Proteomics

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LC-MSMS analyses of purified, recombinant TcJHR complexes


ABSTRACT: Juvenile hormone (JH) serves vital roles in insect reproduction, development, and many aspects of physiology. JH primarily acts at the gene-regulatory level through an intracellular receptor. The JH receptor (JHR) is a ligand-activated complex of transcription factors of the bHLH-PAS family, consisting of the JH-binding protein Methoprene-tolerant (MET) and its partner Taiman (TAI). Initial studies have indicated significance of post-transcriptional modification (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 functional JHR proteins in a purified form. Here we present successful fermentation-level purification of JHR complexes of MET and TAI proteins from two insect species, the beetle Tribolium castaneum and the mosquito Aedes aegypti. The recombinant JHR subunits from each species were co-expressed using a baculovirus system in an insect cell line and purified through affinity steps, yielding soluble proteins, capable of binding both the hormonal ligand (JH III) and DNA bearing cognate JH-response elements. The present shotgun LC-MSMS analyses identified at least ten potential phosphoserine and phosphothreonine residues located in the C-terminal disordered region of TcMET. A functional bipartite nuclear localization signal, straddled by some of these phosphorylated residues, was subsequently identified within the disordered C-terminal region of TcMET.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Tribolium Castaneum (red Flour Beetle)

SUBMITTER: Thomas Nebl  

LAB HEAD: Tom Nebl

PROVIDER: PXD028394 | Pride | 2022-02-16

REPOSITORIES: Pride

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Publications

Purification of an insect juvenile hormone receptor complex enables insights into its post-translational phosphorylation.

Jindra Marek M   McKinstry William J WJ   Nebl Thomas T   Bittova Lenka L   Ren Bin B   Shaw Jan J   Phan Tram T   Lu Louis L   Low Jason K K JKK   Mackay Joel P JP   Sparrow Lindsay G LG   Lovrecz George O GO   Hill Ronald J RJ  

The Journal of biological chemistry 20211107 6


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 (JH 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 tran  ...[more]

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