Prolactin stimulates ubiquitination, initial internalization, and degradation of its receptor via catalytic activation of Janus kinase 2

Abstract

Prolactin (PRL) activates its receptor to initiate signal transduction pathways (including activation of Janus kinases, Jak) but also stimulates downregulation of this receptor to limit the magnitude and duration of signaling. Degradation of the long form of PRL receptor (PRLr) depends on its phosphorylation on Ser349 that is required to facilitate PRLr ubiquitination. Signaling events that mediate PRL-induced degradation of PRLr remain to be elucidated. Here, we investigated the role of Jak2 activity in ligand-triggered increase of PRLr phosphorylation on Ser349, PRLr ubiquitination, endocytosis, and degradation. Using Jak2 reconstitution in Jak2-null cells as well as pharmacologic approaches, we found that treatment with PRL (but not with PRLr antagonist) promotes phosphorylation of PRLr on Ser349 and accelerates endocytosis of PRLr. Furthermore, PRL-stimulated PRLr phosphorylation, endocytosis, and degradation in Jak2-null cells reconstituted with wild type but not with catalytically inactive Jak2. We discuss how Jak2-mediated signaling might be transduced into Ser349 phosphorylation of PRLr as well as its ubiquitination and endocytosis.

Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway

“…2B). These results indicate that PRLr internalization is clathrin dependent and confirm our previous observations that in this experimental system, endocytosis of recombinant HA-tagged PRLr faithfully recapitulates the processes involved in the regulation of the endogenous receptor (39).…”

“…Reversible cell surface biotinylation. Cell surface biotinylation was performed as previously described (20,24,39). This procedure uses immunoblotting to measure levels of biotinylated proteins that are protected from debiotinylation as a result of being internalized.…”

“…Ubiquitination and degradation of PRLr is mainly mediated by the ␤-transducin-repeat-containing protein (␤-TrCP) E3 ubiquitin ligase that is recruited to PRLr in a manner that requires phosphorylation of Ser349 of the receptor (28). Signaling induced by PRL (but not by a PRLr antagonist) and mediated by catalytic activation of Janus kinase 2 (Jak2) stimulates this Ser349 phosphorylation in addition to ubiquitination, initial internalization, and degradation of PRLr (39). Furthermore, the intracellular localization of Ser349-phosphorylated PRLr in human mammary tissues is consistent with its targeting to the lysosomal compartment (27).…”

Ligand-induced EpoR internalization is mediated by JAK2 and p85 and is impaired by mutations responsible for primary familial and congenital polycythemia

“…[30][31][32][33] Importantly, Epo-induced EpoR internalization requires both JAK2 kinase activity and the tyrosine residues of the EpoR cytoplasmic domain. These results are consistent with studies demonstrating that ligand-stimulated internalization of the prolactin receptor 34 and the thrombopoietin receptor 31 also depends on associated JAK2 kinase activities. Moreover, as the EpoR/JAK2 complex functions as a unit that is equivalent to a receptor tyrosine kinase, these results are consistent with experiments showing that kinase activity is necessary for maximal rate of internalization and down-regulation of receptor tyrosine kinases.…”

Prolactin (PRL)-stimulated Ubiquitination of ZnT2 Mediates a Transient Increase in Zinc Secretion Followed by ZnT2 Degradation in Mammary Epithelial Cells

“…PRL Induces Ubiquitination of ZnT2-PRL stimulates ubiquitination (27), and ubiquitin serves as a sorting signal to regulate protein trafficking through the secretory compartment (reviewed in Ref. 22).…”