Dual Mechanism of Signal Transducer and Activator of Transcription 5 Activation by the Insulin Receptor

Maithao N. Le, Ronald A. Kohanski, Lu-Hai Wang and Henry B. Sadowski

From the Departments of Microbiology (M.N.L., L.-H.W.), Biochemistry and Molecular Biology (R.A.K., H.B.S.) and Pharmacology and Biological Chemistry (H.B.S.), Mount Sinai School of Medicine, New York, New York 10029

Address all correspondence and requests for reprints to: Dr. Henry Sadowski, ENZO Life Sciences, 60 Executive Boulevard, Farmingdale, New York 11735. E-mail: hbsadowski{at}ENZObio.com, or Dr. L.-H. Wang, Mount Sinai School of Medicine, New York, New York 10029. E-mail: lu-hai.wang{at}mssm.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin stimulates signal transducer and activator of transcription 5 (Stat5) activation in insulin receptor (IR)-overexpressing cell lines and in insulin target tissues of mice. Stat5b and insulin receptor substrate 1 (IRS-1) interact with the same autophosphorylation site in the IR [phosphotyrosine (pY) 972] in yeast two-hybrid assays, and the IR phosphorylates Stat5b in vitro. These data suggest that Stat5 proteins might be recruited to, and phosphorylated by, the activated IR in vivo. Nevertheless, insulin activates Janus kinases (JAKs) in IR-overexpressing cell lines and in insulin target tissues. To determine whether Stat5 proteins must be recruited to the pY972LSA motif in the IR for insulin-stimulated activation in mammalian cells, we generated and tested a series of IR mutants. The L973R/A975D mutation abolishes the ability of the IR to induce Stat5 activation, whereas IRS-1 phosphorylation is unaffected. In contrast, the N969A/P970A mutation in the IR has no effect on Stat5 activation but significantly reduces IRS-1 phosphorylation. In coimmunoprecipitation assays, insulin-stimulated Stat5 activation correlates with Stat5 recruitment to the IR. We also find that insulin stimulates tyrosine phosphorylation of JAKs that are constitutively associated with the IR. Expression of dominant-negative (DN) JAKs, the JAK inhibitor suppressor of cytokine signaling 1, or pretreatment with the JAK inhibitor, AG490, reduces, but does not eliminate, insulin-induced Stat5 activation. Expression of the appropriate pair of DN JAKs in each of the singly JAK-deficient cell lines further establishes a component of insulin-stimulated Stat5 activation that is JAK independent. This likely represents phosphorylation of Stat5 proteins by the IR, as we find that IR kinase domain phosphorylates Stat5b in vitro on Y699 as efficiently as JAK2. Increasing the concentration of Stat5 proteins in cells favors the direct phosphorylation of Stat5 by the IR kinase where the DN-JAK inhibition of insulin-stimulated Stat5 activation becomes insignificant. At physiological levels of Stat5 however, we propose that JAKs and the IR both contribute to the insulin-induced phosphorylation of Stat5.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE POLYPEPTIDE HORMONE insulin exerts a variety of physiological effects including the regulation of glucose, lipid and protein metabolism, gene expression, cell growth, and differentiation (1, 2). Insulin mediates its pleiotropic effects through the insulin receptor (IR), which possess intrinsic protein tyrosine kinase (PTK) activity. This PTK activity is activated upon insulin binding (3, 4, 5, 6) resulting in autophosphorylation, as well as phosphorylation of specific intracellular substrates. Among these target proteins are insulin receptor substrate (IRS) 1 and 2 (7, 8) and the Src homology/collagen (Shc) proteins (9, 10). These IR substrates are recruited to the NPEpY972 autophosphorylation site in the IR via their pY binding domains (11, 12, 13, 14). Tyrosine phosphorylation of the IRS proteins leads to recruitment of Src homology 2 (SH2) domain-containing adapter/signaling molecules including Grb2-SOS (15, 16) and the phosphatidylinositol 3-kinase-regulatory subunit p85{alpha} (17, 18) resulting in activation of the Ras/MAPK (8, 19) and phosphatidylinositol 3-kinase (20) signaling cascades. Tyrosine-phosphorylated Shc provides an IRS-independent pathway for activation of the Ras/MAPK pathway by recruiting Grb2-SOS as well (15, 21). While many of the biological effects of insulin are dependent upon one or both of these signaling cascades, additional insulin-regulated signaling pathways continue to be identified and in some cases linked to specific actions of insulin, such as stimulation of glucose transporter 4 translocation to the plasma membrane in fat cells (22).

Recently, by using the kinase-active cytoplasmic domain of the IR as bait in yeast two-hybrid screening, we and others have identified a number of potential novel IR-interacting proteins. Among these proteins is the signal transducer and activator of transcription 5b (Stat5b) protein (23, 24). We have shown that insulin stimulates the tyrosine phosphorylation and activation of both Stat5a and Stat5b, as well as Stat3 and Stat1 in cell lines that overexpress the IR (23). More importantly, we have shown that perfusion of mouse liver with insulin stimulates the selective activation of Stat5 proteins and that refeeding of fasting mice, which leads to postprandial secretion of insulin, results in the activation of Stat5 proteins in the major target organs of insulin: liver, adipose tissue, and skeletal muscle (23). Recently, Stat5b has been suggested to play a role in the activation of glucokinase and suppressor of cytokine signaling (SOCS)3 gene transcription after insulin stimulation (25, 26). Taken together, these data suggest that Stat5 proteins likely play a physiological role in insulin-activated gene expression.

The Stat proteins have been most extensively characterized within the context of the Janus kinase (JAK)/Stat pathway, which plays an essential role in type I and II cytokine signaling (27, 28, 29). A model of cytokine signaling via the JAK/Stat pathway has emerged. According to this model, binding of cytokine to its receptor causes the receptor to dimerize. Receptor dimerization brings JAKs that are constitutively associated with the intracellular portion of receptors into close contact, leading to the activation of the intrinsic PTK activity of JAKs and tyrosine phosphorylation of the receptor. The pY residues of the receptor now serve as docking sites for recruitment of the SH2 domain-containing Stats, which are then phosphorylated and activated by the receptor-associated JAKs. Activated Stats form homo- or heterodimers and translocate into the nucleus where they can bind DNA and transactivate Stat-responsive genes (30).

In addition to being activated by a large number of cytokines through receptor-associated JAKs, Stat proteins have been shown to be activated by a number of growth factors that signal through receptor PTKs, such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), colony-stimulating factor-1, hepatocyte growth factor, fibroblast growth factor, and IGF-I (reviewed in Ref. 31). Putative pY docking sites for Stats have been identified for the EGF receptor (32), the PDGF receptor (33), the colony-stimulating factor-1 receptor (34), and the hepatocyte growth factor receptor (35). Once recruited to receptor PTK complexes, it is not clear which kinase phosphorylates a specific Stat, as many of these receptor PTKs can stimulate JAK activation in ligand-treated cells (35, 36, 37, 38, 39, 40, 41, 42). Therefore, defining the exact mechanisms through which a given receptor PTK activates specific Stats has been elusive and further complicated by evidence suggesting that different receptor PTKs potentially activate different Stats via distinct mechanisms. For example, the PDGF receptor appears to activate Stat1 via direct phosphorylation (40, 43), whereas Stat3 activation by EGF receptor, PDGF receptor, or IGF-1 receptor has been reported to depend on either a Src-kinase family member or JAKs (38, 40, 44).

