Insulin induces tyrosine phosphorylation of JAK2 in insulin-sensitive tissues of the intact rat.

The Janus kinase family of protein tyrosine kinases constitutes a novel type of signal transduction pathway activated in response to a wide variety of polypeptide ligands and has four known members: JAK1, JAK2, JAK3, and Tyk2. In this study, we examined the ability of insulin to stimulate JAK2 tyrosine phosphorylation in insulin-sensitive tissues of the intact rat using immunoprecipitation and immunoblotting. The results demonstrate that after an infusion of insulin, JAK2 is rapidly tyrosine phosphorylated (and the kinase is activated) in the liver, adipose tissue, skeletal muscle, heart, and isolated adipocytes. The presence of phosphorylated JAK2 was detectable after an infusion of insulin that increased serum insulin to physiological postprandial levels (40-70 microunits/ml). Co-immunoprecipitation with anti-insulin receptor antibody, anti-JAK2 antibody, and anti-IRS-1 antibody showed that JAK2 interacts with the insulin receptor and IRS-1 to form stable complexes following stimulation by insulin. In two animal models of insulin resistance the regulation of JAK2 tyrosine phosphorylation after insulin infusion paralleled the phosphorylation of the insulin receptor and of IRS-1. In conclusion, our data indicate that after physiological stimulation by insulin in the intact animal, JAK2 associates with the insulin receptor and is tyrosine phosphorylated in insulin-sensitive tissues in a time- and dose-dependent fashion.

The Janus kinase family of protein tyrosine kinases constitutes a novel type of signal transduction pathway activated in response to a wide variety of polypeptide ligands and has four known members: JAK1, JAK2, JAK3, and Tyk2. In this study, we examined the ability of insulin to stimulate JAK2 tyrosine phosphorylation in insulin-sensitive tissues of the intact rat using immunoprecipitation and immunoblotting. The results demonstrate that after an infusion of insulin, JAK2 is rapidly tyrosine phosphorylated (and the kinase is activated) in the liver, adipose tissue, skeletal muscle, heart, and isolated adipocytes. The presence of phosphorylated JAK2 was detectable after an infusion of insulin that increased serum insulin to physiological postprandial levels (40 -70 microunits/ml). Co-immunoprecipitation with anti-insulin receptor antibody, anti-JAK2 antibody, and anti-IRS-1 antibody showed that JAK2 interacts with the insulin receptor and IRS-1 to form stable complexes following stimulation by insulin. In two animal models of insulin resistance the regulation of JAK2 tyrosine phosphorylation after insulin infusion paralleled the phosphorylation of the insulin receptor and of IRS-1. In conclusion, our data indicate that after physiological stimulation by insulin in the intact animal, JAK2 associates with the insulin receptor and is tyrosine phosphorylated in insulin-sensitive tissues in a time-and dosedependent fashion.
The insulin receptor is the principal mediator of insulin action on cellular mitogenic and metabolic processes. The insulin receptor ␤-subunit, which contains an intrinsic tyrosine kinase, undergoes tyrosyl autophosphorylation and is activated in response to insulin binding to the extracellular ␣-subunit. Moreover the discovery of the insulin receptor's tyrosine kinase activity suggested that the mechanism of insulin action involved the tyrosyl phosphorylation of intracellular substrates. Using anti-phosphotyrosine antibodies an insulin-stimulated phosphoprotein called pp185 was identified in many cells and tissues (1,2). One component of the pp185 band was purified and cloned from several sources (3)(4)(5)(6)(7)(8). The cloned protein was called insulin receptor substrate 1 (IRS-1). 1 More recently another constituent of the pp185 band termed IRS-2 was also purified, and its cDNA sequence was determined (9). There is also evidence showing that the protein Shc is tyrosine phos-phorylated in response to insulin (10 -13). Other proteins such as ecto-ATPase (14) and pp60 (15) are also known to be phosphorylated following insulin treatment. Furthermore a direct interaction between insulin receptor and phosphatidylinositol 3-kinase (16,17) in addition to the interaction/activation of the later with insulin receptor substrates 1 and 2 (9,18) has been demonstrated.
