We established that PRL signaling is dependent upon receptor level, indicating that the study of PRLr downregulation and its key determinants is a valuable endeavor in that it will allow us to have insights into how PRL signaling becomes aberrant and subsequently can promote tumorigenesis. The relationship between enhanced PRL signaling and malignancies of the breast have been well-established but poorly understood. Our studies on the role of PRL signaling in the
downregulation of PRLr have shown that treatment with PRL promotes Jak2 activity which works to promote the phosphorylation of the intracellular tyrosines of PRLr (crucial step for PRL signaling) and phosphorylation of Ser 349 of PRLr’s
established that Jak2 is not the direct kinase which phosphorylates Ser 349 (data not shown). This phosphorylation event is necessary for PRLr degradation as
phosphorylated Ser 349 of the conserved phosphodegron motif works to recruit the SCFβ-TrCP E3 ligase complex which binds to PRLr and ubiquitinates the receptor on its target lysines. Additionally, we have shown that the ubiquitination of PRLr occurs in response to PRL in a manner dependent on Jak2 activity. Since Jak2 promotes the phosphorylation of the phosphodegron motif and the subsequent
ubiquitination of the PRLr, both of which are necessary for effective ligand-mediated degradation of PRLr, we have therefore established Jak2 to play an important role in PRLr downregulation (see Model 1). These results may implicate Jak2 in the phosphorylation and activation of the yet to be identified kinase which
phosphorylates PRLr on Ser 349 of the phosphodegron motif. This possible action of Jak2 would explain the importance of Jak2 in regulating the Ser 349
phosphorylation and ubiquitination of PRLr. Future studies aimed at identification of the PRLr Ser 349 kinase should focus on a Jak2-activated kinase.
Furthermore, in the investigation of PRLr downregulation, we have looked to see how the PRLr is internalized and what factors initiate its internalization by using a reliable and highly sensitive fluorescence-based internalization assay which directly studies the rate of receptor that becomes endocytosed as opposed to indirect studies using radiolabeled ligand. These elegant studies showed that PRLr internalization is largely a ligand-induced process. Ligand-induced internalization of PRLr could be abrogated by treatment with a PRLr antagonist which competes with PRL for the
ligand binding site of PRLr. However, treatment with the PRLr antagonist alone did not promote PRLr endocytosis, indicating that PRL-PRLr binding is not sufficient to promote receptor internalization. Furthermore, activation of PRL signaling is needed to activate the internalization of PRLr. Using this fluorescence-based internalization assay, we also identified Jak2 kinase activity, and not Src kinase activity, to be crucial for PRL-mediated internalization of the PRLr (see Model 1). Internalization studies using gamma2A cells, which lack endogenous Jak2, showed that optimal PRLr endocytosis was achieved when wild-type Jak2, and not a kinase inactive version, was introduced into the cell system.
Conclusions of Chapter 2:
- PRL signaling is dependent on levels of PRLr
- Active Jak2 promotes the tyrosine phosphorylation of PRLr in response
to PRL
- Active Jak2 promotes the phosphorylation of Ser 349 of the
phosphodegron motif of PRLr in response to PRL
- Active Jak2 is needed for the efficient PRL-induced ubiquitination of PRLr
- The internalization of PRLr is largely a PRL-driven process
- PRLr antagonist can prevent PRL-induced endocytosis of PRLr
- Jak2 activity is required for the efficient PRL-mediated internalization of PRLr
- Src activity is not required for the efficient PRL-mediated internalization of PRLr
- Active Jak2 protein promotes the PRL-mediated endocytosis of PRLr
Active Jak2 promotes and is necessary for the internalization of PRLr. The importance of Jak2 in the Ser 349 phosphorylation and ubiquitination of PRLr imply that the kinase which phosphorylates PRLr on Ser 349 is likely a Jak2-activated kinase. Identification of this kinase would be a useful next step in the elucidation of the mechanisms by which PRLr levels are regulated.
While Jak2 plays an important role in the downregulation of PRLr, it also is crucial for normal PRL signaling. In cancers driven by aberrant PRL signaling, pharmacological inhibition of Jak2 may not be a viable treatment as this would interfere with IL-3 and GM-CSF signaling, both of which utilize Jak2. This would be counterproductive and hinder the host’s immunological response against the tumor.