We and others (23, 24) have proposed that Stat5b is a direct substrate of the IR. This was based, in part, upon the fact that Stat5b, through its SH2 domain, interacts with the pYLSA motif in the juxtamembrane region of the IR in yeast two-hybrid assays (24) and that a C-terminal fragment of Stat5b can be phosphorylated by the IR kinase domain in vitro (23). Nevertheless, insulin has been shown to stimulate JAK1 and JAK2 activation in vitro in IR-overexpressing cell lines (45, 46) and JAK2 activation in vivo in rats (47). We have therefore carefully examined the molecular mechanisms through which the IR mediates Stat5 protein activation. Here we show that Stat5a and Stat5b are recruited to the IR pYLSA motif in the juxtamembrane region of the IR where activation depends upon this recruitment. Our mutational analysis has also led to the identification of an IR mutant that can no longer activate Stat5 proteins but is otherwise normal for insulin signaling. Consistent with a potential role of JAKs in insulin-stimulated Stat5 activation, we find that insulin stimulates tyrosine phosphorylation of wild-type JAK1 and JAK2 that are constitutively associated with the IR in transiently transfected 293T cells. Insulin-stimulated tyrosine phosphorylation of JAKs appears to be the result of autophosphorylation, as kinase-inactive (KI) JAKs are not phosphorylated in response to insulin. To determine whether JAKs play a role in insulin-stimulated Stat5 activation, we used KI dominant negative (DN) mutants of JAKs and the potent JAK inhibitor protein SOCS1 in transiently transfected COS7 cells. We demonstrate that a significant fraction of Stat5 activation by the IR in this system is JAK dependent. Moreover, in the absence of any transfected proteins, insulin stimulates the activation of endogenous Stat5 proteins in H35 rat hepatoma cells, and this activation is partially inhibited by the JAK inhibitor, AG490. Using transient transfection assays in each of the cell lines lacking an individual JAK family member (JAK1-, JAK2-, or Tyk2-minus), we find that insulin-stimulated Stat5 activation does not require any individual JAK family member, suggesting functional redundancy for the JAK-dependent component. Expression of the appropriate DN JAK proteins in each of the singly JAK-deficient cell lines, however, reduces, but does not abolish, insulin-stimulated Stat5 activation. This JAK-independent component of insulin-induced Stat5 activation is likely to represent direct phosphorylation by the IR kinase. Consistent with this possibility, the purified IR kinase domain phosphorylates purified full-length Stat5b in vitro on Y699 as efficiently as partially purified JAK2. Finally, we show that increasing concentration of Stat5 proteins in cells drives the phosphorylation of Stat5 by IRs that are not engaged with JAKs. Under these conditions of highly overexpressed Stat5, the inhibition of insulin-stimulated Stat5 activation by DN-JAKs becomes insignificant. Based on these results, we propose a dual mechanism for Stat5 activation by the IR. Upon insulin stimulation, the IR recruits Stat5 proteins to the same autophosphorylation site that it uses to recruit IRS-1, -2, and Shc. Once Stat5 proteins arrive at the receptor complex, they can be tyrosine phosphorylated and activated by IR-associated JAKs as well as by the IR itself.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin-Induced Activation of Stat5a and Stat5b in Mammalian Cells Requires the Recruitment of Stat5 Proteins to the pYLSA Motif in the Juxtamembrane of the IR
Previous work in a yeast two-hybrid system suggested that the pY972LSA motif in the juxtamembrane region of the IR played an important role in Stat5b interaction with the phosphorylated IR, whereas the NPEpY972 motif was shown to be important for the interaction of the IR with IRS-1 (24). To determine whether the pY972LSA motif is important for Stat5 activation in mammalian cells, we created three mutant IRs (Fig. 1AGo: L973R, A975D, and L973R/A975D) in which the leucine and the alanine residue have been mutated, respectively, to arginine and aspartic acid. If this motif were important for Stat5 docking and activation, these mutant IRs would lose some or all of their ability to activate Stat5 after insulin stimulation. To demonstrate that the Stat5 binding site is distinct from the IRS-1 recruitment site, we also created the N969A/P970A IR by mutating both the asparagine and proline residues in the NPEpY972 IRS-1 recruitment motif (12, 48) to alanines (Fig. 1AGo). This mutant IR should have a reduced ability to activate IRS-1 but not Stat5. We transiently transfected COS7 cells with the wild-type and mutant IR expression plasmids and assayed for expression of the IRs and their tyrosine phosphorylation upon insulin stimulation by immunoblotting with anti-IR-ß and anti-pY antibodies, respectively. As shown in Fig. 1BGo, no significant differences in insulin-stimulated ß-subunit phosphorylation or ß-subunit expression were observed between the wild-type and mutant IRs. To assess the ability of the wild-type and mutant IRs to support insulin-stimulated tyrosine phosphorylation of Stat5 proteins, Stat5a or Stat5b were coexpressed with either wild-type or different mutant IRs. Stat5 activation was determined by immunoblot analysis with a phosphorylation state-specific monoclonal antibody for tyrosine phosphorylated Stat5a (pY694) and Stat5b (pY699) (49, 50, 51). Both Stat5a and Stat5b were tyrosine phosphorylated in response to insulin in the wild-type IR-expressing cells (Fig. 1BGo). In contrast, insulin-induced tyrosine phosphorylation of Stat5a and Stat5b was dramatically reduced in the cells expressing the L973R IR. Furthermore, in the cells expressing the L973R/A975D IR, insulin stimulation of Stat5a and Stat5b tyrosine phosphorylation was undetectable. As expected, the N969A/P970A IR mediated insulin-induced Stat5a and Stat5b activation as efficiently as the wild-type receptor. Nevertheless, insulin-stimulated tyrosine phosphorylation of IRS-1 was significantly reduced in cells expressing the N969A/P970A mutant. Unlike the N969A/P970A mutant, the L973R and the L973R/A975D mutant IRs display wild-type levels of insulin-stimulated IRS-1 phosphorylation (Fig. 1BGo).



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Figure 1. Activation of Stat5a and Stat5b by the IR Requires the pYLSA Motif

A, Schematic of the amino acid sequence of the juxtamembrane region of the IR depicting the recruitment motifs for IRS-1 (NPEY972) and Stat5 (Y972LSA) as well as the mutations made to generate single- and double-mutant IRs. B, Insulin-induced tyrosine phosphorylation of IR ß-subunit, Stat5a, Stat5b, and IRS-1. For the top six panels, COS7 cells were cotransfected with the following amounts of expression plasmids as indicated in the figure: 0.1 µg FLAG-tagged Stat5a or Flag-tagged Stat5b; and 0.5 µg wild-type IR or mutant IR. For the bottom two panels, COS7 cells were cotransfected with 0.1 µg of IRS-1 and 0.5 µg of either wild-type or mutant IR. After 24 h, the transfected cells were serum starved for 16 h and treated with or without 50 nM insulin for 7 min before lysis in RIPA buffer. Except for the IRS-1 panels, 50 µg of lysates were analyzed directly by immunoblotting. In each pair of panels, the blots were probed first for tyrosine phosphorylation with either antiphospho-Stat5 or anti-pY antibody. The blots were then stripped and reprobed with the indicated antibodies. For IRS-1, 500 µg of lysates were subjected to immunoprecipitation with anti-IRS-1 antibody followed by immunoblot analysis with anti-pY antibody. The blot was stripped and reprobed with anti-IRS-1 antibody.

 
To determine whether the loss of Stat5 activation in these mutant IRs was due to loss of Stat5 recruitment to the IR, we examined the interaction between Stat5 proteins and wild-type or mutant IRs. FLAG-tagged Stat5a or FLAG-tagged Stat5b was transiently coexpressed with wild-type or mutant IRs in COS7 cells and their association measured by coimmunoprecipitation analysis. As shown in Fig. 2Go, both Stat5a and Stat5b exhibited insulin-induced interaction with the wild-type IR. Stat5a and Stat5b were found associated with the wild-type and the N969A/P970A mutant IR in response to insulin. The L973R/A975D mutant IR, however, did not recruit Stat5a or Stat5b in response to insulin. Taken together, these data demonstrate that the pYLSA motif is essential for Stat5 phosphorylation in response to insulin and that phosphorylation requires recruitment to the IR via this motif.