The Janus kinase (JAK) family of protein tyrosine kinases constitute a novel type of signal transduction pathway activated in response to a wide variety of polypeptides ligands. The JAK family of nonreceptor protein tyrosine kinases has four known members: JAK1 (19), JAK2 (20,21), JAK3 (22,23), and Tyk2 (24). Each protein is ϳ130 kDa in mass and has a Cterminal tyrosine kinase domain, an adjacent kinase-related domain, and five further domains with amino acid similarity between members of the family extending toward the N terminus (20). Different cytokines and polypeptides hormones activate different JAKs. The receptors for erythropoietin (25), prolactin (26,27), growth hormone (28), and angiotensin II (29) each have been demonstrated to bind and activate JAK2. However, the effect of insulin on JAK2 tyrosine phosphorylation and its association with the insulin receptor have not yet been investigated. In this report, we have examined the ability of insulin to stimulate JAK2 phosphorylation in insulin-sensitive tissues of the intact rat after injection of the hormone. Our data indicate that following stimulation by insulin JAK2 associates with the insulin receptor and is tyrosine phosphorylated in the liver, heart, adipose tissue, and skeletal muscle in a time-and dose-dependent fashion.

Materials
Reagents for SDS-polyacrylamide gel electrophoresis and immunoblotting were from Bio-Rad. Aprotinin, ATP, dithiothreitol, HEPES, phenylmethylsulfonyl fluoride, angiotensin II, Triton X-100, Tween 20, glycerol, and bovine serum albumin (BSA, fraction V) were from Sigma. Protein A-Sepharose 6MB was from Pharmacia Biotech Inc., 125 I-protein A was from ICN Biomedicals (Costa Mesa, CA), nitrocellulose paper (BA85, 0.2 mm) was from Schleicher & Schuell, and [␥-32 P]ATP was from Amersham Corp. Sodium amobarbital (Amytal) and human recombinant insulin (Humulin R) were from Eli Lilly. Anti-IRS-1 antibodies were raised in rabbits using a synthetic peptide (YIPGATMGT-STALTGDEAA) derived from the last 15 amino acids of the C terminus of rat IRS-1 as described previously (30). Anti-phosphotyrosine monoclonal and anti-JAK2 polyclonal antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-insulin receptor antibody was raised in rabbits using a synthetic peptide derived from the amino acid sequence (KKNGRILTLPRSNPS) corresponding to the C terminus of the protein.

Methods
Animals and Tissue Extracts-Male rats (130 -180 g) were allowed access to standard rodent chow and water ad libitum. Food was withdrawn 12-14 h before the experiments. The rats were anesthetized with sodium amobarbital (15 mg/kg body weight, intraperitoneally) and used 10 -15 min later, as soon as anesthesia was assured by the loss of pedal and corneal reflexes. The abdominal cavity was opened, the portal vein exposed, and 0.5 ml of saline (0.9% NaCl) with or without 2 g of insulin was injected. One minute later, the liver was removed, minced coarsely, and homogenized immediately in approximately 10 volumes of solubilization buffer A at 4°C with a Polytron PTA 20S generator (Brinkmann Instruments model PT 10/35) operated at maximum speed (setting 10) for 30 s. The solubilization buffer A was composed of 1% Triton X-100, 50 mM HEPES (pH 7.4), 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2 mM phenylmethylsulfonyl fluoride, and 0.1 mg/ml aprotinin. In some experiments other tissues such as adipose tissue and skeletal and heart muscle were also extracted. No more than two tissues were processed from the same animal. For the time course and dose-response experiments with adipose tissue, skeletal muscle, and heart tissue, insulin was injected into the vena cava. The extracts were centrifuged at 55,000 rpm at 4°C in a Beckman 70.1 Ti rotor for 60 min to remove insoluble material, and the resulting supernatant was used for immunoprecipitation with anti-JAK2, anti-insulin receptor, and anti-IRS-1 antibodies. The immune complexes were precipitated with protein A-Sepharose 6MB and were washed three times with 50 mM Tris (pH 7.4), 2 mM sodium vanadate, and 0.1% Triton X-100.
Cell Incubations-Epididymal and perirenal fat were harvested from 130 -180-g rats. Adipocytes were isolated by collagenase digestion as described previously (31,32) and after being washed free of enzymes were suspended 1:3 (v/v) in Krebs-Ringer phosphate buffer containing 1% BSA and 5 mM glucose. Aliquots of the cell suspension (600 l) were incubated with insulin (400 ng/ml) or saline in 1.5-ml plastic centrifuge tubes for 1 min. The incubation was terminated by centrifuging for 5 s and aspirating the incubation medium. Proteins were extracted by adding 600 l of the extraction buffer as described above. The extracts were centrifuged and used for immunoprecipitation with anti-JAK2 antibody.
