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Molecular Endocrinology 22 (8): 1825-1841
Copyright © 2008 by The Endocrine Society

JAK2, But Not Src Family Kinases, Is Required for STAT, ERK, and Akt Signaling in Response to Growth Hormone in Preadipocytes and Hepatoma Cells

Hui Jin1, Nathan J. Lanning1 and Christin Carter-Su

Department of Molecular and Integrative Physiology (H.J., C.C.-S.), Graduate Program in Cellular and Molecular Biology (N.J.L., C.C.-S.), University of Michigan Medical School, Ann Arbor, Michigan 48109-5622

Address all correspondence and requests for reprints to: Dr. Christin Carter-Su, Department of Molecular and Integrative Physiology, The University of Michigan Medical School, Ann Arbor, Michigan 48109-5622. E-mail: cartersu{at}umich.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GH IS A PEPTIDE hormone that is secreted into the circulation by the anterior pituitary. It is the primary hormone contributing to postnatal body growth (1, 2). It also regulates carbohydrate, fat, and protein metabolism (1, 2), immune and cardiac function (3), and aging (4) and has been implicated in cellular proliferation, differentiation, and survival (5). GH signaling pathways are initiated by GH binding to its receptor in the plasma membrane. This binding activates the GH receptor-associated tyrosine kinase Janus kinase 2 (JAK2), which in turn phosphorylates multiple tyrosines within both itself and the GH receptor (6, 7, 8). Multiple signaling molecules have been shown to be recruited to the activated GH receptor-JAK2 complex, leading to the activation of a variety of signaling pathways. Among these pathways are the signal transducer and activator of transcription (STAT), phosphatidylinositol 3-kinase (PI 3-kinase)/Akt, and MAPK/ERK signaling pathways (5, 7, 9).

Among the seven known mammalian STATs, STATs 1, 3, 5a, and 5b have been implicated as GH signaling molecules. In response to GH, these STATs become tyrosyl phosphorylated, dimerize, and translocate to the nucleus where they regulate target genes (9, 10). STATs 5a and 5b are thought to be particularly important mediators of GH responses, including body growth, adipose tissue development, and the sexually dimorphic expression of a number of hepatocyte-specific genes (11, 12, 13, 14, 15).

GH activation of ERKs 1/2 and the PI3-kinase/Akt pathway has been observed both in cell culture (16, 17, 18, 19, 20, 21) and in animals (22, 23, 24). Based upon in vitro studies using a number of cell types, several different mechanisms have been proposed by which GH activation of JAK2 leads to activation of ERKs 1 and 2. One proposed mechanism involves Shc as the adapter protein linking Grb2 to the activated GH receptor-JAK2 complex, which in turn initiates a Grb2/son of sevenless/Ras/Raf/ MAPK/ERK/ERK1/2 cascade (25, 26, 27). GH-induced activation of ERKs 1 and 2 has also been reported to involve JAK2 phosphorylation of the Grb2 binding site (tyrosine 1068) in the epidermal growth factor receptor and recruitment of Grb2 (22). Others (18, 28, 29) suggest that protein kinase C and/or PI3-kinase activity are required for GH activation of ERKs 1 and 2.

Similarly, several mechanisms for GH activation of the PI3-kinase/Akt pathway have been suggested. One proposed pathway involves JAK2 phosphorylating insulin receptor substrate (IRS) proteins, which in turn recruit the p85 subunit of PI3-kinase, thereby activating PI3-kinase (5, 19, 20, 30). Others have shown direct binding of the p85{alpha}- and β-subunits of PI3-kinase to phosphorylated tyrosine residues in the C terminus of the GH receptor, raising the possibility that GH may promote direct binding of p85 subunits to GH receptor (31).

Although JAK2 is generally believed to be the major tyrosine kinase initiating GH signaling pathways, several studies have suggested that Src family kinases are also capable of binding to the GH receptor and transducing GH signals. There are eight known members of the mammalian Src kinase family: c-Src, Yes, Fyn, Lyn, Lck, Hck, Fgr, and Blk (32). Like JAK2, c-Src, Yes, and Fyn are expressed in most tissues whereas the other Src family members are expressed predominantly in hematopoietic cells (33). Lck and Lyn are also expressed in neurons (32). Zhu et al. (34) showed that GH could activate Src and Fyn in NIH-3T3 cells and Src in Chinese hamster ovary (CHO) cells ectopically expressing GH receptor (72). Manabe et al. (36) showed that GH can increase Src activity in F-36 human leukemia cells. Based on experiments using the Src family kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), and antisense c-Src oligonucleotides, Manabe et al. also suggest that in F-36 human leukemia cells, Src activates STAT5 in lieu of JAK2. Similarly, Rowlinson et al. (37) report that in FDC-P1 myeloid cells, GH activation of ERKs 1 and 2 is dependent on a Src family kinase. Zhu et al. (34), using NIH-3T3 cells, also concluded that GH-induced activation of ERKs 1 and 2 is mediated by a JAK2-independent pathway involving c-Src.

In this study, we have examined the relative roles played by endogenous JAK2 and Src family kinases in GH signaling in two well-characterized, GH-responsive cell lines, 3T3-F442A preadipocytes and H4IIE hepatoma cells. GH is required for differentiation of 3T3-F442A preadipocytes into mature adipocytes (38) and regulates the actin cytoskeleton (39, 40). In the differentiated, adipocyte form of 3T3-F442A cells, GH regulates lipolysis, hormone-sensitive lipase (41), and rates of glucose transport (42). It also regulates the transcription of multiple genes, including IGF-1, a number of early response genes, and multiple genes encoding proteins that regulate carbohydrate and lipid metabolism (43). Maximal expression of these genes involves a variety of signaling molecules, including STATs 1, 3, and 5a/b, ERKs 1 and 2, and Akt (6, 16, 17, 18, 21, 44, 45, 46, 47, 48, 49). These signaling proteins have also been shown to be activated in H4IIE cells (50, 51, 52). H4IIE cells have been used to study the effect of GH on protein synthesis (53) and insulin responsiveness (52, 54, 55).

