ABSTRACT
The JAK/STAT pathway is an essential signalling cascade required for multiple processes during development and for adult homeostasis. A key question in understanding this pathway is how it is regulated in different cell contexts. Here, we have examined how endocytic processing contributes to signalling by the single cytokine receptor in Drosophila melanogaster cells, Domeless. We identify an evolutionarily conserved di-leucine (di-Leu) motif that is required for Domeless internalisation and show that endocytosis is required for activation of a subset of Domeless targets. Our data indicate that endocytosis both qualitatively and quantitatively regulates Domeless signalling. STAT92E, the single STAT transcription factor in Drosophila, appears to be the target of endocytic regulation, and our studies show that phosphorylation of STAT92E on Tyr704, although necessary, is not always sufficient for target transcription. Finally, we identify a conserved residue, Thr702, which is essential for Tyr704 phosphorylation. Taken together, our findings identify previously unknown aspects of JAK/STAT pathway regulation likely to play key roles in the spatial and temporal regulation of signalling in vivo.
Footnotes
Author contributions
Conceptualization: R.M., K.V., E.S.; Methodology: R.M., K.V., A.E.A.-M., P.S., M.Z., E.S.; Investigation: R.M., K.V., A.E.A.-M., P.S.; Data curation: R.M., K.V., E.S.; Writing - original draft: R.M., A.E.A.-M., E.S.; Writing - review & editing: R.M., K.V., A.E.A.-M., P.S., M.Z., E.S.; Supervision: M.Z., E.S.; Project administration: E.S.; Funding acquisition: E.S.
Funding
R.M. was funded by a University of Sheffield Centre for Membrane Interactions and Dynamics PhD studentship; K.V. by a Cancer Research UK studentship (C12332/A10681); P.S. by a Biotechnology and Biological Sciences Research Council CASE award (BB/M011151/1).
Data availability
Mass spectrometry methods and data have been deposited at the ProteomeXchange Consortium via the PRIDE partner repository http://www.ebi.ac.uk/pride. The identification number of the dataset is PXD020719.