Protein Analysis by Immunoblotting-After washing, the pellet was resuspended in Laemmli sample buffer (33) with 100 mM dithiothreitol and heated in a boiling water bath for 4 min. The samples were subjected to SDS-polyacrylamide gel electrophoresis (6% Tris acrylamide) in a Bio-Rad miniature slab gel apparatus. Electrotransfer of proteins from the gel to nitrocellulose was performed for 2 h at 100 V (constant) in a Bio-Rad miniature transfer apparatus (Mini-Protean) as described by Towbin et al. (34) but with 0.02% SDS added to the transfer buffer to enhance the elution of high molecular mass proteins. Nonspecific protein binding to the nitrocellulose was reduced by preincubating the filter overnight at 4°C in blocking buffer (3% BSA, 10 mM Tris, 150 mM NaCl, and 0.02% Tween 20). The prestained molecular mass standards used were myosin (194 kDa), ␤-galactosidase (116 kDa), bovine serum albumin (85 kDa), and ovalbumin (49.5 kDa).
The nitrocellulose blot was incubated with anti-phosphotyrosine antibodies or with the appropriate antibody diluted in blocking buffer for 4 h at 22°C and washed for 60 min with the blocking buffer without BSA. The blots were then incubated with 2 Ci of 125 I-protein A (30 Ci/g) in 10 ml of blocking buffer for 1 h at 22°C and washed again as described above for 2 h. 125 I-Protein A bound to the anti-phosphotyrosine or other antibodies was detected by autoradiography using preflashed Kodak XAR film with Cronex Lightning Plus intensifying screens at Ϫ70°C for 12-48 h. Band intensities were quantitated by optical densitometry (Molecular Dynamics) of the developed autoradiogram.
JAK2 in Vitro Kinase Assay-Immunoprecipitated proteins bound to protein A-Sepharose were washed with kinase buffer (50 mM NaCl, 5 mM MgCl 2 , 5 mM MnCl 2 , 0.1 mM Na 3 VO 4 , and 10 mM HEPES, pH 7.4) and subsequently incubated for 30 min at 24°C with kinase buffer containing 0.25 mCi/ml [␥-32 P]ATP. After thorough washing with kinase buffer, the proteins were eluted by boiling with Laemmli sample buffer and separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose as described above (19,21).
JAK2 tyrosine kinase activity was also measured by autophosphorylation using a different approach as described previously (29). Insulin was infused into the portal vein to stimulate limited receptor activation and partial JAK2 autophosphorylation. JAK2 was then immunoprecipitated and allowed to autophosphorylate in vitro in the presence of exogenous ATP. Tyrosine phosphorylation was measured by immunoblotting with anti-phosphotyrosine antibody.
Other-Protein determination was performed by the Bradford dye method (35) using the Bio-Rad reagent and BSA as the standard.
Statistical Analysis-Experiments to investigate the regulation of FIG. 1. Insulin-stimulated JAK2 tyrosine phosphorylation in rat tissues. Rats were anesthetized, and the abdominal wall was incised to expose the viscera. Saline, 2 g of insulin, or 10 Ϫ8 M angiotensin II (as depicted in the figure) were administered into the portal vein (liver) or the vena cava (adipose tissue, heart, and skeletal muscle) as a bolus injection. One minute later the tissues were excised and homogenized in extraction buffer at 4°C as described under "Experimental Procedures." After centrifugation, aliquots containing equal amounts of protein were immunoprecipitated with anti-JAK2 antibody and protein A-Sepharose 6MB and then resolved on 6% SDS-polyacrylamide gels. The protein bands were subsequently transferred to a nitrocellulose membrane and detected with anti-phosphotyrosine antibody and 125 I-protein A, after which the membrane was subjected to autoradiography. The data are representative of three experiments.

FIG. 2. Time course and dose-response of insulin-stimulated JAK2 tyrosine phosphorylation in rat liver (panel A) and heart (panel B).
