Protein tyrosine phosphatase 1B attenuates growth hormone-mediated JAK2-STAT signaling

Abstract

Protein tyrosine phosphatase-1B (PTP-1B) attenuates insulin, PDGF, EGF, and IGF-I signaling by dephosphorylating tyrosine residues located in the tyrosine kinase domain of the corresponding receptors. More recently, PTP-1B was shown to modulate the action of cytokine signaling via the nonreceptor tyrosine kinase JAK2. Transmission of the growth hormone (GH) signal also depends on JAK2, raising the possibility that PTP-1B modulates GH action. Consistent with this hypothesis, GH increased the abundance of tyrosine-phosphorylated JAK2 associated with a catalytically inactive mutant of PTP-1B. GH-induced JAK2 phosphorylation was greater in knockout (KO) than in wild-type (WT) PTP-1B embryonic fibroblasts and resulted in increased tyrosine phosphorylation of STAT3 and STAT5, while overexpression of PTP-1B reduced the GH-mediated activation of the acid-labile subunit gene. To evaluate the in vivo relevance of these observations, mice were injected with GH under fed and fasted conditions. As expected, tyrosine phosphorylation of JAK2 and STAT5 occurred readily in the livers of fed WT mice and was almost completely abolished during fasting. In contrast, resistance to the action of GH was severely impaired in the livers of fasted KO mice. These results indicate that PTP-1B regulates GH signaling by reducing the extent of JAK2 phosphorylation and suggest that PTP-1B is essential for limiting the action of GH during metabolic stress such as fasting.

FIG. 1.

PTP-1B D181A binds JAK2 in hGH-stimulated cells. (A) 293LA cells were transiently transfected with GST vector (GST), GST- PTP-1B WT (GST-WT), and GST-PTP-1B D181A (GST-DA). The cells were incubated in the absence (−) or presence (+) of 1 μg of hGH/ml for 10 min. Proteins associated with glutathione-Sepharose (GST) beads were removed by centrifugation (Pulldown). The precipitated proteins were resolved by SDS-PAGE and immunoblotted with antibodies against pY^1007/1008JAK2 (P-JAK2). Immunoblotting with the anti-GST antibody demonstrated that comparable amounts of GST-containing proteins were pulled down from cells transfected with GST-PTP-1B WT and GST- PTP-1B D181A. (B) Precipitated materials (Pulldown) and total cell lysates (TCL; 10 μg) were resolved by SDS-PAGE and immunoblotted (IB) with antibodies against JAK2, JAK1, and JAK3. The results shown are representative of three independent experiments.

FIG. 2.

Hyperphosphorylation of JAK2 in PTP-1B-deficient fibroblasts stimulated with hGH. (A) WT (+/+) and PTP-1B KO (−/−) MEFs were stimulated with hGH (100 ng/ml) for the indicated times. Total cell lysates (20 μg) were subjected to SDS-PAGE and immunoblotted (IB) using antibodies specifically detecting total JAK2, GHR, and PTP-1B or tyrosine-phosphorylated JAK2 (P-JAK2). P-JAK2 signals were quantified by densitometry and are reported as the mean ± standard error of the mean of three independent experiments. (B) WT (+/+) and PTP-1B KO (−/−) MEFs and PTP-1B KO MEFs stably expressing Myc-tagged PTP-1B (rescued) were stimulated for 5 and 20 min with 100 ng of hGH/ml. Analysis of cell lysates by SDS-PAGE and immunoblotting were performed as described for panel A. Similar results were obtained with other rescued cell lines. (C) WT (+/+) and TC-PTP KO (−/−) MEFs were stimulated for 5 and 20 min with 100 ng of hGH/ml. Analysis of cell lysates by SDS-PAGE and immunoblotting were as described for panel A, with the additional detection of TC-PTP using a specific antibody. The results shown are representative of three independent experiments.

FIG. 2.