Using an antibody specific to the activated form of Src family members, we provide evidence that GH does not detectably activate Src family kinases in 3T3-F442A or H4IIE cells. Using Src family kinase inhibitors and short hairpin RNA (shRNA) to JAK2 in 3T3-F442A preadipocytes and H4IIE hepatoma cells, and mouse embryo fibroblasts (MEFs) from control, JAK2 knockout, or Src/Yes/Fyn triple knockout mice, we provide strong evidence that GH activation of STATs 1, 3, and 5, ERKs 1 and 2, and Akt are dependent on JAK2 but not Src family kinases. Our studies also reveal that moderate levels of activated JAK2 are sufficient for maximal GH activation of STAT5 in 3T3-F442A cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GH Does Not Activate Src Family Kinases in 3T3-F442A Preadipocytes or H4IIE Hepatoma Cells
As an initial step in investigating whether Src family kinases mediate actions of GH, we examined whether Src family kinases are activated by GH in the GH-responsive 3T3-F442A preadipocyte and H4IIE hepatoma cell lines. 3T3-F442A preadipocytes and H4IIE hepatoma cells were treated with vehicle alone or with GH (500 ng/ml) for various times. Lysates from these cells were subjected to SDS-PAGE and subsequent immunoblot analysis with anti-pY416-Src antibody ({alpha}pY416-Src), which recognizes the activated form of the Src family members c-Src, Lyn, Fyn, Lck, Yes, and Hck. Thus, it would be expected to recognize all forms of Src found in 3T3-F442A and H4IIE cells. As shown in Fig. 1Go, A and B (third panel, lane 1), Src family kinases were noticeably active in the basal state in both 3T3-F442A preadipocytes and H4IIE hepatoma cells, respectively. However, GH treatment failed to increase Src family kinase activity above basal levels in either cell line (Fig. 1Go, A and B, third panel, lanes 2–5). Levels of Src family proteins in cell lysates were also unchanged by GH, as judged by immunoblotting cell lysates with anti-Src antibody ({alpha}Src) (Fig. 1Go, A and B, bottom panel ). To verify that these cells were responsive to GH, the ability of GH to activate JAK2 in 3T3-F442A cells was assessed by blotting lysates with anti-pY1007/1008-JAK2 antibody ({alpha}pY1007/1008-JAK2) (Fig. 1AGo, top panel ). This antibody recognizes the phosphorylated form of tyrosine(s) 1007 and/or 1008, the activating tyrosines in the kinase domain of JAK2. Phosphorylation of tyrosine 1007 is required for JAK2 activity (56) and generally mirrors overall tyrosyl phosphorylation of JAK2 assessed using an antiphosphotyrosine antibody ({alpha}pY) (57). In contrast to what was observed for Src family kinases, GH caused a rapid and transient phosphorylation of JAK2 on Tyr1007/1008. Phosphorylation of JAK2 was observed as early as 5 min after GH addition, was maximal at 15 and 30 min, and started to decline within 45 min after GH addition (Fig. 1AGo, top panel ). The ability of GH to activate JAK2 in the H4IIE hepatoma cells was assessed by immunoprecipitating JAK2 using {alpha}JAK2 and blotting with {alpha}pY (Fig. 1BGo, top panel ). GH caused a similar rapid and transient tyrosyl phosphorylation of JAK2 in H4IIE cells. A robust signal was evident within 5 min after GH addition and returned to near-basal levels by 60 min. Blotting {alpha}JAK2 immunoprecipitates with {alpha}pY1007/1008-JAK2 revealed a similar time course (see Fig. 4BGo, top panel, lanes 1–5). Immunoblotting cell lysates with {alpha}JAK2 (Fig. 1Go, A and B, second panels) indicated that endogenous levels of JAK2 were similar for all conditions for both cell types. Taken together, the data in Fig. 1Go suggest that whereas GH rapidly and substantially activates JAK2, GH does not appreciably increase total Src family kinase activity in 3T3-F442A preadipocytes or H4IIE hepatoma cells.


Figure 1
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  		Fig. 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 {alpha}pY1007/1008-JAK2 (reprobed with {alpha}JAK2) and {alpha}pY416-Src (reprobed with {alpha}Src) 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 {alpha}JAK2 before blotting with {alpha}pTyr and reprobing with {alpha}JAK2 as indicated. Proteins in aliquots of cell lysates were immunoblotted with {alpha}pY416-Src and reprobed with {alpha}Src as indicated (n = 4). IB, Immunoblot; IP, immunoprecipitation.

 

Figure 4
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  		Fig. 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 {alpha}pY1007/1008-JAK2, {alpha}JAK2, {alpha}pY705-STAT3, {alpha}STAT3, {alpha}pY694-STAT5, and {alpha}STAT5 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 {alpha}JAK2 before blotting with {alpha}pY1007/1008-JAK2 and reprobing with {alpha}JAK2 as indicated. Aliquots of cell lysates were immunoblotted with {alpha}pY701-STAT1, {alpha}STAT1, {alpha}pY694-STAT5, and {alpha}STAT5 as indicated (n = 3). IB, Immunoblot; IP, immunoprecipitation.

 
Inhibition of Src Family Kinases Does Not Affect GH Activation of JAK2 or Tyrosyl Phosphorylation of STATs 1, 3, or 5
Multiple Src family members exist in 3T3-F442A preadipocytes and H4IIE hepatoma cells. Because {alpha}pY416-Src may detect different Src family members with different affinities and Src family kinases are basally active in these cells, it is possible that we were unable to detect a small GH-induced increase in kinase activity of one or more Src kinase family members that might be important for GH signal transduction. Alternatively, GH might increase Src kinase-substrate interactions by altering the subcellular location of already active Src kinases or the availability of Src kinase substrates. We therefore examined whether Src family inhibitors would inhibit GH activation of JAK2 or the activation of a variety of other GH signaling molecules. 3T3-F442A preadipocytes and H4IIE hepatoma cells were pretreated with vehicle [dimethyl sulfoxide (DMSO)], the Src family inhibitors, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) or PP2, or their inactive analog, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3) (58, 59), for 60 min before GH (500 ng/ml) was added for 15 min. As predicted, PP1 and PP2, but not PP3, inhibited Src family kinase activity (assessed using {alpha}pY416-Src) in both untreated (Fig. 2Go, A and B, top panel, lanes 1–4) and GH-treated cells (Fig. 2Go, A and B, top panel, lanes 5–8). In contrast, the number of JAK2 proteins activated in response to GH, assessed using {alpha}pY1007/1008-JAK2 (Fig. 2Go, A and B, third panel, lanes 5–8) and normalized for the amount of JAK2 (Fig. 2Go, A and B, fourth panel, lanes 5–8) present in each lane, was similar for cells pretreated with vehicle or inhibitors. The lack of a change in JAK2 activation with the Src inhibitors suggests that Src family kinases do not play a role in GH activation of JAK2 in 3T3-F442A preadipocytes or H4IIE hepatoma cells.


Figure 2
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  		Fig. 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 {alpha}pY416-Src (reprobed with {alpha}Src), {alpha}pY1007/1008-JAK2, {alpha}JAK2, {alpha}pY705-STAT3, {alpha}STAT3, {alpha}pY694-STAT5, and {alpha}STAT5 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 {alpha}JAK2 before blotting with {alpha}pY1007/1008-JAK2 and reprobing with {alpha}JAK2 as indicated. Proteins in aliquots of cell lysates were immunoblotted with {alpha}pY416-Src (reprobed with {alpha}Src), {alpha}pY701-STAT1 (reprobed with {alpha}STAT1), {alpha}pY694-STAT5, and {alpha}STAT5 as indicated (n = 3). IB, Immunoblot; IP, immunoprecipitation.