Rats were anesthetized, and the abdominal wall was incised to expose viscera. Saline or insulin (at the time and dose indicated) were administered into portal vein or vena cava as a bolus injection. In the dose-response experiment, 1 and 3 min after insulin infusion the liver and the heart, respectively, were excised and homogenized in extraction buffer at 4°C, immunoprecipitated with anti-JAK2 or anti-insulin receptor antibodies, and immunoblotted as described in legend for Fig. 1. The data are representative of three experiments.
JAK2 tyrosine phosphorylation in animal models of insulin resistance were always performed studying the physiological or pathological group of animals in parallel with a control group. Comparisons between fed and fasted (72 h), 2-month-old versus 20-month-old rats were made using paired and unpaired t test.

RESULTS AND DISCUSSION
To investigate whether the protein JAK2 is phosphorylated following stimulation by insulin, we infused insulin into the portal vein of rats and then removed and homogenized the liver and immunoprecipitated the proteins with a polyclonal JAK2 antiserum. The JAK2 immunoprecipitates were analyzed for tyrosyl phosphorylation by immunoblotting with a monoclonal anti-phosphotyrosine antibody. JAK2 was rapidly tyrosine phosphorylated following exposure of the liver to insulin (Fig.  1). The same response was observed when insulin was injected into the vena cava and the adipose tissue, skeletal muscle and heart tissue extracted (Fig. 1). As a control for JAK2 phosphorylation, we infused angiotensin II into the vena cava and then examined the level of tyrosine phosphorylation in the heart. The results show that after stimulation with angiotensin II there was a clear increase in cardiac JAK2 tyrosine phosphorylation (Fig. 1).
To estimate the rate of insulin-induced JAK2 phosphorylation in the liver, we performed a time course experiment after administration of insulin into the portal vein. As shown in Fig.  2A, 1 min after exposure to insulin there was already a substantial increase in the phosphorylation of JAK2, the rate of which decreased after the first minute and had almost vanished after 1 h. A similar rapid phosphorylation was observed for the ␤-chain of the insulin receptor, which was used as a control for insulin receptor activation ( Fig. 2A).
In the heart, the time course of insulin-induced JAK2 phosphorylation was similar to the liver, with a rapid increase at 1 min although the maximal increase was observed 3 min after insulin injection, followed by a decline over the 15 min of the experiment (Fig. 2B). The behavior of cardiac insulin receptor phosphorylation was also similar to the liver. The time courses of JAK2 phosphorylation in skeletal muscle and adipose tissue were similar to those of liver and heart (data not shown).
The insulin-stimulated phosphorylation of JAK2, as determined by anti-JAK2 immunoprecipitates of liver extracts, was dose-dependent ( Fig. 2A). The presence of phosphorylated JAK2 was detectable after the injection of as little as 20 ng of insulin, and half-maximal stimulation occurred with 200 -400 ng of the hormone ( Fig. 2A). Although we could not determine portal insulin levels, in preliminary experiments peripheral insulin levels obtained 90 s after an intraportal injection of 400 ng of insulin ranged between 40 and 70 microunits/ml and were similar to the normal physiological postprandial range in rats. Maximal stimulation was observed with 2 g of insulin and was 7-12 times greater than the basal levels (n ϭ 3).
The dose-response relationships for the insulin stimulation of JAK2 phosphorylation from adipose tissue, skeletal muscle, and heart were similar, when insulin was infused into the vena cava. Fig. 2B shows such a relationship for cardiac tissue and indicates that the maximal effect was observed at 2 g of insulin and decreased thereafter.
Because insulin may affect other mediators in vivo to induce these responses, we performed experiments in which the effects of insulin were analyzed in isolated adipocytes. The results are presented in Fig. 3A. A clear stimulation of JAK2 tyrosine phosphorylation was induced by insulin, showing that insulin has a direct effect on this pathway.