Hyperphosphorylation of JAK2 in PTP-1B-deficient fibroblasts stimulated with hGH. (A) WT (+/+) and PTP-1B KO (−/−) MEFs were stimulated with hGH (100 ng/ml) for the indicated times. Total cell lysates (20 μg) were subjected to SDS-PAGE and immunoblotted (IB) using antibodies specifically detecting total JAK2, GHR, and PTP-1B or tyrosine-phosphorylated JAK2 (P-JAK2). P-JAK2 signals were quantified by densitometry and are reported as the mean ± standard error of the mean of three independent experiments. (B) WT (+/+) and PTP-1B KO (−/−) MEFs and PTP-1B KO MEFs stably expressing Myc-tagged PTP-1B (rescued) were stimulated for 5 and 20 min with 100 ng of hGH/ml. Analysis of cell lysates by SDS-PAGE and immunoblotting were performed as described for panel A. Similar results were obtained with other rescued cell lines. (C) WT (+/+) and TC-PTP KO (−/−) MEFs were stimulated for 5 and 20 min with 100 ng of hGH/ml. Analysis of cell lysates by SDS-PAGE and immunoblotting were as described for panel A, with the additional detection of TC-PTP using a specific antibody. The results shown are representative of three independent experiments.

FIG. 3.

PTP-1B dephosphorylates membrane-associated JAK2. WT (+/+) and PTP-1B KO (−/−) MEFs were stimulated with 100 ng of hGH/ml for 10 min. Cell homogenates from a single 10-cm-diameter dish (Total) were separated in cytosolic (Cytosol) and membrane (Memb) fractions. Aliquots of each fraction (total, 10%; cytosol, 20%; membrane, 50%) were analyzed by SDS-PAGE and immunoblotting using specific antibodies against pY^1007/1008JAK2 (P-JAK2), JAK2, insulin receptor (IR), and PTP-1B. The IR presence was measured as a control for membrane fractionation. The results shown are representative of three independent experiments.

FIG. 4.

JAK2, STAT3, and STAT5 are hyperphosphorylated in PTP-1B-deficient primary fibroblasts upon hGH stimulation. Passage 4 WT (+/+) and PTP-1B KO (−/−) PMEFs were stimulated for 5 and 20 min with hGH (100 ng/ml). Cell lysates (20 μg) were analyzed by SDS-PAGE and immunoblotting (IB) using specific antibodies against total JAK2, STAT3, and STAT5 or their tyrosine-phosphorylated forms (P-JAK2, P-STAT3, and P-STAT5). The levels of the GHR and PTP-1B were also determined using specific antibodies. The results shown are representative of three independent experiments.

FIG. 5.

Overexpression of PTP-1B reduces the ability of GH to increase ALS promoter activity in H4-II-E cells. The luciferase plasmid mALS703WT driven by the mouse ALS promoter was transfected in H4-II-E cells with either an empty expression vector (Ctrl) or expression vectors encoding PTP-1B, TC-PTP, or PTP-PEST. The plasmid pRL-TK encoding Renilla luciferase was used to correct for variation in transfection efficiency. Transfected cells were incubated in serum-free medium in the absence or presence of 100 ng of GH/ml. After 24 h, firefly luciferase activity was measured in cell extracts and corrected for Renilla luciferase activity. The n-fold stimulation (mean ± standard error of two experiments) was calculated as the ratio of luciferase activity in the presence and absence of GH. The bar marked with an asterisk differs at P < 0.05, using one-way analysis of variance followed by the Fisher protected least-significant test.

FIG. 6.

Fasting-induced GH resistance is impaired in the livers of PTP-1B KO mice. PTP-1B WT (+/+) and PTP-1B KO (−/−) male mice were injected with PBS or hGH (0.5 μg of hGH/g of body weight) when fed or after a 48-h period of fasting. At the indicated time points after hGH injection, the livers were collected and extracts were prepared. The liver extracts were analyzed by SDS-PAGE and immunoblotting (IB) using specific antibodies against total JAK2, STAT3, STAT5, or their tyrosine-phosphorylated forms (P-JAK2, P-STAT3, and P-STAT5), as well as PTP-1B and TC-PTP. The results shown are representative of three independent experiments.

FIG. 7.

GH induced a higher expression of SOCS2 in fasted PTP-1B KO mouse livers. WT (+/+) and PTP-1B KO (−/−) mice fasted for 48 h, followed by hGH administration (1 μg/g of body weight). Livers were obtained from individuals before (zero time) and after (30 and 90 min) GH administration. Total RNA was isolated and analyzed by Northern blotting for the abundance of SOCS mRNA using the corresponding mouse cDNA probes. Signals detected with the various probes corresponded to an mRNA of 2.5 kb for CIS, 3.4 kb for SOCS2, and 3.2 kb for SOCS3. Each lane represents RNA from one mouse.