JAK2, but not Src family kinases, is required for STAT, ERK, and Akt signaling in response to growth hormone in preadipocytes and hepatoma cells

Abstract

Janus kinase 2 (JAK2), a tyrosine kinase that associates with the GH receptor and is activated by GH, has been implicated as a key mediator of GH signaling. Several published reports suggest that members of the Src family of tyrosine kinases may also participate in GH signaling. We therefore investigated the extent to which JAK2 and Src family kinases mediate GH activation of signal transducers and activators of transcription (STATs) 1, 3, and 5a/b, ERKs 1 and 2, and Akt, in the highly GH-responsive cell lines 3T3-F442A preadipocytes and H4IIE hepatoma cells. GH activation of Src family kinases was not detected in either cell line. Further, blocking basal activity of Src kinases with the Src inhibitors PP1 and PP2 did not inhibit GH activation of STATs 1, 3, or 5a/b, or ERKs 1 and 2. When levels of JAK2 were depressed by short hairpin RNA in 3T3-F442A and H4IIE cells, GH-stimulated activation of STATs 1, 3, and 5a/b, ERKs 1 and 2, and Akt were significantly reduced; however, basal activity of Src family kinases was unaffected. These results were supported genetically by experiments showing that GH robustly activates JAK2, STATs 3 and 5a/b, ERKs 1 and 2, and Akt in murine embryonic fibroblasts derived from Src/Yes/ Fyn triple-knockout embryos that lack known Src kinases. These results strongly suggest that JAK2, but not Src family kinases, is critical for transducing these GH signals in 3T3-F442A and H4IIE cells.

Figure 1

GH Does Not Activate Src Family Member Proteins A, 3T3-F442A preadipocytes were treated with either vehicle for 0 min (lane 1) or with GH (500 ng/ml) for the indicated times (lanes 2–5). Cell lysates were immunoblotted with αpY1007/1008-JAK2 (reprobed with αJAK2) and αpY416-Src (reprobed with αSrc) as indicated (n = 3). B, H4IIE hepatoma cells were treated with either vehicle for 0 min (lane 1) or with GH (500 ng/ml) for the indicated times (lanes 2–5). Proteins in an aliquot of H4IIE cell lysates were immunoprecipitated with αJAK2 before blotting with αpTyr and reprobing with αJAK2 as indicated. Proteins in aliquots of cell lysates were immunoblotted with αpY416-Src and reprobed with αSrc as indicated (n = 4). IB, Immunoblot; IP, immunoprecipitation.

Figure 2

GH-Stimulated Activation of STATs Is Not Blocked by Src Kinase Inhibitors A, 3T3-F442A preadipocytes were treated with vehicle (DMSO) (−) or 100 μm PP1, PP2, or PP3 for 60 min before addition of vehicle (−GH) or 500 ng/ml GH (+GH) for 15 min as indicated. Proteins in cell lysates were immunoblotted with αpY416-Src (reprobed with αSrc), αpY1007/1008-JAK2, αJAK2, αpY705-STAT3, αSTAT3, αpY694-STAT5, and αSTAT5 as indicated (n = 3). B, H4IIE hepatoma cells were treated with vehicle (DMSO) (−) or 100 μm PP1, PP2, or PP3 for 60 min before addition of vehicle (−GH) or 500 ng/ml GH (+GH) for 15 min as indicated. Proteins in an aliquot of H4IIE cell lysates were immunoprecipitated with αJAK2 before blotting with αpY1007/1008-JAK2 and reprobing with αJAK2 as indicated. Proteins in aliquots of cell lysates were immunoblotted with αpY416-Src (reprobed with αSrc), αpY701-STAT1 (reprobed with αSTAT1), αpY694-STAT5, and αSTAT5 as indicated (n = 3). IB, Immunoblot; IP, immunoprecipitation.

Figure 3

Effect of Src Kinase Inhibitors on GH Activation of ERKS 1 and 2 and Akt Proteins in aliquots of cell lysates from 3T3-F442A preadipocytes (A) and H4IIE hepatoma cells (B) used in Fig. 2 were immunoblotted with αpT202/pY204-ERK1/2 (reprobed with αERK1/2), αpS473-Akt, and αAkt as indicated (n = 3). IB, Immunoblot.

