INTRODUCTION

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Members of the cytokine receptor family do not have any enzymatic activity but instead associate constitutively or ligand inducibly with members of the Janus family of cytoplasmic tyrosine kinases (JAK1, JAK2, JAK3, and tyk2). Upon ligand stimulation, the receptor-associated JAKs are activated. The activated JAKs phosphorylate both the receptors and themselves on multiple tyrosines, generating docking sites for downstream signaling molecules that contain SH2 or other phosphotyrosine-interacting domains. Recruitment of these signaling molecules into the receptor-JAK complexes activates these signaling molecules or enables the activated JAKs to phosphorylate and activate them, thereby initiating a variety of downstream signaling pathways that lead to a wide range of biological responses. One class of well-studied substrates of JAKs is the signal transducers and activators of transcription (Stats). Stats are latent transcription factors present in the cytoplasm. In response to hormones and cytokines, Stats are recruited to receptor-JAK complexes and phosphorylated by JAKs on a conserved C-terminal tyrosine. This phosphotyrosine binds to the SH2 domain in other Stats, thus forming Stat homo- or heterodimers that migrate into the nucleus, bind to their response elements, and regulate expression of their target genes (5, 15). Stats play an essential role in cytokine signaling. For example, Stat1, -3, -4, -5A, -5B, and -6 are required for many of the actions of gamma interferon (2, 19), interleukin-6 (IL-6) (20, 22, 38), IL-12 (40), prolactin (18, 39), growth hormone (GH) (39, 41), and IL-4 (35), respectively.

Activation of JAKs is an obligatory step for cytokine action (13, 14). In the presence of ligands, one or more JAKs bind to a membrane proximal proline-rich region of cytokine receptors (1, 13, 14). It is thought that ligand binding causes homo- or hetero-oligomerization of cytokine receptor subunits. As a result, receptor-associated JAKs are brought into proximity, enabling JAKs to transphosphorylate each other on tyrosines within the kinase domain, resulting in activation (9, 26). Among ligands known to bind to members of the cytokine receptor family, more than two-thirds are known to activate JAK2; these include GH, erythropoietin, leptin, prolactin, gamma interferon, leukemia inhibitory factor, cardiotropin, ciliary neurotrophic factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, thrombopoietin, oncostatin M, IL-2, IL-3, IL-5, IL-6, IL-11, and IL-12 (1). Factors other than cytokine receptors also regulate JAK2. For instance, oxidized JAK2 is inactive whereas the reduced form of JAK2 is active (6), suggesting that oxidation is a regulatory mechanism for JAK2. SOCS-1/JAB, a cytokine-inducible protein, binds and inhibits JAK2, thereby serving as a negative feedback regulator of cytokine responses (7, 21, 37).

Recently, we identified SH2-B as a JAK2-interacting protein and a potent activator of JAK2 that is likely to play an important role in signaling by cytokines that activate JAK2 (30, 34). Three isoforms of SH2-B (, , and ) have been described to date (23, 25, 29, 34). They are identical except for the short C-terminal portion after the SH2 domain and are thought to be a result of alternative splicing of a single gene. SH2-B has multiple protein-protein interaction motifs, including an SH2 domain, a pleckstrin homology (PH) domain, multiple proline-rich regions, and numerous potential phosphorylation sites. Because of these motifs, SH2-B is thought to be an adapter protein in addition to being an activator of JAK2 (27, 31, 33, 34, 43). Because of the ability of SH2-B to activate JAK2 and presumably mediate additional functions of ligands that activate JAK2, it is important to determine how SH2-B interacts with JAK2. In this work, we provide evidence that SH2-B binds to JAK2 via multiple sites: the SH2 domain of SH2-B (amino acids 526 to 620) that binds to one or more phosphotyrosines in JAK2; and one or more sites within amino acids 269 to 555 that bind to JAK2 independent of the kinase activity and tyrosyl phosphorylation of JAK2. The SH2 domain is necessary and sufficient for SH2-B to stimulate JAK2. In contrast, the N-terminal 555 amino acids of SH2-B inhibit JAK2. We propose a model in which the interaction of the N-terminal region of SH2-B with JAK2 not only increases the subcellular local concentration of SH2-B but also inhibits basal and/or abnormal activation of JAK2. The former enables the SH2 domain of SH2-B to bind to JAK2 and enhance JAK2 activity as soon as JAK2 is activated by ligand binding, leading to a more robust cellular response to hormones and cytokines that activate JAK2.