 
The contribution of Src family kinase activity to GH activation of STAT proteins was assessed using antibodies that recognize the tyrosyl-phosphorylated, activated forms of STAT1 ({alpha}pY701-STAT1), STAT3 ({alpha}pY705-STAT3), or STATs 5a and 5b ({alpha}pY694-STAT5). Phosphorylation of tyrosines 701 (60), 705 (61), and 694/699 (62, 63) is required for activation of STATs 1, 3, and 5a/5b, respectively. Because neither {alpha}pY694-STAT5 nor {alpha}STAT5 can distinguish between the very similar STAT5a and STAT5b, we shall use the term STAT5 to indicate both STAT5a and STAT5b. Figure 2AGo (lanes 5–8) reveals similar levels of GH-stimulated tyrosyl phosphorylation of STAT3 (fifth panel ) and similar levels of GH-stimulated tyrosyl phosphorylation of STAT5 (seventh panel ) in 3T3-F442A cells treated with or without the Src family inhibitors PP1 and PP2. PP1 and PP2 also had no effect on the ability of GH to activate STAT1 (Fig. 2BGo, fifth panel, lanes 5–8) or STAT5 in H4IIE cells (Fig. 2BGo, seventh panel, lanes 5–8) or levels of the different STATs in either 3T3-F442A or H4IIE cells (Fig. 2Go, A and B, sixth and bottom panels). Thus, Src family kinases appear not to play a significant mediator role in GH activation of STAT proteins in either 3T3-F442A preadipocytes or H4IIE hepatoma cells.

Effect of Src Family Inhibitors on GH Activation of ERK 1, ERK 2, and Akt
We next examined whether Src family kinases are important for GH activation of ERK1, ERK2, or Akt. Dual phosphorylation of ERK1 on T202 and Y204 and ERK2 on T185 and Y187 (numbering system of human ERKs) is required for their activation. Proteins in aliquots of 3T3-F442A and H4IIE cell lysates from Fig. 2Go were blotted with an antibody that specifically recognizes ERKs 1 and 2 that are phosphorylated on both the activating Thr and Tyr ({alpha}pT202/pY204-ERK1/2). As seen in Fig. 3Go, A and B (top panel, lanes 1 and 5), GH activated ERKs 1 and 2 in 3T3-F442A preadipocytes and H4IIE hepatoma cells. In 3T3-F442A cells, this activation was not reduced when cells were pretreated with PP1, PP2, or PP3 (Fig. 3AGo, top panel, lanes 5–8). In H4IIE cells, PP1 and PP3 reduced GH activated ERKs 1 and 2, whereas PP2 had no effect (Fig. 3BGo, lanes 5–8, first panel ). When comparing these results to the results seen in Fig. 2BGo (lanes 5–8, top panel ), it becomes apparent that the effect of PP1 on ERK activation in H4IIE cells is not specific to inhibition of Src family kinases. This comparison shows that when Src family kinase activity is undetectable due to pharmacological inhibition by PP2, GH is still able to fully activate ERKs 1 and 2 (compare Fig. 2BGo, lane 7, top panel, to Fig. 3BGo, lane 7, top panel ). Therefore, the ability of PP1 to inhibit GH-mediated ERK 1 and 2 activation in H4IIE cells cannot be ascribed to a lack of Src family kinase activity. Taking this together with the observation that the negative control (PP3) also significantly inhibits GH-mediated activation of ERKs 1 and 2 but not Src family kinase activity, the conclusion can be drawn that in rat hepatoma cells, PP1 and PP3 inhibit ERK 1 and 2 activation in a non-Src family kinase-specific manner. Thus, these inhibitor studies fail to implicate Src family kinases in GH-mediated activation of ERKs 1 and 2 in 3T3-F442A or H4IIE cells. Whether the PP1/2/3 pattern of inhibition indicates the direct or indirect contribution of some other enzyme to the activation of ERKs 1 and 2 in H4IIE cells, but not 3T3-F442A cells, is not known. However, the pattern of inhibition does not fit that of tested kinases (64, 65).


Figure 3
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  		Fig. 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. 2Go were immunoblotted with {alpha}pT202/pY204-ERK1/2 (reprobed with {alpha}ERK1/2), {alpha}pS473-Akt, and {alpha}Akt as indicated (n = 3). IB, Immunoblot.

 
Finally, we sought to determine whether Src family kinases are important for GH-stimulated activation of PI3-kinase signaling. Akt, a downstream signaling molecule of PI3-kinase, requires phosphorylation on Ser 473 to be active (66). As shown in Fig. 3Go, A and B (third panel, lane 1 vs. 5) phosphorylation of Akt on Ser 473 was robust in both 3T3-F442A preadipocytes and H4IIE hepatoma cells stimulated with GH. In both cell types, this level of phosphorylation was substantially reduced in cells pretreated with PP1 and PP2 (Fig. 3Go, A and B, third panel, lanes 5–8). A more modest inhibition was observed in cells pretreated with PP3. The ability of PP1 and PP2 to inhibit phosphorylation of Ser473 in Akt in response to GH raises the possibility that Src family kinases are important for GH activation of Akt. However, PP1 and PP2 (and to a lesser extent PP3) were also found to reduce basal levels of Akt phosphorylation in both 3T3-F442A and H4IIE cells (Fig. 3Go, A and B, third panel, lanes 1–4), indicating that Src family kinase activity may not mediate GH activation of Akt per se. Rather, GH activation of Akt may depend on basal priming of Akt by basally active Src. Taken together, the results of Figs. 2Go and 3Go, in which Src family kinases were inhibited in 3T3-F442A preadipocytes and H4IIE hepatoma cells, do not support the hypothesis that Src family kinases play a mediator role in the ability of GH to activate JAK2, STATs 1, 3, or 5, or ERKs 1 and 2. However, they do suggest that GH activation of Akt may require basal Src family kinase activity. Because of the inhibition of basal Akt activity by inhibitors of Src family kinases, these experiments are unable to address whether or not Src family kinases play a mediator role in GH activation of Akt.

GH-Stimulated Activation of STATs 1, 3, and 5, ERKs 1 and 2, and Akt Is Diminished When Endogenous JAK2 Levels Are Reduced in 3T3-F442A Preadipocytes and H4IIE Hepatoma Cells
To determine the degree to which JAK2 is required for GH-mediated activation of STAT proteins, ERKs 1 and 2, and Akt, we examined the ability of GH to activate these signaling molecules when endogenous JAK2 levels were reduced in both 3T3-F442A preadipocytes and H4IIE hepatoma cells. 3T3-F442A preadipocytes and H4IIE hepatoma cells stably expressing control shRNA (Fig. 4Go, A and B, second panel, lanes 1–5) or JAK2 shRNA (Fig. 4Go, A and B, second panel, lanes 6–10) were treated with vehicle or GH (500 ng/ml) for various times. Immunoblotting cell lysates with {alpha}JAK2 indicated at least an 80% reduction of endogenous JAK2 protein levels in 3T3-F442A preadipocytes (83% ± 6%, n = 3) and H4IIE hepatoma cells (89% ± 4%, n = 3), as determined by quantification of JAK2 bands. Immunoblotting with {alpha}pY1007/1008-JAK2 confirmed that levels of activated JAK2 are decreased to a similar extent as levels of total JAK2 in the JAK2 shRNA expressing 3T3-F442A and H4IIE cells (Fig. 4Go, A and B, top panels).