To determine whether JAK2 kinase activity was stimulated by insulin, we measured enzyme autophosphorylation in vitro using two approaches (Fig. 3, B and C). In the first approach, FIG. 3. A, insulin induces JAK2 tyrosine phosphorylation in isolated adipocytes. Adipocytes isolated from normal rats were incubated in the absence or the presence of 400 ng/ml insulin for 1 min, and then the extracts were immunoprecipitated with anti-JAK2 antibody and immunoblotted with anti-phosphotyrosine antibody. B, insulin activates JAK2 kinase activity in vitro in immunoprecipitates. Liver extracts (from rats infused with insulin or saline) were immunoprecipitated with anti-JAK2 antibody. The immunoprecipitates were used for in vitro kinase assays, and the products were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and detected by autoradiography. The blots were subsequently probed with anti-JAK2 antibody (data not shown). C, JAK2 tyrosine kinase activity measured by autophosphorylation. Insulin (20 ng) was injected into the portal vein to stimulate partial JAK2 autophosphorylation. JAK2 was then immunoprecipitated and allowed to autophosphorylate in vitro in the presence of exogenous ATP (lane 4). Tyrosine phosphorylation was measured by immunoblotting with an anti-phosphotyrosine antibody. Control conditions are shown in lanes 1-3. Lane 1, the liver extract was not exposed to insulin, nor was exogenous ATP added to the in vitro autophosphorylation reaction. Lane 2, the liver extract was not exposed to insulin, but ATP was added to the in vitro autophosphorylation reaction. Lane 3, insulin (20 ng) was infused into the portal vein, and the liver then extracted, but no exogenous ATP was added during the in vitro autophosphorylation step. The small signal seen in this lane probably represents JAK2 autophosphorylation using endogenous ATP. we immunoprecipitated liver extracts (with or without insulin treatment) with anti-JAK2 antibodies and performed an in vitro kinase assay using [␥-32 P]ATP as described above. The results are presented in Fig. 3B and show that the JAK2 kinase activity was significantly increased in liver extracts after a portal infusion of insulin. In the second approach, a low dose of insulin (20 ng) was infused into the portal vein to obtain limited tyrosine phosphorylation of JAK2, which was then immunoprecipitated and reacted with ATP to permit autophosphorylation. The phosphorylation of tyrosine was quantified by Western blot analysis using an anti-phosphotyrosine antibody, which showed that insulin induces JAK2 autophosphorylation (Fig. 3C, lane 4). The small signal seen in the liver extract previously infused with a low dose of insulin but with no exogenous ATP added during the in vitro autophosphorylation step (Fig. 3C, lane 3) probably represents JAK2 autophosphorylation using endogenous ATP.
The rapid induction of JAK2 tyrosine phosphorylation by insulin suggests that JAK2 may associate with the insulin receptor. To test this possibility, liver extracts were immuno-precipitated with the anti-JAK2 antibody before and after insulin stimulation, and the precipitated proteins were probed with anti-receptor antibody. The experiments show that JAK2 co-precipitates with insulin receptor. Although small amounts of insulin receptor were bound to JAK2 before insulin stimulation, the addition of the hormone increased the insulin receptor-JAK2 association within 1 min. Anti-insulin receptor coimmunoprecipitation was also performed with the same tissue extracts. As shown in Fig. 4, JAK2 protein was co-immunoprecipitated by anti-insulin receptor antibody, with much higher affinity following insulin stimulation. These results were also reproduced in rat skeletal muscle, adipose tissue, and cardiac tissue and demonstrate that JAK2 physically associates with the insulin receptor clearly upon insulin stimulation.
Co-immunoprecipitation between JAK2 and IRS-1 in liver was also observed. In immunoprecipitates of IRS-1 that were blotted with anti-JAK2 antibody, a band corresponding to JAK2 was evident after insulin stimulation (Fig. 4). When the liver extracts were immunoprecipitated with anti-JAK2 antibody and blotted with anti-IRS-1 antibody, a band correspond-  (panel B). A, liver samples from fed or 72 h fasted rats were extracted and homogenized as described in the legend for Fig. 1, after an intraportal injection of saline or 2 g of insulin (as depicted in the figure). Aliquots containing equal amounts of protein were immunoprecipitated (IP) with anti-IRS-1 anti-JAK2 or anti-insulin receptor antibody and protein A-Sepharose 6MB and then resolved on 6% SDS-polyacrylamide gels. The proteins were transferred to nitrocellulose membranes and detected with anti-phosphotyrosine antibody and 125 I-protein A before undergoing autoradiography. The lower panel demonstrates the mean Ϯ S.E. of four separate experiments, determined by scanning densitometry. *, differences from control (2 months old) at p Ͻ 0.05. B, heart samples from of 2-month-old or 20-month-old rats were extracted and homogenized as described in the legend for Fig. 1 after an intraportal injection of saline or 2 g of insulin (as depicted in the figure). Aliquots containing equal amounts of protein were immunoprecipitated (IP) with anti-IRS-1, anti-JAK2, or anti-insulin receptor antibodies and protein A-Sepharose 6MB and then resolved on 6% SDS-polyacrylamide gels. The proteins were transferred to nitrocellulose membranes and detected with anti-phosphotyrosine antibody and 125 I-protein A before undergoing autoradiography. The lower panel demonstrates the mean Ϯ S.E. of four separate experiments, determined by scanning densitometry. *, differences from control (2 months old) at p Ͻ 0.05. ing to IRS-1 was detected in basal state, and after insulin stimulation there was an increase in this band (Fig. 4, lower  panel). As a control we performed also immunoprecipitation of liver extracts with anti-insulin receptor and anti-IRS-1 antibody, and blotted with anti-IRS-1 antibody. There was a band corresponding to IRS-1 in anti-insulin receptor immunoprecipitates in the basal state and an increase in this band after insulin stimulation. These results clearly demonstrated that JAK2 interacts with insulin receptor and IRS-1 and forms stable complexes following insulin stimulation.
