnt–Sim Insect Proteins HSP heat shock protein λPP lambda protein phosphatase Tc Tribolium castaneum Research Article FDR false discovery rate Protein subunit nuclear translocation Receptors Cell Surface Spodoptera FBS fetal bovine serum Animals TcTAI Tribolium castaneum taiman Molecular Biology PAS domain juvenile hormone Cell Biology hormone receptor USP ultraspiracle basic helix–loop–helix/transcription factor protein phosphorylation TCEP Tris (2-carboxyethyl) phosphine AaMET Aedes aegypti methoprene-tolerant NLS nuclear localization signal Juvenile hormone ARNT aryl hydrocarbon receptor nuclear translocator insect BSA bovine serum albumin methoprene TSAP thermosensitive alkaline phosphatase Ecdysone receptor Protein Processing Post-Translational Sf9 Spodoptera frugiperda 9 cell line |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X |
| Popis: | Juvenile hormone (JH) plays vital roles in insect reproduction, development, and in many aspects of physiology. JH primarily acts at the gene-regulatory level through interaction with an intracellular receptor (JHR), a ligand-activated complex of transcription factors consisting of the JH-binding protein Methoprene-tolerant (MET) and its partner Taiman (TAI). Initial studies indicated significance of post-transcriptional phosphorylation, subunit assembly, and nucleocytoplasmic transport of JHR in JH signaling. However, our knowledge of JHR regulation at the protein level remains rudimentary, partly due to the difficulty of obtaining purified, functional JHR proteins. Here we present a method for high-yield expression and purification of JHR complexes from two insect species, the beetle Tribolium castaneum and the mosquito Aedes aegypti. Recombinant JHR subunits from each species were co-expressed in an insect cell line using a baculovirus system. MET-TAI complexes were purified through affinity chromatography and anion exchange columns to yield proteins capable of binding both the hormonal ligand (JH III) and DNA bearing cognate JH-response elements. We further examined the beetle JHR complex (TcJHR) in greater detail. Biochemical analyses and mass spectrometry confirmed that TcJHR was a 1:1 heterodimer consisting of TcMET and TcTAI proteins, stabilized by the JHR agonist ligand methoprene. Phosphoproteomics uncovered multiple phosphorylation sites in the TcMET protein, some of which were induced by methoprene treatment. Finally, we report a functional bipartite nuclear localization signal, straddled by phosphorylated residues, within the disordered C-terminal region of TcMET. Our present characterization of the recombinant JHR is an initial step towards understanding JHR structure and function. |
| Databáze: | OpenAIRE |
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