In control shRNA 3T3-F442A cells, GH-stimulated phosphorylation of STAT3 on Y705 was detectable within 5 min and maximal at 30 min (Fig. 4AGo, third panel, lanes 1–5). GH-stimulated tyrosyl phosphorylation of STAT5 on Y694 was also detectable within 5 min but remained elevated even after 45 min (Fig. 4AGo, fifth panel, lanes 1–5). Reduction of levels of endogenous JAK2 using shRNA resulted in a substantially reduced GH-dependent phosphorylation of both STAT3 (by 68% ± 4%, n = 3) and STAT5 (by 47% ± 7%, n = 3) (Fig. 4AGo, third and fifth panels, respectively). Reduction of endogenous JAK2 did not alter levels of STAT3 or STAT5 (Fig. 4AGo, fourth and sixth panels, respectively). In control shRNA expressing H4IIE cells, GH-stimulated phosphorylation of both STAT1 on Y701 and STAT5 on Y694 was detectable within 5 min, was maximal at 15 min, and returned to near-basal values by 60 min. In the shRNA-JAK2 H4IIE cells, GH-dependent phosphorylation of both STAT1 and STAT5 was substantially reduced at all time points (Fig. 4BGo, third and fifth panels, respectively). Reduction of endogenous JAK2 did not alter levels of STAT1 or STAT5 (Fig. 4BGo, fourth and sixth panels, respectively). Thus, reduction of endogenous JAK2 substantially reduces the ability of GH to activate STAT proteins in both 3T3-F442A preadipocytes and H4IIE hepatoma cells.

We next determined the importance of JAK2 for GH-mediated activation of ERKs 1 and 2. GH stimulation of control shRNA 3T3-F442A preadipocytes resulted in the transient activation of ERKs 1 and 2, which was evident within 5 min and over by 45 min (Fig. 5AGo, top panel, lanes 1–5). Activation of ERKs 1 and 2 was almost eliminated in cells expressing JAK2 shRNA, being detectable above basal values only at the 5-min time point (Fig. 5AGo, top panel, lanes 6–10). GH stimulation of control shRNA H4IIE hepatoma cells resulted in a relatively modest increase in activation of ERKs 1 and 2, visible in Fig. 5BGo (top panel, lanes 1–5) at the 15- and 30-min time points. No GH stimulation of ERKs 1 and 2 was detectable in the JAK2 shRNA hepatoma cells (Fig. 5BGo, top panel, lanes 6–10). Reduction of endogenous JAK2 did not alter levels of ERKs 1 or 2 in either cell line (Fig. 5Go, A and B, second panels). Thus, reduction of endogenous JAK2 substantially reduces the ability of GH to activate ERKs 1 and 2 in both 3T3-F442A preadipocytes and H4IIE hepatoma cells.


Figure 5
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  		Fig. 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. 4Go were immunoblotted with {alpha}pT202/pY204-ERK1/2, {alpha}ERK1/2, {alpha}pS473-Akt, and {alpha}Akt as indicated (n = 3). IB, Immunoblot.

 
For Akt, GH (500 ng/ml) caused a robust increase in phosphorylation of Ser473 within 5 min in both control shRNA 3T3-F442A preadipocytes and H4IIE hepatoma cells (Fig. 5Go, A and B, third panels, lanes 1–5). Phosphorylation was sustained for 30 min in the 3T3-F442A cells before declining, whereas it started declining within 15 min in the H4IIE cells. For both cell types, however, phosphorylation of Ser473 was reduced to barely detectable values at all time points by the reduction of JAK2 with shRNA JAK2 (Fig. 5Go, A and B, third panels, lanes 6–10). Akt levels were not similarly reduced in either control or shRNA JAK2 expressing cells (Fig. 5Go, A and B, fourth panels). The fact that reducing JAK2 is so effective in reducing levels of GH activation of Akt supports the hypothesis that JAK2 is the primary kinase responsible for GH activation of the PI3 kinase-Akt pathway.

To rule out the possibility that reduction of endogenous JAK2 reduces the level of Src family kinases, lysates from both 3T3-F442A and H4IIE cells were blotted with {alpha}pY416-Src and {alpha}Src (Fig. 6Go, A and B, third and fourth panels). No differences in levels of Src activation or Src protein were observed between control and JAK2 knockdown cells, indicating that reducing the level of JAK2 does not affect basal Src family kinase activity. Thus, basal Src family kinase activity appears to be independent of JAK2. To test whether the decreased responsiveness to GH of the JAK2 shRNA cells compared with the control shRNA cells could be a result of reduced expression of the GH receptor, control and shRNA-expressing 3T3-F442A cells were treated with GH for 15 min. GH receptor levels were similar in control and JAK2 shRNA-expressing cells as shown by blotting lysates with antibody to the intracellular domain of the GH receptor (Fig. 6CGo, middle panel ). When GH receptor was immunoprecipitated using antibody to the extracellular domain of the GH receptor and blotted with {alpha}PY, tyrosyl phosphorylation of GH receptor was found to be reduced in JAK2 shRNA cells (Fig. 6CGo, bottom panel ) compared with control shRNA cells, as one would predict from the decreased levels of JAK2. Taken together, the data of Figs. 4–6GoGoGo indicate that JAK2 is required for maximal GH-mediated activation of STATs 1, 3, and 5, ERKs 1 and 2, and Akt. In contrast, Src family kinase activity is independent of JAK2.


Figure 6
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  		Fig. 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. 4Go were immunoblotted with {alpha}pY1007/1008-JAK2, {alpha}JAK2, and {alpha}pY416-Src (reprobed with {alpha}Src) 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. 4Go were immunoprecipitated with {alpha}JAK2 before blotting with {alpha}pY1007/1008-JAK2 and reprobing with {alpha}JAK2 as indicated (n = 3). Proteins in aliquots of cell lysates were immunoblotted with {alpha}pY416-Src and reprobed with {alpha}Src as indicated (n = 3). The top two panels for A and B are the same as the top two panels in Fig. 4Go, 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 {alpha}GHBP before blotting with {alpha}pTyr. Aliquots of cell lysates were immunoblotted with {alpha}GHR (AL47) as indicated (n = 3). GHR, GH receptor; IB, immunoblot; IP, immunoprecipitation.

 
GH Activates JAK2, STATs 1, 3, and 5, ERKs 1 and 2, and Akt in Cells Lacking Src Family Members
To provide genetic evidence that JAK2, but not Src family kinases, plays a direct and essential role in GH-induced activation of signaling molecules, mouse embryo fibroblasts derived from embryos from src/yes/fyn (SYF) triple knockout mice (67) were tested for their responses to GH. SYF MEF cells also lack Lyn expression. Thus, these cells lack all Src family members reported to be present in murine fibroblasts (67). This absence of Src family members was confirmed by the absence of any detectable signal in {alpha}Src or {alpha}pY416 Src immunoblots of SYF MEF cells (Fig. 7Go, top two panels; compare with wild-type MEFs shown in Fig. 8AGo, fifth and sixth panels, lanes 1–5). SYF MEFs were treated with vehicle alone or with GH (500 ng/ml) for various times. Immunoblots of lysates from these cells revealed that JAK2, STAT3, STAT5, ERK1, ERK2, and Akt are all robustly phosphorylated in response to GH (Fig. 7Go). These results provide strong evidence that Src family kinases are dispensable for GH activation of these signaling proteins.