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Figure 2. Insulin Activation of Stat5 Proteins by the IR Requires Recruitment to the pYLSA Motif

COS7 cells were cotransfected with the following amounts of expression plasmids as indicated in the figure: 0.1 µg FLAG-tagged Stat5a or 0.1 µg Flag-tagged Stat5b; and either 0.5 µg wild-type IR or 0.5 µg mutant IR. After 24 h, the transfected cells were serum starved for 16 h and treated with or without 50 nM insulin for 7 min. Cells were lysed in NP-40 buffer. Lysates (500 µg) were subjected to immunoprecipitation with anti-IR ß-subunit antibody followed by immunoblotting with anti-FLAG antibody. The blots were then stripped and reprobed with anti-IR ß-subunit antibody.

 
JAK1 and JAK2 Constitutively Associate with the IR and Become Tyrosine Phosphorylated in Response to Insulin
Previous work has shown that in IR-overexpressing cells, insulin stimulates the tyrosine phosphorylation of JAK1 and JAK2 and their interaction with the IR (45, 46). Furthermore, insulin rapidly stimulates JAK2 tyrosine phosphorylation and association with the IR in insulin target tissues in rats (47). Together, these results suggested the possibility that IR-associated JAKs are the kinases that phosphorylate Stat5 proteins in response to insulin. To investigate this possibility, we first determined whether insulin stimulates tyrosine phosphorylation of JAK1 or JAK2 in transiently transfected 293T cells. As shown in Fig. 3Go, wild-type JAK1 and JAK2 proteins are constitutively tyrosine phosphorylated, most likely due to their overexpression. This constitutive activation of JAKs has been consistently observed by others in transient transfection analysis (52, 53). Nevertheless, insulin treatment increased the tyrosine phosphorylation of both JAK1 and JAK2. To determine whether JAK1 or JAK2 associates with the IR, we performed coimmunoprecipitation analysis with antibodies recognizing the IR-ß subunit. Surprisingly, both JAK1 and JAK2 were detected in the anti-IR immunoprecipitates even in the absence of insulin stimulation. This result suggests that in transiently transfected 293T cells, overexpressed JAK1 and JAK2 are constitutively associated with the IR in a manner similar to that of JAKs and cytokine receptors.



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Figure 3. JAK1 and JAK2 Constitutively Associate with the IR and Become Tyrosine Phosphorylated upon Insulin Stimulation

293T cells were transfected with 0.5 µg of plasmid expressing wild-type (WT) or KI JAK1 (panel A) or WT or KI JAK2 (panel B). After 24 h, the transfected cells were serum starved for 16 h and treated with or without 50 nM insulin for 7 min before lysis in NP-40 buffer. Protein (1 mg) of each lysate was subjected to immunoprecipitation with anti-IR ß-subunit, anti-JAK1, or anti-JAK 2 antibody as indicated in the figure. The immunoprecipitates were analyzed by immunoblotting with anti-pY, anti-JAK1, or anti-JAK2 antibody as indicated. The blots were then stripped and reprobed with anti-JAK1, anti-JAK2, or anti-IR ß-subunit antibody as indicated.

 
To determine whether JAK1 and JAK2 are direct substrates for the IR kinase, we overexpressed KI JAK1 or JAK2 in 293T cells and assayed for insulin-stimulated tyrosine phosphorylation of the mutant JAKs. Insulin-stimulated tyrosine phosphorylation of the KI JAKs was not observed, consistent with previous results (46). Like wild-type JAK1 and JAK2, coimmunoprecipitation analysis showed that the KI JAKs also constitutively associated with the IR. Thus, the ability of JAKs to associate with the IR does not depend on their kinase activity. Taken together, these findings suggest that IR-associated JAKs are not substrates for the IR and, in fact, may become activated by autophosphorylation when IR-ß subunits undergo conformation changes after insulin binding.

KI JAK1 and JAK2 Partially Inhibit Insulin-Induced Stat5 Activation
Like the wild-type JAK1 and JAK2, the KI JAK1 and JAK2 constitutively associate with the IR but are clearly not substrates for the IR kinase. Thus, the KI JAK1 and JAK2 can function as DN proteins. To determine whether JAK activity is required for insulin-stimulated Stat5 activation, we cotransfected increasing amounts of the KI DN-JAK1 or DN-JAK2 expression plasmids in COS-7 cells with constant amounts of expression plasmids encoding the wild-type IR and Stat5a or Stat5b. While there was little inhibition of insulin-stimulated Stat5 protein activation at the level of 10 ng of DN-JAK1 or DN-JAK2 plasmids, at 100 ng, insulin-stimulated phosphorylation of both Stat5a and Stat5b was significantly reduced (Fig. 4Go). Although insulin-stimulated phosphorylation of Stat5a and Stat5b was completely inhibited at higher levels of either DN-JAK1 or DN-JAK2 expression plasmid (1 µg), the resulting level of DN-JAK expression resulted in a significant decrease in IR-ß subunit autophosphorylation as well as decreased Stat5 expression (data not shown). This result suggests that at high expression levels, DN-JAK1 and DN-JAK2 exert a toxic or nonspecific inhibitory effect. Nevertheless, at a low expression level, DN-JAK1 or DN-JAK2 partially inhibits IR-mediated Stat5 activation.



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Figure 4. DN Mutants of JAK1 and JAK2 Inhibit Stat5 Activation by the IR

COS7 cells were transiently cotransfected with 0.5 µg of plasmid expressing wild-type IR, 0.1 µg of plasmid expressing either Stat5a or Stat5b, and 0.01 µg or 0.1 µg of plasmid expressing either DN-JAK1 (panel A) or DN-JAK2 (panel B). After 24 h, the transfected cells were serum starved for 16 h and treated with or without 50 nM insulin for 7 min before lysis in RIPA buffer. Lysates (50 µg) were analyzed by immunoblotting with the indicated antibodies. In each pair of panels, the blots were probed first for tyrosine phosphorylation with either antiphospho-Stat5 or anti-pY antibody. The blots were then stripped and reprobed with the indicated antibodies for measuring the expression levels of the transfected proteins.

 
SOCS-1 Partially Inhibits Stat5 Activation by the IR
As an alternative approach to assess the role of JAKs in insulin-stimulated Stat5 activation, we examined Stat5 protein tyrosine phosphorylation in the presence of the suppressor of cytokine signaling 1 (SOCS1) (54, 55). SOCS1 has been shown to inhibit cytokine-mediated JAK-Stat activation by binding to phosphorylated tyrosine residues in the activation loops of all JAK family members and thereby inhibiting their kinase activity (56, 57). In this experiment, COS7 cells were transiently cotransfected with plasmids expressing wild-type IR, Stat5a or Stat5b, and SOCS1, and insulin-induced Stat5a and Stat5b phosphorylation in these cells was determined (Fig. 5AGo). While having no effect on IR autophosphorylation, expression of SOCS1 caused a significant reduction in insulin-induced phosphorylation of Stat5a and Stat5b. To demonstrate that the level of SOCS1 expressed in these cells was sufficient to inhibit JAK activity, we cotransfected COS7 cells with Stat5b, wild-type JAK1 or JAK2, and SOCS1 expression plasmids and measured Stat5b tyrosine phosphorylation. As shown in Fig. 5BGo, coexpression of either wild-type JAK1 and JAK2 with Stat5b leads to robust Stat5b tyrosine phosphorylation via the constitutively active JAKs (see Fig. 2Go), and this JAK-driven Stat5 phosphorylation is completely blocked by expression of SOCS1. Nevertheless, in cotransfection experiments with the IR and Stat5 expression vectors, increasing the amount of SOCS1 expression vector did not completely eliminate insulin-stimulated tyrosine phosphosphorylation of Stat5a and Stat5b (data not shown). To address the possibility of incomplete JAK inhibition by SOCS1, we measured interferon-{gamma} (IFN{gamma})-stimulated Stat1/3 reporter gene activity and insulin-stimulated Stat5 reporter gene activity in the absence or presence of SOCS1. While IFN{gamma} stimulation of the Stat1 reporter gene is completely inhibited by SOCS1 expression, this level of SOCS1 has little or effect on insulin stimulation of the Stat5 reporter gene (Fig. 5CGo). Combined with results from the expression of DN-JAKs (Fig. 4Go), these data suggest that IR-associated JAKs can phosphorylate Stat5 in response to insulin but that an additional JAK-independent component also exists.



