Jak2 is a negative regulator of ubiquitin-dependent endocytosis of the growth hormone receptor

Abstract

Background: Length and intensity of signal transduction via cytokine receptors is precisely regulated. Degradation of certain cytokine receptors is mediated by the ubiquitin ligase SCF(βTrCP). In several instances, Janus kinase (Jak) family members can stabilise their cognate cytokine receptors at the cell surface.

Principal findings: In this study we show in Hek293 cells that Jak2 binding to the growth hormone receptor prevents endocytosis in a non-catalytic manner. Following receptor activation, the detachment of phosphorylated Jak2 induces down-regulation of the growth hormone receptor by SCF(βTrCP). Using γ2A human fibroblast cells we show that both growth hormone-induced and constitutive growth hormone receptor endocytosis depend on the same factors, strongly suggesting that the modes of endocytosis are identical. Different Jak2 RNA levels in HepG2, IM9 and Hek293 cells indicate the importance of cellular concentration on growth hormone receptor function. Both Jak2 and βTrCP bind to neighbouring linear motifs in the growth hormone receptor tail without the requirement of modifications, indicating that growth hormone sensitivity is regulated by the cellular level of non-committed Jak2.

Conclusions/significance: As signal transduction of many cytokine receptors depends on Jak2, the study suggests an integrative role of Jak2 in cytokine responses based on its enzyme activity as well as its stabilising properties towards the receptors.

Figure 1. Jak2 inhibits GHR endocytosis.

A. GHR-expressing Hek293 cells were transfected with empty vector (EV), Flag-Jak2, Flag-Jak2(Y119E) or Flag-Jak2(1-525). One coverslip was incubated for 15 min with Cy3-GH at 37°C, fixed and stained with anti-Flag (left panels). For EV- and Jak2-transfected cells, cells were also incubated with Cy3-GH and Alexa488-transferrin (Tf) for 15 min at 37°C (right panel). Bar, 5 µm. B. Hek293 GHR or GHR(UbE mutant)-expressing cells were transfected with Jak2 DNA or empty vector and, after 2 days, incubated for 2h on ice with 180 ng/ml ¹²⁵I-GH. To measure uptake kinetics unbound label was removed and the cells were incubated at 37°C for 10 min. C. Hek293 cells were transfected as in 1A and treated with GH for 15 min at 37°C as indicated. Total cell lysates (TCL, upper panel) and GHR immunoprecipitations (lower panel) were analysed on western blot (WB) using anti-Flag, or anti-phosphotyrosine (pY). 130, mature GHR; 110, precursor GHR. Data are representative of three independent experiments. D. Diagram showing the different binding motifs in the dimerised GHR; Jak2 binds Box1, TrCP binds UbE). The transmembrane domains are the dimerisation domains.

Figure 2. SCF(βTrCP) and Clathrin act downstream of Jak2.

A. GHR-expressing Hek293 cells were either silenced for GFP (negative control), βTrCP or clathrin, and then treated for 15 min with GH at 37°C as indicated. GHR immunoprecipitations were analysed for ubiquitination. In the lower part of the figure the efficiency of silencing is visualised using anti-βTrCP or anti-Clathrin. B. Hek293 cells were transiently transfected with HA-tagged wild-type ubiquitin, ubiquitin K48R or ubiquitin K63R. Total cell lysates (TCL) and GHR immunoprecipitates were analysed on western blot using the indicated antibodies. EV, empty vector. C. Cells were transfected as in Fig. 1A and then treated for 15 min with GH at 37°C as indicated. GHR was immunoprecipitated from SDS-boiled lysates, and analysed for ubiquitination. 130, mature GHR, 110, precursor GHR. At the bottom of the figure the ratios of ubiquitinated versus 130kD-GHR are given. All data in this figure are representative of three independent experiments.

Figure 3. GH induces Jak2 phosphorylation and release from the GHR.

A. IM9 cells were treated with GH for 1 h at 37°C as indicated, followed by cell fractionation. Membrane (M) and cytosol (C) fractions were immunoprecipitated for Jak2 and analysed with the indicated antibodies. The ratio of phosphorylated Jak2 (pJak2) and total Jak2 was calculated for each fraction. Total cell lysates were analysed for Hsp90, a cytosolic marker. B. IM9 cells were pretreated with vehicle (dimethylsulfoxide), staurosporin or MG132 for 1 h and stimulated with GH at 37°C as indicated. Total cell lysates (TCL) and GHR immunoprecipitations were analysed on western blot with the indicated antibodies. C. IM9 cells were pretreated with staurosporin (Stau) for 1 h followed by incubation with GH at 37°C as indicated. Jak2 immunoprecipitations were analysed with the indicated antibodies. Data in A, B, and C are representative of three independent experiments.