Figure 7
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  		Fig. 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 {alpha}pY416-Src (reprobed with {alpha}Src), {alpha}pY1007/1008-JAK2 (reprobed with {alpha}JAK2), {alpha}pY705-STAT3, {alpha}STAT3, {alpha}pY694-STAT5, {alpha}STAT5, {alpha}pT202/Y204-ERK1/2 (reprobed with {alpha}Erk1/2), {alpha}pS473-Akt, and {alpha}Akt as indicated (n = 3). IB, Immunoblot.

 

Figure 8
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  		Fig. 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 {alpha}pY1007/1008-JAK2 (reprobed with {alpha}JAK2), {alpha}pY694-STAT5, {alpha}STAT5, and {alpha}pY416-Src (reprobed with {alpha}Src) 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 {alpha}JAK2, {alpha}pY694-STAT5, or {alpha}STAT5 as indicated (n = 2). IB, Immunoblot.

 
GH Is Unable to Activate STAT5 in JAK2–/– MEFs
The finding that in shRNA-JAK2 3T3-F442A cells, GH stimulation of STAT5 phosphorylation was reduced to a lesser extent than levels of JAK2 (Fig. 4AGo) allows for the possibility that some other kinase contributes to that stimulation. The SYF MEF and Src family inhibitor studies suggest that members of the Src family of tyrosine kinases do not contribute. However, those results do not allow us to rule out another, unidentified kinase. To test the possibility that some kinase other than JAK2 mediates GH activation of STAT5, we examined to what degree deleting all JAK2 would block GH activation of STAT5. Wild-type and JAK2–/– MEFs were treated with vehicle alone or with GH (500 ng/ml) for various times. Immunoblotting cell lysates with {alpha}JAK2 confirmed the complete loss of JAK2 expression in the JAK2–/– MEF cells (Fig. 8AGo, second panel ). As predicted, blotting the lysates with {alpha}pY1007/1008-JAK2 showed that, similar to what we observed in 3T3-F442A and HEII4 cells, JAK2 was rapidly and transiently phosphorylated on Tyr1007/1008 in wild-type MEFs whereas no phosphorylated JAK2 was detectable in JAK2–/– MEFs (Fig. 8AGo, top panel ). Similar to what we observed with lysates from SYF MEF cells, immunoblots of the wild-type MEF lysates with {alpha}pY694-STAT5 revealed a robust activation of STAT5 that was evident at 5 min and maximal at 10–15 min (Fig. 8AGo, lanes 1–5, third panel ). However, there was no detectable phosphorylated STAT5 in the lysate from JAK2–/– MEFs (Fig. 8AGo, lanes 6–10, third panel ), suggesting a total dependency on JAK2 of STAT5 activation. Blotting the lysates with {alpha}pY416-Src showed no difference in Src kinase phosphorylation after GH addition or between the wild-type and JAK2–/– MEFs (Fig. 8AGo, fifth and sixth panels). Levels of STAT5 and Src were similar in wild-type and JAK2–/– MEFs (Fig. 8AGo, fourth and sixth panels). Therefore, the loss of activation of STAT5 in JAK2–/– cells is most likely due to the lack of JAK2, and not indirectly to changes in the activity of Src family kinase activity or any other kinases. Reintroducing wild-type JAK2 into JAK2–/– MEFs rescued GH-stimulated STAT5 activation, to a level commensurate with the level of reintroduced JAK2 (Fig. 8BGo), providing further support for the hypothesis that JAK2 is required for GH activation of STAT5.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Because members of the cytokine family of receptors do not have intrinsic kinase activity, the signaling events initiated by cytokines are achieved through activation of receptor-associated tyrosine kinases, primarily members of the JAK family of tyrosine kinases (reviewed in Ref. 68). However, some cytokine receptors have been reported to bind to and activate members of the Src family of tyrosine kinases (32). In the case of GH, JAK2 has classically been thought to be the major kinase responsible for initiating downstream signaling events although JAK1 and JAK3 have been shown to be minimally activated in some cells (69, 70). Multiple studies have demonstrated an interaction between GH receptor and JAK2 and a robust activation of JAK2 after GH binding of GH receptor (6, 7, 71). However, several studies have suggested that GH may also activate members of the Src family of tyrosine kinases and that activation of Src family members may contribute to activation of signaling molecules downstream of GH, including STAT5 and ERKs 1 and 2 (34, 36).

In this study, we sought to determine the relative contribution of Src family kinases to GH signaling by assessing the activation status of endogenous GH-signaling proteins in cell lines that have been well characterized for GH signaling and GH responses. Using an antibody to the phosphorylated form of the activating tyrosine in JAK2 to assess levels of activated JAK2, we found that JAK2 was inactive in both 3T3-F442A and H4IIE cells that had not seen GH. Upon GH treatment, JAK2 was rapidly and transiently activated, as reported previously (6, 50). In contrast, using an antibody that recognizes the phosphorylated form of the activating tyrosine in Src family kinases to assess levels of activated Src family kinases, we found that Src kinases are basally active in 3T3-F442A and H4IIE cells. GH treatment did not appreciably enhance that activity at early time points (30 sec and 2 min) (data not shown) or over an extended period of treatment (up to 60 min) (Fig. 1Go). Thus, it seems unlikely that in these cell types, Src kinases can substitute for JAK2 as important mediators of GH action, unless GH activates only a small, undetectable, subset of the Src family kinases or increases Src kinase-substrate interactions (e.g. by altering the subcellular location of already active Src kinases or the availability of Src kinase substrates). Similarly, Yamauchi et al. (22) reported seeing no GH-induced increase in Src activity. The reason why these results differ from those of the groups observing a GH-induced increase in the activity of Src family kinases is not clear. Possible explanations include differences in culture conditions or cell type. Relevant to the former, we observed no GH-induced increase in Src kinase activity in either subconfluent (70–80%) or confluent 3T3-F442A cells (data not shown). Regarding the latter, all the different groups used different cell types. Zhu et al. reported GH induced activity of Src kinases in CHO cells overexpressing GH receptor (c-Src and c-Fyn) (72) and in NIH-3T3 cells (c-Src) (34), whereas Manabe et al. (36) and Rowlinson et al. (37) reported GH induction of Src using F-36P human leukemia cells (c-Src) and FDC-P1 myeloid cells (Lyn), respectively. Circulating cells, such as the F-36P and FDC-P1 cells, in general seem to have a greater propensity for utilizing Src family kinases for cytokine signaling compared with noncirculating cell types (32), raising the question of whether the ratio of Src family kinases to JAK2 is higher in these cells or they have accessory proteins that enable or enhance cytokine activation of Src kinases. It is interesting to note that even when GH was observed to activate Src family kinases, the degree of stimulation when assessed quantitatively, was quite modest, between 30–70% (72), in contrast to the degree of GH stimulation of JAK2 that generally shows a robust on/off type of response. It is also important to note that we found the activity of Src family kinases to be unaffected by the level of JAK2 and vice versa. Thus, reducing levels of JAK2 in 3T3-F442A and H4IIE cells by shRNA to JAK2 or in MEFs genetically deleted for JAK2 did not decrease the level of activity of Src kinases, nor did reducing the activity of Src family kinases using PP1 and PP2 alter the ability of GH to activate JAK2. This independence of Src and JAK2 activity supports the previous findings of Zhu et al. (34) in NIH-3T3 cells using both pharmacological inhibitors (PP1, PP2, and AG490) and dominant-negative constructs of Src and JAK2. It also argues against Src being recruited to GH receptor-JAK2 complexes and being activated as a consequence of binding to tyrosines within JAK2 or GH receptor that are phosphorylated by JAK2 in response to GH.