The studies contained in Chapter 2 are significant because they show that PRLr downregulation is a mainly a PRL-mediated process that is dependent on active Jak2. This is counter to the work by Piazza TM et al. that claimed PRLr endocytosis is a Src-dependent process (Piazza TM et al., 2009). However, these studies were performed using radiolabeled ligand and did not directly study the endocytosis of PRLr. Our studies, using the fluorescence-based internalization assay, accurately and reproducibly study the direct internalization of the receptor, allowing us to gain
accurate insights into the regulation of PRLr endocytosis. Our work showed that the PRL-mediated internalization of PRLr is Jak2-dependent and not Src-dependent. The presence of a minimal rate of PRLr endocytosis in the absence of ligand raises the possibility of an alternative yet minor ligand-independent internalization
mechanism which may be Src-dependent. Such an alternative mechanism may allow for the slow turnover and subsequent renewal of cell surface PRLr even in the
absence of active PRL signaling. In spite of any alternative internalization mechanisms, our studies show that ligand-mediated PRLr endocytosis is a Jak2 dependent process. These data propose Jak2 to not only be crucial for signaling downstream of the PRLr, as has been previously established, but also to be a key regulator of PRLr levels. We propose that upon hyperactive PRL signaling, the activation of Jak2 works to promote PRLr downregulation and thereby keep aberrant signaling in check. Jak2 is both needed for active PRL signaling and for the
effective execution of a negative feedback loop.
PRL treatment and subsequent activation of Jak2 both work to promote receptor endocytosis and these events both promote Ser 349 phosphorylation, receptor
ubiquitination, and receptor degradation. These facts lead us to believe that receptor ubiquitination may be the key linking factor which connects PRL and Jak2 to
receptor internalization and subsequent degradation. While it has been previously shown that PRLr is ubiquitinated, the importance, nature, and specificity of PRLr ubiquitination and its importance for the internalization of PRLr have not been
established. These questions must be properly answered so that we may understand how the cell maintains and regulates PRLr levels.
Elucidating the mechanism by which PRLr levels are regulated will help us to understand how this regulation may go awry, resulting in aberrant PRL signaling and the induction of a disease state. Therefore, we will seek to investigate the nature of PRL-mediated PRLr endocytosis and the role of ubiquitination in this process in
Figure 2.1
Figure 2.1: PRL-induced CISH promoter–driven luciferase activity in indicated T47D cell lines was performed as described in Materials and Methods.
Figure 2.2
Figure 2.2: Phosphorylation of PRLr on Tyr residues (pY, upper panel) or on Ser349 (pS349, middle panel) in gamma2A cells expressing indicated Jak2 and treated as indicated with PRL (200 ng/ml for 30 min) was analyzed by immunoprecipitation of Flag-PRLr using anti-Flag antibody followed by immunoblotting using indicated phospho-specific antibodies. Ratios between pS349 and Flag signals corresponding to cell types and treatments are calculated as an average from four independent experiments (GS.E.M.) and depicted in the graph below. Asterisk denotes P<0.05 in the t-test relative to untreated Jak2WT cells. Experiment performed by Gayathri
Figure 2.3
Figure 2.3: Flag-PRLr stringently immunopurified from denatured lysates of
gamma2A cells expressing the indicated Jak2 and treated where indicated with PRL (200 ng/ml for 30 min) was analyzed by immunoblotting using either anti-ubiquitin (upper panel) or anti-Flag (lower panel) antibodies. Experiment performed by Gayathri Swaminathan.
Figure 2.4
Figure 2.4: PRL signaling via Jak2 regulates initial internalization of PRLr. Effect of PRL (open squares and open triangles) or PRLr antagonist (PRL∆1-9,G129R, closed and open triangles) on the initial rate of internalization of amino-terminally HA-tagged PRLr expressed in 293T cells measured by a fluorescent assay as described in Materials and Methods.
Figure 2.5
Figure 2.5: Effect of Jak inhibitor AG490 (50 mM, diamonds) or Src inhibitor PP1 (10 mM, squares) on the initial rate of internalization of PRLr in 293T cells in the presence (closed symbols) or absence (open symbols) of PRL (50 ng/ml) measured by a fluorescent assay. Inhibitors were added to cells 40 min before the
internalization start. Controls are represented by treatment of cells with vehicle (DMSO, closed or open circles).
Figure 2.6
Figure 2.6: Effect of transient expression of Jak2WT (circles) or Jak2KD (squares) on the initial rate of internalization of PRLr in γ2A cells in the presence (closed
symbols) or absence (open symbols) of PRL (50 ng/ml) measured by a fluorescent assay. Controls are represented by internalization of PRLr in cells that did not receive any Jak2 (closed or open diamonds circles). Bottom panel depicts immunoblotting analysis of Jak2 expression in these cells.