Figure 4

GH-Mediated STAT Activation Is Significantly Diminished When JAK2 Levels Are Reduced Using shRNA to JAK2 A, 3T3-F442A preadipocytes stably expressing either control shRNA or JAK2 shRNA were treated with either vehicle for 0 min or with GH (500 ng/ml) for the indicated times. Cell lysates were immunoblotted with αpY1007/1008-JAK2, αJAK2, αpY705-STAT3, αSTAT3, αpY694-STAT5, and αSTAT5 as indicated (n = 3). B, H4IIE hepatoma cells stably expressing either control shRNA or JAK2 shRNA were treated with either vehicle for 0 min or with GH (500 ng/ml) for the indicated times. Proteins in aliquots of H4IIE cell lysates were immunoprecipitated with αJAK2 before blotting with αpY1007/1008-JAK2 and reprobing with αJAK2 as indicated. Aliquots of cell lysates were immunoblotted with αpY701-STAT1, αSTAT1, αpY694-STAT5, and αSTAT5 as indicated (n = 3). IB, Immunoblot; IP, immunoprecipitation.

Figure 5

GH Activation of ERK 1, ERK 2, and Akt Is Diminished Substantially When JAK2 Levels Are Reduced Using shRNA to JAK2 Proteins in aliquots of the lysates of (A) 3T3-F442A preadipocytes stably expressing either control shRNA or JAK2 shRNA or (B) H4IIE hepatoma cells stably expressing either control shRNA or JAK2 shRNA used in Fig. 4 were immunoblotted with αpT202/pY204-ERK1/2, αERK1/2, αpS473-Akt, and αAkt as indicated (n = 3). IB, Immunoblot.

Figure 6

Src Family Kinase Activity Is Not Affected by Reducing Levels of JAK2 A, Proteins in aliquots of the lysates from 3T3-F442A preadipocytes stably expressing either control shRNA or JAK2 shRNA used in Fig. 4 were immunoblotted with αpY1007/1008-JAK2, αJAK2, and αpY416-Src (reprobed with αSrc) as indicated (n = 3). B, Proteins in aliquots of the lysates from H4IIE hepatoma cells stably expressing either control shRNA or JAK2 shRNA used in Fig. 4 were immunoprecipitated with αJAK2 before blotting with αpY1007/1008-JAK2 and reprobing with αJAK2 as indicated (n = 3). Proteins in aliquots of cell lysates were immunoblotted with αpY416-Src and reprobed with αSrc as indicated (n = 3). The top two panels for A and B are the same as the top two panels in Fig. 4, A and B, respectively. C, 3T3-F442A preadipocytes stably expressing either control shRNA or JAK2 shRNA were treated with either vehicle for 0 min or GH (500 ng/ml) for the indicated time. Aliquots were immunoprecipitated with αGHBP before blotting with αpTyr. Aliquots of cell lysates were immunoblotted with αGHR (AL47) as indicated (n = 3). GHR, GH receptor; IB, immunoblot; IP, immunoprecipitation.

Figure 7

GH Activates JAK2, STATs 1, 3, and 5, ERKs 1 and 2, and Akt in SYF MEF Cells SYF MEF cells were treated with either vehicle (0 min) or with GH (500 ng/ml) for the indicated time. Proteins in cell lysates were blotted with αpY416-Src (reprobed with αSrc), αpY1007/1008-JAK2 (reprobed with αJAK2), αpY705-STAT3, αSTAT3, αpY694-STAT5, αSTAT5, αpT202/Y204-ERK1/2 (reprobed with αErk1/2), αpS473-Akt, and αAkt as indicated (n = 3). IB, Immunoblot.

Figure 8

GH Is Unable to Activate STAT5 in MEFs Lacking JAK2 A, Wild-type MEFs or MEFs from JAK2^−/− mice were treated with either vehicle (0 min) or with GH (500 ng/ml) for the indicated times. Cell lysates were immunoblotted with αpY1007/1008-JAK2 (reprobed with αJAK2), αpY694-STAT5, αSTAT5, and αpY416-Src (reprobed with αSrc) as indicated (n = 3). B, MEFs from JAK2^−/− mice or the same MEFs in which JAK2 was stably reintroduced were treated with either vehicle (0 min) or with GH (500 ng/ml) for the indicated times. Cell lysates were immunoblotted with either αJAK2, αpY694-STAT5, or αSTAT5 as indicated (n = 2). IB, Immunoblot.