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Figure 5. Expression of SOCS1 Protein Partially Inhibits Stat5 Activation by the IR

A, COS7 cells were transiently cotransfected with 0.5 µg of pEF-hIR, and 0.1 µg of either pRK5-FlagStat5a or pRK5-FlagStat5b; and 0.1 µg of either pEF or pEF-FlagSOCS1. After 24 h, the transfected cells were serum starved for 16 h and treated with or without 50 nM insulin for 7 min before lysis in RIPA buffer. For the top three panels, 50 µg of lysates were analyzed directly by immunoblotting with the indicated antibodies. In the lower two panels, 100 µg of lysates were immunoprecipitated with anti-IR ß-subunit antibody followed by immunoblotting with anti-pY antibody. The blot was then stripped and reprobed with anti-IR ß-subunit antibody. B, SOCS1 completely inhibits JAK1- and JAK2-mediated Stat5b phosphorylation. COS7 cells were transiently cotransfected with 0.1 µg of pRK5-FlagStat5b; and 0.05 µg of either pRK5-JAK1 or pRK5-JAK2; and 0.1 µg of either pEF or pEF-FlagSOCS1. After 24 h, the transfected cells were serum starved for 16 h before lysis in RIPA buffer. Lysates (50 µg) were analyzed by immunoblotting with the indicated antibodies. C, The level of SOCS1 that completely blocks IFN{gamma} induction of a Stat1 reporter gene only partially inhibits insulin induction of a Stat5 reporter gene. For the left half, 2fTGH cells were transiently cotransfected with 0.5 µg of a Stat5 luciferase reporter (LHRR), 0.05 µg of CMV-Renilla luciferase reporter, 1.0 µg of pEF-hIR, 0.025 µg of pRK5-FlagStat5a, 0.025 µg of pRK5-FlagStat5b, and 1.0 µg of either pEF or pEF-FlagSOCS1. For the right half, 2fTGH cells were transiently cotransfected with 0.5 µg of Stat1 luciferase reporter (m67luc), 0.05 µg of CMV-Renilla luciferase reporter, and 1.0 µg of either pEF or pEF-FlagSOCS1. Sixteen hours after transfection, the cells were serum-starved for 6 h and then left untreated or treated with hIFN{gamma} or insulin for 6 h. Results represent the mean ± SEM of triplicate estimations.

 
Insulin-Stimulated Tyrosine Phosphorylation of Endogenous Stat5 Proteins in H35 Rat Hepatoma Cells Is Partially Inhibited by the JAK Inhibitor, AG490
While both the JAK-DN and SOCS1 experiments suggest that JAKs can play a role in insulin-stimulated Stat5 activation, these data were generated in transient cotransfection systems. To determine whether insulin-stimulated Stat5 activation is JAK dependent in the absence of overexpressed proteins, we identified an insulin-responsive cell line, H35 rat hepatoma cells (58), in which Stat5 proteins become activated by insulin through the endogenous complement of IRs. In serum-starved H35 cells, a 10-min treatment with 50 nM insulin leads to increased tyrosine phosphorylation of both Stat5a and Stat5b (Fig. 6AGo). Importantly, the insulin-stimulated tyrosine phosphorylation of Stat5a and Stat5b in these cells is partially inhibited by the JAK inhibitor, AG490 (59), whereas insulin-stimulated tyrosine phosphorylation of the IR-ß subunit is unaffected (Fig. 6BGo). The incomplete inhibition of insulin-stimulated Stat5 phosphorylation at the highest concentration of AG490 tested (400 µM) may represent a JAK-independent component, incomplete JAK inhibition, or both, as GH-stimulated Stat5 tyrosine phosphorylation is not completely blocked at this dose of AG490 (Fig. 6CGo). Nevertheless, these data clearly suggest a role for JAKs in insulin-stimulated Stat5 activation in the absence of any overexpressed proteins and support the results obtained in transient transfection experiments.



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Figure 6. Insulin-Stimulated Tyrosine Phosphorylation of Endogenous Stat5 Proteins in H35 Rat Hepatoma Cells Is Partially Blocked by the JAK Inhibitor, AG490

A, Insulin stimulates tyrosine phosphorylation of endogenous Stat5 proteins in H35 rat hepatoma cells. After 16 h of serum starvation, H35 cells were treated with or without 50 nM insulin for 10 min before lysis in NP-40 buffer. Cell lysates were immunoprecipitated with the indicated antibodies and immunoblotted with anti-pY antibody. After stripping, the blots were reprobed with the indicated antibodies. B, AG490 partially inhibits insulin-stimulated Stat5 activation in H35 cells. After 2 h of serum starvation, H35 cells were pretreated with either DMSO or 50, 100, 200, or 400 µM AG490 for 4 h, before treatment with or without 50 nM insulin for 10 min. Cells were lysed with NP-40 buffer and analyzed as described in panel A. C, The JAK2-dependent tyrosine phosphorylation of Stat5 in GH-treated H35 cells is dramatically inhibited by AG490. Cells were serum starved and pretreated as described in panel B with DMSO or 50 or 400 µM AG490 before treatment with or without 500 ng/ml GH for 10 min. After cell lysis in NP-40 buffer, 50 µg of total cell lysates were immunoblotted with antiphospho-Stat5 antibodies, and then stripped and reprobed with anti-Stat5 antibody.

 
JAK1, JAK2, and Tyk2 Appear to Play Redundant Roles in Insulin-Stimulated Stat5 Activation
We have shown that the IR activates JAK1 and JAK2 to similar extents and that DN-JAK1 and DN-JAK2 exert similar inhibitory effects on insulin-induced Stat5 phosphorylation. To determine whether a specific JAK family member is required for insulin-stimulated Stat5 activation, or whether JAKs play an interchangeable role, we performed experiments in cell lines lacking individual JAK family members. The JAK1-minus (U4A), the JAK2-minus ({gamma}2A), and Tyk2-minus (U1A) cell lines were derived from an HT1080 human fibrosarcoma cell clone (2fTGH) that expresses all three JAKs (60, 61, 62, 63). These cell lines were transiently cotransfected with plasmids expressing wild-type IR and either Stat5a or Stat5b, and insulin-induced Stat5 protein phosphorylation was measured. Insulin induced Stat5a and Stat5b activation in the parental 2fTGH cells (Fig. 7AGo). This activation requires cotransfection of both the IR and Stat5 expression plasmids (data not shown). As shown in Fig. 7Go, the ability of the IR to mediate Stat5a and Stat5b phosphorylation in response to insulin remains intact in the U4A, {gamma}2A, and U1A cell lines. These results demonstrate that no individual JAK family member is solely required for IR-mediated Stat5 activation and are therefore redundant in this pathway.