Over the past several years, considerable progress has been made in elucidating the signaling pathways downstream from tyrosine phosphorylation. It is becoming increasingly clear that these pathways are complex and contain many branches that often diverge from a common point. One tyrosine kinase may phosphorylate many proteins. For example, in addition to autophosphorylation, the insulin receptor phosphorylates IRS-1 (3)(4)(5)(6)(7)(8), Shc (10 -13), ecto-ATPase (14), and IRS-2 (9). Moreover, converging branches and intracellular cross-talk between different signaling pathways have been demonstrated. Thus, there are two pathways from the insulin receptor that lead to the activation of GRB-2/m-SOS; one involves the phosphorylation of IRS-1, whereas the other involves the phosphorylation of Shc (13). It is interesting that hormones that do not contain an intrinsic tyrosine kinase, as well as cytokines, are able to phosphorylate IRS-1. This further suggests the presence of converging branches with different ligands and receptors. Because JAK2 is an obvious candidate for the tyrosine kinase responsible for growth hormone-and some cytokines-dependent tyrosyl phosphorylation of IRS-1 (36), it is interesting that JAK2 forms a stable complex with the insulin receptor and IRS-1 after stimulation with insulin. Whether JAK2 is able to phosphorylate IRS-1 in response to insulin remains to be determined.
Because JAK2 binds the insulin receptor and is phosphorylated after exposure to insulin, it is also a candidate for a role in the regulation of physiological and pathophysiological states of insulin resistance. Previous studies have shown that the insulin receptor is up-regulated in insulin-sensitive tissues after a period of fasting (37,38) and that this correlates with an increase in insulin receptor and IRS-1 phosphorylation (39). In order to investigate the effect of fasting on JAK2 phosphorylation in insulin-sensitive tissues, we injected insulin into the portal vein of fed and fasted rats, extracted the liver, and performed immunoprecipitation with anti-insulin receptor, anti-IRS-1, and anti JAK2 antibodies, followed by immunoblotting with antiphosphotyrosine antibody. The results of these experiments showed a remarkable increase in insulin receptor and IRS-1 phosphorylation during fasting, thus confirming previous studies, and also indicated that after insulin stimulation, JAK2 tyrosine phosphorylation increased more dramatically in the liver of rats fasted for 72 h (Fig. 5A).
We have recently demonstrated that in aging rats (20 months old) there is a decrease in insulin-stimulated insulin receptor and IRS-1 phosphorylation in insulin-sensitive tissues (40). We therefore evaluated the effect of aging on the tyrosine phosphorylation of JAK2 in the heart in response to insulin administered via the vena cava. The hearts were excised and homogenized, and the extracts used for immunoprecipitation and immunoblotting as described previously. The results demonstrated that JAK2 tyrosine phosphorylation in response to insulin paralleled that of insulin receptor and IRS-1 phosphorylation, because there was also a decrease in JAK2 tyrosine phosphorylation in the heart of old rats compared with 2-month-old rats (Fig. 5B).
In various cells and tissues, JAK activation has been proposed to be the signaling pathway that mediates the transcrip-tional activation of early growth response genes by cell surface cytokines (41). The JAK2 pathway may play a similar role in the control of insulin-induced cell growth. The shared use of JAK2 by multiple receptors is likely to reveal important connections between various hormones and cytokines that were previously unrecognized or that had been observed but remained unexplained.