Figure 4. GH induces Jak2 release from GHR.

Hek293-TR stable cell lines expressing GHR-Rluc and YFP-Jak2 (wt or Y119E) were seeded 24 h before the measurements; doxycycline was added in order to stimulate the expression of YFP-Jak2 constructs. BRET measurements were started immediately after GH addition (interval t = 0–4 min), or 10 min later (interval t = 10–14 min). A. Cells were lysed, and the proteins expression levels of the BRET pair in the cell lysates, were assessed by western blot, using the indicated antibodies. Data are representative for three independent experiments. Panel B shows the BRET ratios expressed in mBU, representative of 8 independent measurements ± S.E.M. (* significantly different from GHR-Rluc+YFP-Jak2 wt, minus GH condition, p<0.01; Φ significantly different from GHR-Rluc+YFP-Jak2 Y119E condition, p<0.01; Ψ significantly different from GHR-Rluc+YFP-Jak2 wt, plus GH [0–4 min] condition, p<0.01).

Figure 5. Decreased Jak2 activity in coated pit.

A. GHR-expressing Hek293 cells were pretreated with MβCD for 1 h, incubated with Cy3-GH for 30 min at 37°C and the fluorescence was visualised with a confocal microscope. Bar, 5 µm. B. Cells were pretreated as in Fig. 4A, followed by incubation for 15 min with GH at 37°C as indicated. Total cell lysates (TCL, upper panel) and GHR immunoprecipitations (lower panel) were analysed on western blot with the indicated antibodies. 130, mature GHR; 110, precursor GHR. C. Cells were silenced either for clathrin or for GFP (as a negative control), followed by incubation for 15 min with GH at 37°C as indicated. Total cell lysates (TCL, upper panel) and GHR immunoprecipitations (IP, lower panel) were analysed on western blot with the indicated antibodies. 130, mature GHR; 110, precursor GHR. Data are representative for two independent experiments (B).

Figure 6. GH-induced and constitutive GHR endocytosis share the same characteristics.

A. γ2A_GHR_Jak2 cells (left panel) and γ2A_GHR cells (right panel) were stimulated with 500 ng/ml GH for 90 min at 37°C as indicated. Total cell lysates (TCL) (lower panels) and GHR immunoprecipitates (upper panels) were analysed on western blot with the indicated antibodies. B. γ2A_GHR_Jak2 cells were transfected with control siRNA (lanes 1 and 2), clathrin heavy chain siRNA (CHC) (lanes 3 and 4) or TrCP siRNA (lane 5 and 6). Cells were stimulated with 500 ng/ml GH for 90 min at 37°C as indicated, after which total cell lysates (TCL) (3 lower panels) and GHR immunoprecipitates (upper panel) were analysed on western blot with the indicated antibodies. C. γ2A_Jak2 cells, stably transfected with wild-type GHR (WT), GHR mutated in its DSGxxS motif (DSG), GHR mutated in its UbE motif (UbE) and GHR with all cytosolic tyrosine mutated into phenylalanine residues (Y-less) were stimulated with 500 ng/ml GH at 37°C as indicated, after which total cell lysates (TCL) (2 lower panels) and GHR immunoprecipitates (upper panel) were analysed on western blot with the indicated antibodies. Cell lines used in C are stably transfected with the indicated GHR constructs, resulting in clonal variation of Jak2 levels. Data in A and B are representatives of three independent experiments. Data in C are representatives of two independent experiments. D. Quantification of the GH-induced GHR degradation (Y-less from experiment in C, and the data from the experiment in B), expressed as percentage of mature GHR.

Figure 7. mRNA levels of Jak2 and βTrCP2 in IM9, Hek293 and HepG2 cells.

mRNA levels were measured using qRT-PCR. Ratios of Jak2/βTrCP2 are displayed of IM9, Hek293 and HepG2 cells as mean values of six independent experiments ± SD.