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Figure 7. Stat5 Proteins Become Phosphorylated After Insulin Stimulation in Wild-Type and JAK-Deficient Cell Lines

2fTGH (panel A), U4A (panel B), {gamma}2A (panel C), or U1A (panel D) cells were cotransfected with 1 µg of pEF-hIR; and 0.2 µg of either pRK5-FlagStat5a or pRK-FlagStat5b; and 1 µg of either pRK5 or pRK5-DN-JAK1, pRK5-DN-JAK2 or pMT2-DN-Tyk2 as indicated. After 24 h, the transfected cells were serum starved for 16 h and treated with or without 50 nM insulin for 7 min before lysis in RIPA buffer. Lysates (50 µg) were analyzed by immunoblotting with antiphospho-Stat5 antibody, and then stripped and reprobed with either anti-Stat5a or anti-Stat5b antibody. Lysates (100 µg) were immunoprecipitated with anti-IR ß-subunit antibody and immunoblotted with anti-pY antibody. The blot was then stripped and reprobed with anti-IR ß-subunit antibody.

 
Insulin Can Stimulate Stat5 Phosphorylation in JAK-Minus Cells
While our results suggest that JAKs are either the kinases that phosphorylate Stat5 in response to insulin or play an important positive role in this response, we have been unable to completely inhibit insulin-stimulated Stat5 phosphorylation by DN-JAK expression, SOCS1 expression, or the drug AG490. These results suggest the possibility that we are unable to generate completely JAK-minus cells. To determine whether insulin can stimulate Stat5 phosphorylation in cells deficient for JAK1, JAK2, and Tyk2 activity, we transiently cotransfected the appropriate pairs of DN-JAK expression plasmids into each of the single JAK-deficient cell lines and assayed for insulin-induced Stat5a and Stat5b phosphorylation. As shown in Fig. 7Go, U4A, {gamma}2A, and U1A cells overexpressing the appropriate pairs of DN-JAK proteins display decreased insulin-stimulated tyrosine phosphorylation of Stat5a and Stat5b. Nevertheless, insulin-stimulated Stat5 phosphorylation was still detectable in all of these assays. This result is consistent with the partial inhibition observed with the expression of individual DN-JAKs or SOCS1, or AG490 treatment in H35 cells. These data support the possibility that another kinase can also phosphorylate Stat5 proteins in response to insulin. The most obvious candidate is the IR itself.

Both the IR and JAK2 Phosphorylate Stat5b in Vitro
We have previously shown that purified IR kinase domain (IR-KD) can phosphorylate a C-terminal fragment of human Stat5b (23). The His-tagged Stat5b C-terminal fragment used as substrate in those assays, however, was likely to be partially denatured, and denatured proteins often function as nonphysiological substrates of tyrosine kinases. Furthermore, this Stat5b fragment contained seven tyrosine residues and the phosphorylation site(s) were not mapped. Therefore, to determine whether the IR phosphorylates full-length Stat5b on the activating tyrosine residue (Y699), murine Stat5b was expressed to high levels in insect cells, and soluble unphosphorylated Stat5b was purified under nondenaturing conditions to greater than 95% purity. For comparison, we expressed JAK2 in insect cells and partially purified the heavily tyrosine phosphorylated kinase active JAK2. In vitro kinase reactions were performed with Stat5b alone, Stat5b plus IR-KD, or Stat5b plus JAK2. The phosphorylation of Stat5b on the activating tyrosine residue was assessed by immunoblotting with the antiphospho-Stat5 antibody, whereas tyrosine phosphorylation of all the kinase reaction components was monitored by immunoblotting with anti-pY antibody. As shown in Fig. 8Go, tyrosine-phosphorylated Stat5b was not detectable in the absence of added kinase, demonstrating the lack of contaminating tyrosine kinases in the Stat5b preparation. The IR-KD phosphorylated Stat5b on the activating tyrosine residue as efficiently as JAK2 under the conditions of this assay (substrate excess). Although we have not yet determined the apparent Michaelis-Menten constant (Km) of each kinase for the Stat5b substrate, our results support the hypothesis that Stat5 proteins recruited to the IR can be phosphorylated and activated by the IR itself as well as by IR-associated JAKs.



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Figure 8. In Vitro Phosphorylation of Full-Length Stat5b by the IR and JAK2

The human IR-KD phosphorylates full-length murine Stat5b on the activating tyrosine residue (Y699) as efficiently as murine JAK2. Purified baculovirus-expressed murine Stat5b (3 µg protein) was subjected to in vitro kinase reactions (30 µl) with buffer alone, purified baculovirus-expressed human IR-KD (10 nM), or partially purified baculovirus-expressed murine JAK2 (~16 nM kinase) for 30 min at 25 C. An aliquot (20 µl) of each reaction was mixed with an equal volume of 2x SDS sample buffer and boiled for 8 min. Aliquots of each quenched reaction (10 µl) were analyzed by immunoblotting with either antiphospho-Stat5 or anti-pY antibody as indicated.

 
At High Levels of Stat5 Protein Expression, IR-Mediated Stat5 Activation Is Effectively JAK Independent
The data we have presented support a model of insulin-stimulated Stat5 tyrosine phosphorylation in which Stat5 proteins can be phosphorylated by either the IR-associated JAKs or the IR itself. In contrast, Sawka-Verhelle et al. (25) were unable to detect a JAK-dependent component, despite reporting that JAKs can become activated in insulin-treated cells (45, 46). To reconcile these conflicting results, we performed transient cotransfections in COS7 cells with a fixed amount of IR expression plasmid, the absence or presence of a fixed amount of DN-JAK1 or DN-JAK2 expression plasmid, and increasing amounts of Stat5a or Stat5b expression plasmids. As shown in Fig. 9Go, at low amounts of Stat5a or Stat5b expression plasmid (0.01 µg), cotransfection of DN-JAK1 or DN-JAK2 was able to reduce insulin-stimulated Stat5 phosphorylation by more than 2-fold. This result suggests that at a limited level of Stat5 proteins JAKs contribute to insulin-induced phosphorylation of Stat5 proteins. When higher amounts of Stat5 expression plasmids (0.1–1.0 µg) were transfected, cotransfection of DN-JAK1 or DN-JAK2 expression plasmids had little or no effect on insulin-stimulated Stat5 tyrosine phosphorylation. These results suggest that as the amount of Stat5 substrate increases, the JAK-dependent pathway becomes saturated, and more Stat5 proteins become available to be phosphorylated and activated directly by the IR. These levels of Stat5 proteins, however, are 5- to 20-fold higher than what we observe in most cell lines and tissues (data not shown). Therefore, at physiological levels of Stat5 expression, the JAK-dependent pathway is likely to be significant.



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Figure 9. At High Levels of Stat5 Protein Expression, IR-Mediated Stat5 Activation Is Effectively JAK Independent

