JHSIGNAL Project

Lenka Bittova received a prestigious individual fellowship from the Marie Sklodowska-Curie Actions of the H2020 program of the European union. 

Project Title: Modulation of juvenile hormone signaling by receptor phosphorylation

Juvenile hormones (JHs) govern many aspects of insect and related arthropod development and reproduction. Species relying on regulation by JH range from beneficial pollinators to agricultural pests and disease vectors. Thorough understanding of JH signaling is therefore a prerequisite for development of targeted JH-based insecticides, a direct benefit for the society. The intracellular JH receptor has been identified only recently and its actions are still poorly understood. Current evidence suggests that activity of JH receptor is modulated by JH-induced phosphorylation. The main research objective of this MSCA project was to identify the phosphorylation target sites of JH receptor and to determine their significance for its function. An equally important objective of this Fellowship was to facilitate a full reintegration of the Fellow, an erudite biochemist with 20 years of experience from top-ranking US institutions, back into Czech and European science environment. We believe that these goals have been achieved.

The main research objective of this project was to identify the phosphorylation target sites of JH receptor (JHR) and to determine their significance for its function. An equally important goal of this fellowship was to facilitate a full reintegration of Lenka back into Czech and European science environment.

Now that the project is near its completion, we believe that it has fully achieved its objectives and milestones for the period.

Three specific aims have been proposed to study - (1) Effects of JHR phosphorylation on JH-dependent transcription and JH binding (2) Effects of JHR phosphorylation on nuclear localization and protein-protein interactions (3) Role of JHR phosphorylation in JH-signaling in vivo. Lenka has made a major progress on all these aims (see Figure):

• In collaboration with the IOCB in Prague we hawe characterized requirements for active JHR agonists in terms of the receptor binding, dimerization, and biological activity (Bittova et al., 2019); in collaboration with the CSIRO (Australia), the JHR phosphorylation sites have been identified.
• We have developed and tested a new luciferase reporter system designed to measure transcriptional activation mediated by JHRs of Tribolium castaneum and Drosophila melanogaster in a cell-based assay.
• We have designed and generated an extensive set of mutated Tribolium and Drosophila JHR proteins (TcMet and Gce) in order to preclude or to mimic constitutive phosphorylation of these targets. She tested the capacity of the mutated receptors to activate luciferase reporter in response to JH in cell-based assays, to bind radiolabeled JH in vitro as well as their ability to interact with their known binding partner, Taiman (Tai).
• We have designed and prepared CRISPR-based DNA tools to replace the endogenous JHR gene Met in Tribolium by its phosphorylation-mutant counterparts to study the effect of these mutations in a living model organism.
• We investigated cellular localization of the epitope-tagged Met/Gce phosphorylation-mutants expressed in cell lines in the presence or absence of JH by immunofluorescence. In the course of this research, novel nuclear localization signal (NLS) motifs in both Tribolium and Drosophila JHRs were discovered. Lenka prepared a series of NLS mutants with a disrupted nuclear targeting of Met/Gce in cells. She is now focusing on their closer characterization.
• We prepared a set of transgenic Drosophila lines expressing selected TcMet or Gce mutant proteins and screened them for altered phenotypes. These mutants were also expressed in the Met/Gce-null genetic background and tested for the ability to rescue the otherwise lethal Drosophila mutant strain. Very exciting findings came from mutations introduced in the PAS-A domain of Gce. The flies with the mutated JHR displayed strong developmental defects, such as rotated genitalia in males or reduced fertility. The mechanism of this action is now under investigation.

Together, these promising results have already started to reveal novel and significant aspects of JHR signaling mechanism (Figure). They will certainly improve our understanding of the role of post-translational modifications in regulation of insect development.