The fact that GH did not appear to activate Src family kinases in our experiments does not a priori exclude them from being mediators of GH signaling, because it is possible that GH elicits a small, undetectable increase in the activity of one of more Src kinases, alters the subcellular location of already active Src kinases, or alters the availability of Src kinase substrates. However, our data using the Src family kinase inhibitors PP1 and PP2 reveal that blocking the activity of Src family kinases in 3T3-F442A preadipocytes and H4IIE hepatoma cells does not attenuate GH-mediated activation of STATs 1, 3, or 5, indicating that activation of these signaling molecules by GH is independent of Src in these cells. The inability of PP1 and PP2 to block GH activation of STAT5 is consistent with the previous report of Guren et al. (73) showing no reduction in cultured rat hepatocytes of GH-mediated STAT5 activation by a different Src kinase inhibitor, CGP77675. It is also consistent with the finding that STAT5 is activated by GH in CHO cells stably expressing wild-type GH receptor but not in CHO cells stably expressing a mutated GH receptor lacking the binding site for JAK2 (48, 69). However, it contrasts with the finding of Manabe et al. (36), who showed a PP2-dependent, src antisense oligonucleotide-sensitive, inhibition of GH-mediated STAT5 phosphorylation in F-36P cells. One explanation for the apparent discrepancy between these studies is a difference in cell type, with fibroblasts, preadipocytes, and hepatocytes relying solely on JAK2 for GH activation of STAT5 and circulating cells being able to utilize Src family kinases in addition to, or in place of, JAK2. Unfortunately, in the latter study, the authors did not explore the relative contributions of JAK2 and Src kinases to the GH activation of STAT5, so that it is unclear whether in F-36P cells, Src family kinases mediate or modulate GH activation of STAT5, and whether that action is independent of JAK2. In further support of JAK2, and not Src kinases, being responsible for GH activation of STAT5, we observed a robust activation of STAT5 by GH in SYF MEFs that are genetically deleted for Src family kinases and the absence of STAT5 activation in MEFs genetically deleted for JAK2. The latter was rescued upon reintroduction of JAK2. GH activation of STAT5 was also significantly decreased in 3T3-F442A and H4IIE cells with reduced levels of JAK2 due to expression of shRNA to JAK2.

Similar to our results with STAT5, our findings with STATs 1 and 3 suggest that their activation by GH is highly dependent upon JAK2 and independent of Src family kinases. The independence from Src family kinases is supported by the findings that PP1 and PP2 eliminated Src activity but had no effect on the ability of GH to activate STATs 1 and 3 in H4IIE and 3T3-F442A cells, respectively. In addition, STAT3 was robustly activated by GH in SYF MEFs that are genetically deleted for Src family kinases. In support of their activation being dependent upon JAK2, GH activation of STATs 1 and 3 above basal values was severely depressed in H4IIE and 3T3-F442A cells, respectively, in which JAK2 levels were reduced using shRNA to JAK2. The dependence of GH activation of STATs 1 and 3 on JAK2 is consistent with the finding of Han et al. (75) that STATs 1 and 3 are activated by GH in wild-type H1080 cells but not in H1080 cells lacking intact JAK2.

Our Src family kinase chemical inhibitor and SYF MEF experiments also demonstrate that GH-mediated activation of ERKs 1 and 2 is not dependant on Src family kinases in 3T3-F442A, H4IIE, or MEF cells. This finding is consistent with the previous findings that Shc phosphorylation and MAPK activity are stimulated by GH in CHO-GH receptor cells but not in CHO cells stably expressing a mutated GH receptor lacking the binding site for JAK2 (25, 76). It is also consistent with the report that Shc phosphorylation is stimulated by GH in wild-type H1080 cells but not in H1080 cells lacking JAK2 (75). Zhu et al. (34) and Ling et al. (77) proposed a JAK-independent, Src-dependent mechanism for activation of ERKs 1 and 2 based on the observations that 1) GH activates c-Src (as well as JAK2) in NIH-3T3 cells, 2) GH stimulates RalA and RalB, 3) GH-activated RalA results in an increase in phospholipase D activity and the production of phosphatidic acid, and 4) RalA, phospholipase D activity, and phosphatidic acid are all required for GH-stimulated activation of ERKs 1 and 2 as assessed using an Elk-1 reporter assay. However, this group did not actually test directly the effects of decreasing levels of JAK2 or Src family kinase activity (by use of pharmacological inhibitors or decreasing levels of expression) on the ability of GH to activate ERKs 1 and 2. Thus, the relative contributions of JAK2 and Src kinases to GH activation of ERKs 1 and 2 are not clear, nor is it clear from those studies whether Src is sufficient, or simply necessary, for GH activation of ERKs 1 and 2. Finally, Gu et al. (78) raise the possibility of Src regulating GH-mediated activation of ERK2 by showing that overexpression of Csk (a protein that inactivates Src family kinases) in cardiac myocytes inhibits the ability of GH to activate overexpressed ERK2. Unfortunately, Src family kinase and JAK2 activities were not assessed in the context of Csk overexpression, raising the possibility that this effect of Csk overexpression was not Src family kinase specific. Furthermore, inhibitors of JAK2, epidermal growth factor receptor, and Src all blocked GH stimulation of ERKs 1 and 2 in these cells, confounding the assessment of the role of Src kinases in the process.

In the case of Akt, we observed in both 3T3-F442A and H4IIE cells a PP1- and PP2-dependent inhibition of GH-mediated phosphorylation on Ser473, raising the possibility that GH activation of Akt may require Src family kinases. This would be consistent with studies in human neutrophils and BAF3 cells that suggest that the cytokines granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor may signal to STATs and MAPKs through JAKs but signal to Akt through Src family kinases (79, 80). However, we found that GH robustly stimulates the phosphorylation of Ser-473 in Akt in MEFs genetically deleted for Src family kinases. Furthermore, reduction of endogenous JAK2 levels by shRNA reduced GH-mediated Akt activation to barely detectable levels in both 3T3-F442A and H4IIE cells, indicating that JAK2 is essential for GH-mediated Akt activation in these cells. Consistent with Akt activation requiring JAK2, Yamauchi et al. (23) found, in {gamma}2A-GHR cells lacking JAK2, that GH is unable to stimulate the tyrosyl phosphorylation of IRS-1, IRS-2, and IRS-3, their association with p85 subunit of PI3-kinase, and the activation of PI3-kinase, events that are thought to link GH receptor to Akt activation. Similarly, Argetsinger et al. (19, 30) found that GH stimulated tyrosyl phosphorylation of IRS 1 and 2 in CHO-GHR cells but not in CHO cells stably expressing a mutated GH receptor lacking the binding site for JAK2. Thus, our finding that PP1 and PP2 inhibit GH-induced Ser473 phosphorylation of Akt raises the possibility that Src activity, rather than being a necessary component linking GH receptor to Akt, may be indirectly required for GH to activate Akt. Supporting this hypothesis, Qui and associates (82) and Jiang and Qui (83) have described a potential mechanism whereby Src must phosphorylate Akt on Tyr315 and Tyr326 before growth factor-dependent phosphorylation of Thr308 and Ser473. Consistent with this, our data show that both PP1 and PP2 inhibit basally active Akt (Fig. 3Go, A and B), raising the possibility that maximal phosphorylation of Akt Ser473 by any factor is unachievable when Src activity is decreased. In support of this idea, we found that epidermal growth factor is also unable to stimulate phosphorylation of Ser473 in Akt when 3T3-F442A preadipocytes are pretreated with PP1 or PP2 (data not shown). Because PP3 at the concentrations used did not inhibit Src kinase activity, the finding that PP3 inhibits both basal and GH-stimulated phosphorylation of Ser473 in Akt also raises the possibility that the effects of PP1 and PP2 are not mediated exclusively via Src family kinases. PP1 and PP2 have been reported to have significant off-target effects (64, 65).