COS7 cells were cotransfected with 0.5 µg of plasmid expressing wild-type IR and the indicated amounts of plasmids expressing either DN-JAK1 (panel A), DN-JAK2 (panel B), and Stat5a or Stat5b. After 24 h, the transfected cells were serum starved for 16 h, and treated with or without 50 nM insulin for 7 min before lysis in RIPA buffer. Lysates (50 µg) were analyzed directly by immunoblotting with antiphospho-Stat5, anti-JAK1, or anti-JAK2 antibody as indicated. The blots were stripped and reprobed with either anti-Stat5a or anti-Stat5b antibody. Lysates (100 µg) were immunoprecipitated with anti-IR ß-subunit antibody followed by immunoblotting analysis with anti-pY antibody. The blot was then stripped and reprobed with anti-IR ß-subunit antibody. Note that in the anti-JAK2 blot, the lower arrow points to a protein band that nonspecifically cross-reacts with anti-Jak2 antibody. The upper arrow points to the band corresponding to endogenous JAK2 and DN-JAK2 protein.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our previous study suggests that Stat5 proteins are physiological substrates of IR signaling (23). We have shown that perfusion of mouse liver leads to rapid Stat5 phosphorylation and activation of Stat5 DNA binding activity and that refeeding of fasting mice causes rapid Stat5 phosphorylation in liver, muscle, and adipose tissue. We and others (23, 24) have shown that Stat5b interacts with the IR in the yeast two-hybrid system and Van Obberghen and colleagues (24) have gone on to show that the pYLSA sequence in the juxtamembrane domain of the IR is important for this interaction. In the present study, we have focused on the potential mechanism of how the IR activates Stat5 proteins in mammalian cells. We have shown that both Stat5a and Stat5b associate with the receptor and become phosphorylated in response to insulin stimulation. This receptor interaction and activation require the pY972LSA motif. Mutation of the leucine (+1) and the alanine (+3) residues in this SH2 domain binding motif effectively renders the IR incapable of recruiting Stat5a and Stat5b to the receptor complex for activation but is still capable of recruiting and phosphorylating IRS-1. Thus, we created a mutant IR (L973R/A975D) that is selectively defective in Stat5 phosphorylation and have used this mutant IR to identify insulin-regulated genes that require Stat5 activation in C2C12 skeletal muscle cells (50). Our data also suggest that IRS-1 and Stat5 may compete for association with the IR, as IRS-1, through its pY binding domain, interacts with pY972 and amino acids N terminal to pY972 for binding, and Stat5, through its SH2 domain, interacts with pY972 and amino acids C terminal to pY972 for binding.

Existing evidence suggests that both JAK1 and JAK2 are phosphorylated in response to insulin stimulation, and that both JAKs can interact with the IR directly (45, 46, 47). This implies that JAK1 and JAK2 could play a role in Stat5 activation downstream of the IR. In this study, we show that Stat5a and Stat5b activation by the IR can be JAK dependent, as expression of either DN-JAK1 or DN-JAK2 can partially inhibit Stat5a and Stat5b activation in response to insulin. SOCS1, a potent inhibitor of all JAKs, also partially inhibits insulin-mediated Stat5 activation, even at levels that completely block IFN{gamma} stimulation of Stat1 reporter gene. Furthermore, insulin-stimulated tyrosine phosphorylation of endogenous Stat5 proteins in H35 hepatoma cells is partially inhibited by the JAK inhibitor, AG490. Because the inhibition of insulin-induced Stat5 phosphorylation by all three types of JAK inhibitors was partial, we concluded that there is a JAK-independent component that also mediates insulin-stimulated Stat5 phosphorylation. The most obvious candidate for the JAK-independent pathway would be direct phosphorylation of recruited Stat5 proteins by the IR kinase. In support of this hypothesis, we demonstrate that in vitro, the IR kinase phosphorylates Stat5b on the activating tyrosine residue as efficiently as JAK2. The existence of a JAK-independent component is supported by a recent report demonstrating that Stat5b activation by the IR can be completely JAK independent (25). While our evidence supports a role of JAK1 and JAK 2 in insulin-stimulated Stat5 phosphorylation, when we transfect high levels of Stat5a or Stat5b expression plasmids, we also find that the insulin-stimulated activation of Stat5 proteins becomes effectively JAK independent. We speculate that the JAK-dependent pathway becomes saturated with an increasing amount of substrates such that direct phosphorylation of Stat5 proteins by the IR is the dominant pathway. Nevertheless, at physiological levels of Stat5 expression, a significant fraction of insulin-stimulated Stat5 activation is likely to be JAK dependent.

JAKs play a pivotal role in cytokine signaling. Because cytokine receptors lack intrinsic kinase activity, they rely on the constitutively associated JAKs to phosphorylate and activate their Stat protein substrates (reviewed in Refs. 28, 29 , and 64). In addition, JAKs are known to become phosphorylated in response to EGF (36, 37, 38), PDGF (39, 40), and insulin (46, 47). We have shown that both JAK1 and JAK2 constitutively associate with the IR. Insulin stimulates tyrosine phosphorylation of both JAKs but does not increase the amount of JAKs that associate with the IR. Furthermore, the KI JAKs are also constitutively associated with the IR, suggesting that the association of JAKs with the IR does not depend on JAK activity. This finding supports their use as DN inhibitors. Our results differ from earlier reports in which the interaction between the IR and JAK1 or JAK2 could only be detected when the IR was activated (45, 46, 47). In the study by Gual et al. (46), both the glutathione-S-transferase (GST)-fused C-terminal and GST-fused N-terminal domains of JAK1 were found to interact with phosphorylated IR in GST pull-down assays. It is possible that the full-length native JAK1 constitutively associates with the IR but that significant interaction of the partial domains in GST-pull-down assays requires the IR to be phosphorylated. However, Saad et al. (47) were able to demonstrate interaction between JAK2 and the IR only after insulin stimulation in vivo. Therefore, it is possible that the high level of expression of JAK1 and JAK2 in our experiments resulted in a constitutive association of JAKs with the IR that masks an insulin-stimulated increase in their association. As there is no detectable insulin-induced phosphorylation of the KI JAKs, we suspect that IR-associated JAKs become activated by JAK autophosphorylation. Taken together, these results suggest that JAK1 and JAK2 interact with the IR and become tyrosine phosphorylated and activated in response to insulin stimulation in a manner similar to JAK-cytokine receptor interaction and activation.

This model predicts that a specific region of the IR mediates the constitutive association with JAKs. Examination of the IR sequence revealed a region N-terminal to Y972 (PDGPLGPL, AA 957–964) that is homologous to JAK binding sites in cytokine receptors (29). Further experiments, however, will be necessary to determine whether this motif represents a bona fide JAK binding site. However, it is noteworthy that robust insulin-stimulated JAK activation has not been routinely observed in cultured cell lines unless either the IR or JAKs are overexpressed (45, 46, 65). These findings suggest that the interaction between the IR and JAKs is either relatively weak and/or that in most cultured cell lines, the majority of JAKs are already engaged by cytokine receptor subunits. Nevertheless, insulin injection into rats leads to the rapid tyrosine phosphorylation of JAK2 in insulin target tissues (47), suggesting that the interaction between IR and JAK2 could be meaningful in vivo.

The interaction of the IR with JAK2 in vivo suggested that JAK2 might be the primary JAK that interacts with the IR, but other JAKs were not examined in this study. For single-subunit cytokine receptors, a single unique JAK is used for signaling (e.g. JAK2 for GH, prolactin, and erythropoietin receptors), whereas for multiple-subunit receptors, specific pairs of JAKs are required for signaling (e.g. JAK1 and JAK3 for IL-2 and IL-4 receptors) (29). In contrast to most cytokine receptors, JAK1 and JAK2 (46), as well as Tyk2 (Le, M. N., and H. B. Sadowski, unpublished observations), can all interact with the IR in overexpression studies. These results suggest that the JAK-dependent component of IR-mediated Stat5 activation might not depend upon a specific JAK. Consistent with this possibility, both DN-JAK1 and DN-JAK2 could inhibit Stat5 activation to similar extents in COS7 cells. Moreover, experiments with JAK1-, JAK2-, and Tyk2-deficient cell lines revealed that no single JAK family member is absolutely required for insulin-stimulated Stat5 phosphorylation. Therefore, it is likely that JAK1, JAK2, or Tyk2 could play an equivalent role in Stat5 activation, and that the lack of one JAK can be compensated by the others. The same approach has yielded similar results for PDGF receptor-mediated activation of Stat3 proteins (39, 40).