	MATERIALS AND METHODS

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Reagents. Recombinant human GH was a gift of Eli Lilly and Co. Recombinant protein A-agarose was from Repligen. Aprotinin, leupeptin, and Triton X-100 were purchased from Boehringer Mannheim. The enhanced chemiluminescence detection system and [-³²P]ATP were from Amersham Corp. Polyclonal antibodies to rat SH2-B (SH2-B) were raised against a glutathione S-transferase (GST) fusion protein containing the C-terminal portion of SH2-B as described previously (34) and used at dilutions of 1:100 for immunoprecipitation and 1:15,000 for immunoblotting. Anti-JAK2 antiserum (JAK2) was raised in rabbits against a synthetic peptide corresponding to amino acids 758 to 776 (36) and was used at dilutions of 1:500 for immunoprecipitation and 1:15,000 for immunoblotting. Monoclonal antiphosphotyrosine antibody 4G10 (PY) was purchased from Upstate Biotechnology Inc. and used at a dilution of 1:7,500 for immunoblotting. Monoclonal antibody against Myc-tag (9E10; Myc) and polyclonal anti-Stat5B antibody (Stat5B) were from Santa Cruz Biotechnology Inc. Myc was used at dilutions of 1:100 for immunoprecipitation and 1:1,000 for immunoblotting. Stat5B was used at dilutions of 1:100 for immunoprecipitation and 1:5,000 for immunoblotting. Monoclonal anti-phospho-Stat5 (pStat5) was from Zymed Laboratories Inc. and used at 1 µg/ml in immunoblotting.

Plasmids. cDNAs encoding both wild-type murine JAK2 [JAK2 (WT)] and mutant murine JAK2(K882E), in which the critical lysine in the ATP binding domain is mutated to glutamate, were previously cloned into a mammalian expression vector and provided by J. Ihle and B. Witthuhn (St. Jude Children's Research Hospital). Plasmid encoding rat Stat5B was from L. Yu-Lee (Baylor College of Medicine). Construction of vectors encoding SH2-B or SH2-B(R555E) (with a defective SH2 domain) with a Myc tag at the N terminus (30) and Stat5B with a green fluorescent protein (GFP) tag at the N terminus (GFP-Stat5B) (12) has been described previously. To generate a series of N- or C-terminally truncated SH2-B, restriction sites were inserted at appropriate positions in SH2-B by using a QuickChange site-directed mutagenesis kit (Stratagene). The correspondent restriction enzyme was used to delete the N- or C-terminal portion of SH2-B. A detailed protocol will be provided upon request.

Cell culture and transfection. 3T3-F442A cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1 mM L-glutamine, 100 U of penicillin per ml, 100 µg of streptomycin per ml, 0.25 µg of amphotericin B per ml, and 9% calf serum. COS 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 10% fetal bovine serum. COS cells were transiently transfected using calcium phosphate precipitation (4) or FuGENE 6 transfection reagents (Boehringer Mannheim) and assayed 48 h after transfection.

GST fusion protein pull-down assay. 3T3-F442A cells were deprived of serum overnight 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 1% bovine serum albumin and treated for 10 min with 500 ng of GH per ml. Cells were then rinsed three times with 10 mM sodium phosphate (pH 7.4)-150 mM NaCl-1 mM Na₃VO₄ 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, 1 mM phenylmethylsulfonyl fluoride, 10 µg of aprotinin per ml, 10 µg of leupeptin per ml). Cell lysates were centrifuged at 14,000 × g for 10 min at 4°C. Proteins in cell lysates were quantified using the Pierce bicinchoninic assay protein assay reagent and incubated with the indicated immobilized GST fusion protein as described previously (34).