An interesting byproduct of our studies is the observation that some signaling pathways are more tightly coupled to the level of activation of JAK2 than others. Thus, when JAK2 levels were reduced by approximately 80% by shRNA against JAK2 in 3T3-F442A preadipocytes, the ability of GH to activate JAK2, ERKs 1 and 2, Akt, and STAT3 was reduced to a similar extent. In contrast, the ability of GH to stimulate the tyrosyl phosphorylation of STAT5 was reduced by only approximately 50%. In JAK2 shRNA 3T3-F442A cells that exhibited only a 50–60% reduction of JAK2 (as quantified from all time points in two independent experiments), GH activation of ERKs 1 and 2 and Akt was again almost abolished, whereas GH stimulation of STAT5 activity was relatively unaffected (data not shown). Although one could argue that this apparent discrepancy is because another kinase is necessary for maximal GH activation of STAT5, the MEF data argue that JAK2 is required for GH activation of STAT5 because we detected no GH stimulation of STAT5 when JAK2 was completely absent. These results therefore suggest that in the case of ERKs 1 and 2, Akt, and STAT3, levels of activated JAK2 are rate limiting, whereas they are not for STAT5. The MEF data also show that replacement of only a small amount of JAK2 is able to reconstitute substantial GH activation of STAT5. That levels of STAT5 rather than levels of JAK2 appear to be rate limiting in 3T3-F442A cells and MEFs is not so surprising, given that STAT5 is known to be recruited to multiple binding sites in the GH receptor (69), where it is rapidly phosphorylated by JAK2 and released from the GH receptor. It then migrates to the nucleus where it is thought to undergo dephosphorylation and then recycle back to the GH receptor for reactivation (9). The conclusion that levels of STAT5 rather than levels of JAK2 are sometimes rate limiting for GH activation of STAT5 would be consistent with the finding of Yang et al. (84) using both 3T3-F442A cells and {gamma}2A cells expressing ectopic GH receptor and JAK2. When these cells were treated with a dimerized form of the GH antagonist G120R, GH activation of STAT5 was maintained at normal levels even though levels of JAK2 activation are greatly suppressed. These results emphasize the need to consider the rate-limiting step in instances in which one GH signaling pathway (e.g. GH activation of STAT5) is inhibited to a lesser extent than other GH signaling pathways. We also noticed that in contrast to the 3T3-F442A cells, in H4IIE cells, the ability of GH to activate STAT5 appears to be more closely linked to levels of JAK2. In JAK2 shRNA cells, the reduction in levels of GH-activated STAT5 was similar to the reduction in levels of JAK2. This finding suggests that the rate-limiting step for a particular GH-signaling pathway may vary between cell types.

In conclusion, our results using pharmacological inhibitors of Src family kinases and cells with reduced levels of JAK2 using shRNA suggest that JAK2 and not a Src family kinase, is the primary kinase responsible for GH activation of STATs 1, 3, and 5, ERKs 1 and 2, and Akt in the well-characterized, highly GH-responsive 3T3-F442A preadipocytes and H4IIE hepatoma cells. Studies using JAK2 and Src-deficient MEFs further support the hypothesis that GH is capable of activating STATs 3 and 5, ERKs 1 and 2, and Akt in the absence of Src family kinases and is incapable of activating STAT5 in the absence of JAK2. It is conceivable, however, that in different cell lines, perhaps where the ratio of Src family kinases to JAK2 is naturally or artificially high, Src family kinases are able to substitute for some or all of the actions of JAK2. One can also envision the levels of some as-yet-unidentified accessory proteins shifting the balance between JAK2 and Src in ways that we do not yet understand. Finally, our data provide a reminder that some signaling pathways are more tightly coupled to the level of activation of JAK2 than others and that this level of coupling is likely to vary between cell types. Thus, titrating the level of JAK2 activity should enable one to preferentially stimulate or inhibit some pathways more than others in different cell types.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
Recombinant 22,000-Da human GH was a kind gift from Eli Lilly & Co. (Indianapolis, IN). DMEM was from Cambrex Corp. (East Rutherford, NJ). Swims S-77 powder, L-cystine, and L-glutamine were from United States Biological, Inc. (Swampscott, MA). Fetal bovine serum (FBS) was from Hyclone Laboratories, Inc. (Logan, UT). Calf serum was from Atlanta Biologicals, Inc. (Norcross, GA). Sodium bicarbonate powder was from Mallinckrodt (Chesterfield, MO). Calcium chloride dihydrate, puromycin, and polybrene (hexadimethrine bromide) were from Sigma (St. Louis, MO). The antibiotic-antimycotic solution, trypsin-EDTA and Magic Mark XP western standards were from Invitrogen (Carlsbad, CA). Aprotinin, leupeptin, and Triton X-100 were from Roche (Indianapolis, IN). Recombinant protein A-agarose was from Repligen Corp. (Waltham, MA). Hybond-C Extra nitrocellulose was from Amersham Biosciences (Piscataway, NJ). Src family kinase inhibitors PP1 and PP2 were from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). PP3 was from Calbiochem (La Jolla, CA). The mammalian expression vector prk5 encoding wild-type murine JAK2 (GI: 309463) was a generous gift from Dr. J. Ihle (St. Jude Children’s Hospital, Memphis, TN) (85).