Based on the evidence presented here, we have proposed a model for IR-mediated tyrosine phosphorylation of Stat5 proteins. In this model, Stat5 proteins are recruited to the same autophosphorylation site pY972 in the IR that is used to recruit other signaling molecules such as IRS-1. Once recruited to the receptor, Stat5 proteins become phosphorylated by IR-associated JAKs or the IR itself. In our transfection system, the JAK-dependent mechanism is quite evident at low levels of Stat5, whereas at higher levels of Stat5 the direct IR component predominates. Neither of these pathways is mutually exclusive. Our model predicts that the magnitude of the JAK-dependent component of insulin-stimulated Stat5 activation in a given cell will be related to the percentage of insulin receptors with JAKs bound to each ß-subunit. It is therefore not clear which of these kinases play the most important role in vivo. To determine the physiological importance of JAKs in IR signaling, it would be necessary to identify the JAK binding site on the IR and demonstrate that this site is required for JAK association and activation. Future studies will address these issues.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents and Antibodies
Porcine insulin and BSA were purchased from Sigma (St. Louis, MO). Recombinant human GH was purchased from Genentech, Inc. (South San Francisco, CA). AG490 was obtained from Calbiochem (San Diego, CA). The generation, specificity, and use of the antiphospho-Stat5 monoclonal antibody 18E5 has already been described (49, 51). Anti-Stat5a and anti-Stat5b polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The monoclonal anti-pY antibody, RC20, was purchased from BD Transduction Laboratories (San Diego, CA). The monoclonal anti-pY antibody, 4G10, as well as JAK1 and JAK2 polyclonal antibodies, was purchased from Upstate Biotechnology, Inc. (Lake Saranac, NY). Anti-FLAG monoclonal antibody (M2) was purchased from Sigma.

Cell Culture and Transfections
All cell lines were cultured in a 37 C, 5% CO2 humidifed incubator. COS7 cells were grown in high-glucose DMEM supplemented with 10% calf serum. 293T cells, 2fTGH, U1A, U4A, and {gamma}2A cells were grown in high-glucose DMEM supplemented with 10% fetal bovine serum. H35 rat hepatoma cells were grown in low-glucose DMEM supplemented with 10% fetal bovine serum. Transfection of 2fTGH, U1A, U4A, and {gamma}2A cells were performed with Fugene 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s protocol. Transfection of COS7 and 293T cells was carried out by the calcium phosphate method (66, 67). Approximately 16 h before transfection, cells were seeded at 80% confluence in 6-cm diameter dishes. Fifteen hours after the addition of calcium phosphate-DNA precipitates or Fugene-DNA complexes, the cells were washed twice with PBS, incubated in growth media for 24 h, and then placed in serum-free media (DMEM supplemented with 0.1% BSA) for 16 h before insulin stimulation. For detection of tyrosine phosphorylation of endogenous Stat5 proteins in H35 cells, cultures were grown to 80% of confluence and serum starved overnight before insulin stimulation. To determine the effect of JAK inhibition by AG490 on insulin-stimulated Stat5 activation, nearly confluent cultures of H35 cells were serum starved for 2 h and placed in serum-free DMEM containing either dimethylsulfoxide (DMSO) or 50, 100, 200, or 400 µM of AG490 for 4 h before insulin or GH stimulation.

Plasmids and Preparation of IR Mutant Constructs
The construction of the N969A/P970A, L973R, A975D, and L973R/A975D mutant plasmids was performed using two DNA fragments obtained by PCR. Briefly, a 5' XhoI site-containing oligonucleotide and a 3'-oligonucleotide containing the appropriate mutation coupled with a restriction site unique for each mutation were used to generate a 5'-DNA fragment using pECE-IR (5) containing the full-length IR cDNA as the template. A 5'-oligonucleotide containing the respective restriction site unique for each mutation and a 3'-BstXI site-containing oligonucleotide were used to generate a 3'-DNA fragment using pECE-IR as the template. The two DNA fragments were digested with XhoI or with BstXI and the restriction enzyme that cuts the unique site, and ligated to each other. The resulting DNA fragment is cloned into pECE-IR replacing the original IR cDNA by using the XhoI and BstXI sites.

The expression plasmids containing Stat5a, Stat5b, Stat5a-FLAG, and Stat5b-FLAG were kindly provided by Dr. Jim Ihle. The expression plasmid containing FLAG-SOCS1 was a generous gift from Drs. Tracy Wilson and Douglas Hilton. The DN mutant of JAK1 contained a three-amino acid change in the conserved region VIII of the kinase domain (FWYAPE -> LTWAPV) that impairs its catalytic function. The DN mutant of JAK2 contained a single-amino acid change (Lys -> Ala) at the ATP binding site, impairing the ATP binding and catalytic activity of the enzyme. These mutants were generated in the laboratory of Dr. David Levy and are generous gifts from him. The DN mutant of Tyk2 contained a single-amino acid change (Lys -> Ala) at the ATP binding site, impairing the ATP binding and catalytic activity of the enzyme. This mutant was kindly provided by Dr. John Krolewski. The human IRS-1 expression vector was kindly provided by Dr. Wei-Quin Li (National Institutes of Health).

Western Blotting and Immunoprecipitation
Cellular proteins were extracted either with radioimmunoprecipitation analysis (RIPA) buffer [50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 5 mM EDTA, pH 8.0; 1% sodium deoxycholate; 10 µg/ml aprotinin (Sigma-Aldrich, St. Louis, MO), 1% Triton X-100; 1 mM sodium orthovanadate; 1 mM phenylmethylsulfonyl fluoride (PMSF)], or Nonidet P-40 (NP-40) buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 5 mM EDTA, pH 8.0, 1% NP-40; 1% Trasylol; 1 mM sodium orthovanadate; 1 mM PMSF). Cell lysates were either fractionated directly by SDS-PAGE followed by transfer to nitrocellulose or immunoprecipitated with indicated antibodies in RIPA or NP-40 buffer. The immunoprecipitates were washed three times, after which bound proteins were eluted by boiling in 2x sodium dodecyl sulfate (SDS) sample buffer, fractionated by SDS-PAGE, and electroblotted onto nitrocellulose filters. The filters were blocked overnight at 4 C in either 1% BSA (anti-pY) or 5% nonfat milk (all other antibodies) in 10 mM Tris-HCl, pH 7.5; 150 mM NaCl; and 0.1% Tween 20 [Tris-buffered saline (TBS)-Tween]. Binding of the primary antibody was performed in either 1% BSA or 5% nonfat milk in TBS-Tween for 4 h at room temperature or overnight at 4 C. After antibody binding, the filters were washed in TBS-Tween three times for 5 min each at room temperature. If needed, the filters were incubated with a secondary antibody (either goat antirabbit or rabbit antimouse IgG) conjugated with either horseradish peroxidase or alkaline phosphatase. The filters were washed extensively to remove nonspecific binding and developed with the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ) or by adding alkaline phosphatase substrates, 5-bromo-4-chloro-3-indonyl phosphate and nitroblue tetrazolium in 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 5 mM MgCl2.

Reporter Gene Assays
For reporter gene assays, 2fTGH cells were transfected with Superfect reagent (QIAGEN, Chatsworth, CA) according to the manufacturer’s method with a cytomegalovirus (CMV)-Renilla luciferase plasmid as a control for transfection efficiency, a Stat-responsive firefly luciferase reporter gene, and either empty vector or the cDNA expression plasmids indicated in the figure legends. The IFN{gamma}-responsive Stat1 reporter gene (m67Luc) contains four copies of the m67-sis-inducible element linked to a TATA box and the firefly luciferase open reading frame. The insulin-responsive Stat5 reporter gene (LHRR) contains three copies of the ß-casein-prolactin-inducible element linked to a minimal thymidine kinase promoter and the firefly luciferase open reading frame (50). After 16 h, the cells were rinsed with PBS, placed in DMEM containing 0.5% fetal calf serum for 6 h, and left untreated or treated with either hIFN{gamma} (1000 U/ml, Roche) or insulin (50 nM) for 6 h. Cells were harvested in passive lysis buffer and both firefly and Renilla luciferase activity were measured with the Dual Luciferase Reporter kit according to the manufacturer’s protocol (Promega Corp., Madison, WI). For all conditions, triplicate transfections were performed, the values for reporter (firefly) luciferase activity were normalized to Renilla luciferase activity, and the results were expressed as the average fold stimulation by insulin or IFN.

Protein Purification
Baculovirus-expressed human IR kinase domain (IR-KD) was purified to homogeneity as described (68). A recombinant baculovirus expressing full-length murine Stat5b was generated and used to infect High Five cells (Invitrogen, San Diego, CA). Forty-eight hours post infection, the cells were harvested by low-speed centrifugation (1000 x g for 10 min), resuspended in ice-cold homogenization buffer (50 mM Tris-HCl, pH 7.5; 20 mM NaCl; 250 mM sucrose; 1 mM dithiothreitol; and 0.5 mM PMSF) and lysed by 25 strokes with a Potter-Elvehjem homogenizer at 4 C. Unless otherwise stated, all purification steps were performed at 4 C. The homogenate was subjected to high-speed centrifugation (100,000 x g for 90 min), the resulting supernatant filtered (0.45 µm, Durapore) and applied to an FPLC Source Q-15 column (Amersham Pharmacia Biotech) that had been preequilibrated with homogenization buffer minus sucrose (buffer A). The column was developed with a linear gradient of NaCl (20–500 mM) in buffer A. Fractions were screened for the presence of Stat5b by immunoblotting with anti-Stat5b and antiphospho-Stat5 antibodies. Full-length Stat5b fractionated into two major peaks. The first peak (I) contained large amounts of unphosphorylated Stat5b. The second peak (II), which eluted at slightly higher salt concentration, contained large amounts of Stat5b phosphorylated on Y699. Peak I fractions were pooled and diluted with an equal volume of 20 mM Tris-HCl, pH 8.9 buffer, concentrated on a HiTrap Q column. The unphosphorylated Stat5b was eluted with 0.5 M NaCl in buffer A and the eluate applied to an FPLC Super-Dex 200 gel filtration column (Amersham Pharmacia Biotech). The major protein peak eluted at a volume consistent with a Stat5b monomer (>100,000 kDa) and contained virtually homogenous Stat5b (>95% full-length Stat5b). The eluate containing the Stat5b was concentrated by centrifugation in a centriprep unit (Amicon, Inc., Beverly, MA), stabilized against freezing by the addition of glycerol (35% wt/vol), and stored at -20 C.

For JAK2, High Five cells were infected with a recombinant baculovirus expressing full-length murine JAK2 (kindly provided by J. Krolewski, University of California, Irvine, CA). Forty-eight hours post infection, the cells were harvested by low-speed centrifugation (1000 x g for 10 min), resuspended in ice-cold homogenization buffer (50 mM Tris-HCl, pH 8.9; 20 mM NaCl; 250 mM sucrose; 1 mM dithiothreitol; 0.5 mM PMSF; and 0.5 mM activated sodium orthovanadate) and lysed by 25 strokes with a Potter-Elvehjem homogenizer. All protein purification steps were performed at 4 C. The homogenate was subjected to high-speed centrifugation (100,000 x g for 90 min), and the resulting supernatant was filtered (0.45 µm, Durapore) and applied to an FPLC Source Q-15 column (Amersham Pharmacia Biotech) that had been preequilibrated with homogenization buffer minus sucrose (buffer A, pH 8.9). The column was developed with a linear gradient of NaCl (20–500 mM) in buffer A, pH 8.9. Fractions containing significant amounts of tyrosine-phosphorylated JAK2 were identified by Western blotting with both anti-JAK2 and anti-pY (4G10) antibodies and Coomassie blue staining. Fractions containing the highest amount of tyrosine-phosphorylated JAK2 (active JAK2) were pooled and diluted with an equal volume of buffer A, pH 8.9. This fraction was then concentrated by binding to a HiTrap Q column and eluting with a small volume of 0.5 M NaCl in buffer A and directly applied to an FPLC Super-Dex 200 gel filtration column. The tyrosine-phosphorylated JAK2 peak eluted at a volume consistent with its molecular mass (>100,000 kDa) and contained no more than 10% JAK2 protein by Coomassie blue staining. After concentration by centrifugation in a centriprep unit (Amicon, Inc.), glycerol was added (35% wt/vol), and the partially purified preparation of full-length tyrosine phosphorylated JAK2 was stored at -20 C.

In Vitro Kinase Reaction
The IR-KD apoenzyme (nonphosphorylated) was subjected to a preactivation reaction in the absence of other substrates. IR-KD (2 µM) was incubated for 45 min at 25 C in a reaction containing 50 mM Tris-acetate (pH 7.5), 5 mM ATP, 20 mM Mg-acetate, and 0.05 mg/ml BSA. To prevent dephosphorylation of the IR-KD, the reaction was then diluted 20-fold with ice-cold buffer A containing 35% glycerol and kept on ice. Kinase reactions (30 µl) were performed as follows: either buffer alone (3 µl), preactivated IR-KD (3 µl) or tyrosine phosphorylated JAK2 (3 µl) were added to tubes containing a Stat5b substrate cocktail (27 µl) and incubated for 30 min at 25 C. The final concentrations of individual components was: 1 µM Stat5b, 1 mM ATP, 10 mM Mg-acetate, 0.1 mg/ml BSA, 50 mM Tris-acetate (pH 7.5), approximately 7% glycerol, and either no kinase, 10 nM IR-KD, or about 16 nM JAK2. At the end of the 25 C incubation, aliquots were removed and either added to tubes containing an equal volume of 2x SDS sample buffer and boiled for 8 min (SDS-PAGE and Western blotting analysis).

Assessment of Stat5b Activation
One sixth (10 µl) of each of the kinase reactions was run on duplicate 7.5% SDS-PAGE gels. After transfer to nitrocellulose, phosphorylation of Stat5b on Y699 was assessed by immunoprobing with the antiphospho-Stat5 monoclonal antibody 18E5, and overall tyrosine phosphorylation of both kinases and Stat5b was assessed by immunoprobing with the anti-pY monoclonal antibody 4G10 (Upstate Biotechnology, Inc.).


    ACKNOWLEDGMENTS
 
We thank J. Ihle for the gifts of Stat5a and Stat5b expression plasmids; T. Wilson and D. Hilton for the gift of SOCS1 expression plasmid; J. Krolewski and D. Levy for the gifts of wild-type and DN JAK expression plasmids; I. Kerr and G. Stark for the JAK-deficient cell lines; R. Taub for the H35 rat hepatoma cells; M. Goldfarb for his critical reading of this manuscript; and C. Sadowski, T.-S. Choi, J. Chan, C. S. Zong, P. Sachdev, and K. T. Nguyen for their assistance in the preparation of this manuscript.


    FOOTNOTES
 
This work was supported by grants from the American Diabetes Association and NIH Grant DK-53000 (to H.B.S.) and in part by NIH Grants CA-29339 and CA-55054 (to L.H.W.).

Abbreviations: CMV, Cytomegalovirus; DMSO, dimethylsulfoxide; DN, dominant negative; EGF, epidermal growth factor; FPLC, forced pressure liquid chromatography; GST, glutathione-S-transferase; IR, insulin receptor; IR-KD, IR kinase domain; IRS, insulin receptor substrate; JAK, Janus kinase; KD, kinase domain; KI, kinase inactive; NP-40, Nonidet P-40; PDGF, platelet-derived growth factor; PMSF, phenylmethylsulfonyl fluoride; pStat5, phospho-Stat5; pY, phosphotyrosine; PTK, protein tyrosine kinase; RIPA, radioimmunoprecipitation analysis; SDS, sodium dodecyl sulfate; Shc, Src homology collagen; SH2, Src homology 2; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; TBS, Tris-buffered saline.

Received for publication January 14, 2002. Accepted for publication August 15, 2002.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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