Antibodies
Antibodies recognizing a peptide containing phosphorylated tyrosines 1007 and 1008 of JAK2 ({alpha}pY1007/1008, catalog no. 07-606); phosphotyrosines ({alpha}PY) (4G10, catalog no. 05-321); and phosphor-STAT1 ({alpha}pY701-STAT1, catalog no. 06-657) were from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibody recognizing total STAT1 was from Transduction Laboratories, Inc. (Lexington, KY; catalog no. S21120). Antibody recognizing both total STAT5b and total STAT5a ({alpha}STAT5, catalog no. sc-1656) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody recognizing both phosphor-STAT5a and phosphor-STAT5b ({alpha}pY694-STAT5, catalog no. 71-6900) was from Zymed Laboratories, Inc. (South San Francisco, CA). Antibodies recognizing phosphor-STAT3 ({alpha}pY705-STAT3, catalog no. 9131), total STAT3 ({alpha}STAT3, catalog no. 4904), phosphor-ERKs 1 and 2 ({alpha}pT202/pY204-ERK1/2, catalog no. 9106), total ERKs 1 and 2 ({alpha}ERK1/2, catalog no. 4695), phosphor-Akt ({alpha}pS473-Akt, catalog no. 4058), total Akt ({alpha}Akt, catalog no. 9272), and phosphor-Src ({alpha}pY416-Src, catalog no. 2113) were from Cell Signaling Technology (Danvers, MA). Mouse monoclonal antibody recognizing Src was from Dr. Tony Hunter (Salk Institute, La Jolla, CA). Mouse monoclonal antibody recognizing total JAK2 and used for immunoblotting was from BioSource International, Inc. (Camarillo, TX). Polyclonal antibody used for JAK2 immunoprecipitation was raised against a peptide corresponding to amino acids 758–776 of murine JAK2 and prepared by our laboratory in conjunction with Pel-Freez Biologicals (Rogers, AR) (6). Polyclonal {alpha}GHBP antibody used for GH receptor immunoprecipitation was from Dr. William Baumbach (American Cyanamid Co., Princeton, NJ). Polyclonal antibody (AL47) used for GH receptor immunoblot was a kind gift from Dr. Stuart Frank (University of Alabama) (86). IRDye 800-conjugated affinity-purified antimouse IgG and antirabbit IgG were from Rockland Immunochemicals (Philadelphia, PA).

Gene Silencing by shRNA and Retroviral Infection
The target sequences of murine and rat JAK2 were 5'-GGAGAGTATCTGAAGTTTC-3' (87) and 5'-GGAATGGCTTGCCTTACAA-3' (88), respectively. Oligonucleotides were annealed and subcloned into pSuperior.retro.puro (Oligoengine) at BglII and XhoI sites. A control sequence of 5'-UUCUCCGAACGUGUCACGU-3' with no known target (QIAGEN-Xeragon, Germantown, MD) was also cloned into the same vector. Retroviral infection was performed according to Erickson et al. (35). In brief, the recombinant plasmids were transfected into HEK 293T cells by calcium phosphate coprecipitation together with the viral packaging vectors SV-E-MLV-env and SV{psi}-E-MLV (74). Virus-containing medium was collected 16 h after transfection and passed through a 0.45-µm syringe filter. Polybrene was added to a final concentration of 8 µg/ml. This medium was then applied to subconfluent (~30%) 3T3-F442 cells or H4IIE cells. The infection protocol was repeated twice with intervals of 8–16 h. When cells achieved approximately 80% confluence, they were trypsinized, and cells expressing JAK2 shRNA were stably selected in medium containing 2 µg/ml (3T3-F442A cells) or 40 µg/ml (H4IIE cells) puromycin.

Cell Culture and Transfection
The stock of murine 3T3-F442A preadipocytes was kindly provided by H. Green (Harvard University, Cambridge, MA). H4IIE rat hepatoma cells were a kind gift from J. Messina (University of Alabama Birmingham School of Medicine). SYF (src/yes/fyn) triple knockout MEFs were kindly provide by P. Soriano (University of Washington, Seattle, WA) (67). JAK2–/– MEFs were a kind gift of J. Ihle (St. Jude Children’s Hospital, Memphis, TN) (81). 3T3-F442A cells and HEK 293T cells were grown in DMEM supplemented with 1 mM L-glutamine, 100 U of penicillin per ml, 100 µg of streptomycin per ml, 0.25 µg of amphotericin per ml, and 8% calf serum. H4IIE cells were grown in SWIMS 77 medium supplemented with 5% FBS, 26.2 mM sodium bicarbonate, 4 mM L-glutamine, 98 µM L-cystine, and 1.8 mM calcium chloride dihydrate. MEFs were grown in DMEM supplemented with 8% FBS, 100 U of penicillin/ml, 100 µg of streptomycin/ml, and 0.25 µg of amphotericin/ml. MEFs were transiently transfected using lipofectamine 2000 (Invitrogen). All cells were incubated overnight in serum-free medium containing 1% BSA before treatment with 100 µM PP1, PP2, or PP3 and/or GH (500 ng/ml). All experiments were carried out at 37 C.

Immunoprecipitation and Immunoblotting
For all experiments, cells were grown in 10-cm culture dishes. After GH treatment, cells were washed and solubilized in lysis buffer [50 mM Tris (pH 7.5), 0.1% Triton X-100, 150 mM NaCl, 2 mM EGTA, 1 mM Na3VO4 (pH 7.5)], containing 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Triton X-100 (1%) was used in place of 0.1% Triton X-100 to lyze cells for GH receptor studies. The supernatant was collected. For H4IIE cells, 50% of the supernatant was incubated with {alpha}JAK2 on ice for 2 h followed by protein G-agarose beads (General Electric, Fairfield, CT) rotating at 4 C for 1 h. For GH receptor immunoprecipitation, 60% of the supernatant was incubated with {alpha}GHBP on ice for 2 h followed by protein G-agarose beads (General Electric) rotating at 4 C for 1 h. The beads were washed three times with lysis buffer and boiled for 5 min in a mixture (80:20) of lysis buffer and SDS-PAGE sample buffer [250 mM Tris-HCl (pH 6.8); 10% sodium dodecyl sulfate; 10% β-mercaptoethanol; 40% glycerol; 0.01% bromophenol blue]. Eluted proteins as well as proteins in cell lysates prepared in the same buffer were separated by SDS-PAGE, using 10% polyacrylamide gels and an acrylamide:bis acrylamide ratio of 30:0.5. Bands on Western blots represent 12.5% of the total lysate from a 10-cm culture plate. For immunoblotting, proteins in the gel were transferred to nitrocellulose and detected by immunoblotting with the indicated antibody using the ODYSSEY Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). The intensity of the bands in immunoblots was quantified using LI-COR Odyssey 2.1 software. Values for phosphorylated proteins were normalized for total levels of that protein. For the shRNA experiments, JAK2 protein levels were normalized for total Src (H4IIE) or total ERK1/2 (3T3-F442A) protein levels. Every experiment was carried out at least twice with similar results. Most were performed three or more times (number indicated in the figure legends) with similar results.


   ACKNOWLEDGMENTS
 
We thank Matthew Lee for his help with Western blot analysis and Barbara Hawkins for her help in the preparation of this manuscript.


   FOOTNOTES
 
This work was supported in part by National Institutes of Health Grants RO1-DK34171 (to C.C.-S.) and K01-DK077915 (to H.J.). N.J.L. was supported by the Training Program in Organogenesis National Institutes of Health Grant T32-HD007505. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Current address for H.J.: Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 22, 2008

1 H.J. and N.J.L. contributed equally to this work. Back

Abbreviations: CHO, Chinese hamster ovary; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; IRS, insulin receptor substrate; JAK2, Janus kinase 2; MEFs, murine embryonic fibroblasts; PI3-kinase, phosphatidylinositol 3-kinase; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PP3, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine; shRNA, short hairpin RNA; STAT, signal transducer and activator of transcription; SYF, Src/Yes/Fyn triple knockout.

Received for publication January 11, 2008. Accepted for publication May 15, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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