Immunoprecipitation and immunoblotting. Transfected COS cells were solubilized in lysis buffer. The cell lysates were incubated with the indicated antibody on ice for 2 h. The immune complexes were collected on protein A-agarose (50 µl) during 1 h of incubation at 4°C. The beads were washed three times with washing buffer (50 mM Tris [pH 7.5], 0.1% Triton X-100, 150 mM NaCl, 2 mM EGTA) and boiled for 5 min in a mixture (80:20) of lysis buffer and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (250 mM Tris-HCl [pH 6.8], 10% SDS, 10% -mercaptoethanol, 40% glycerol, 0.01% bromophenol blue). The solubilized proteins were separated by SDS-PAGE (5 to 12% gradient or 7.5% acrylamide gels). Proteins in the gel were transferred to a nitrocellulose membrane (Amersham) and detected by immunoblotting with the indicated antibody using enhanced chemiluminescence.

In vitro kinase assay. The in vitro kinase assay was performed as described previously (30). Briefly, JAK2 was immunoprecipitated using JAK2 from COS cells coexpressing JAK2 and the indicated wild-type and/or mutant SH2-B. After being washed twice with lysis buffer and twice with kinase buffer (50 mM HEPES [pH 7.6], 5 mM MgCl₂, 5 mM MnCl₂, 0.5 mM dithiothreitol, 100 mM NaCl, 1 mM Na₃VO₄), JAK2 immunoprecipitates were incubated at 30°C for 30 min in 50 µl of kinase buffer supplemented with aprotinin (10 µg/ml), leupeptin (10 µg/ml), and 20 µCi of [-³²P]ATP. After the in vitro kinase assay, proteins in the reaction mixture were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and visualized by autoradiography or immunoblotting with JAK2. In some experiments, the amount of ³²P incorporated into JAK2 was quantified. Autoradiographs were scanned using an Agfa or UMAX scanner and Fotolook SA or VistaScan DA software, and results were quantified using the Molecular Analyst image software from Bio-Rad. These values were then normalized for amount of immunoprecipitated JAK2, judged by scanning of JAK2 immunoblots. Multiple film exposure times were made to ensure that all bands were scanned within the linear range of the film.

Stat5B nuclear localization assay. 3T3-F442A cells were plated on glass coverslips and transfected with Transfast (Promega) according to the protocol recommended by the manufacturer. Cells were cotransfected with 1.3 µg of cDNA encoding GFP-Stat5B and either 2.5 µg of control plasmid or plasmid encoding Myc-tagged C-terminally truncated SH2-B (amino acids 1 to 555; C555). Following overnight incubation in serum-free medium, cells were stimulated for 40 min with GH (500 ng/ml) where appropriate, fixed (4% paraformaldehyde in phosphate-buffered saline [PBS] for 15 min at room temperature), and permeabilized (1% Triton X-100 and 4% paraformaldehyde in PBS for 15 min at room temperature). Nonspecific sites were blocked with 10% goat serum in PBS overnight at 4°C. Myc-tagged truncated SH2-B was visualized by immunostaining using Myc (1:200) followed by anti-mouse immunoglobulin G-Texas red (Jackson ImmunoResearch). Confocal imaging was performed with a Noran OZ laser scanning confocal microscope equipped with a 60× Nikon objective. GFP was excited at 488 nm by a krypton-argon laser, and fluorescence at 500 ± 12 nm was captured. Texas red was excited at 568 nm, and fluorescence above 590 nm captured. The nuclear-to-cytosol GFP fluorescence intensity ratios for 20 cells per condition were calculated as reported previously (12). Data were analyzed using a one-tailed unpaired t test. Differences were considered to be statistically significant at P < 0.05. Results are expressed as the mean ± standard error of the mean (SEM).

	RESULTS

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SH2-B binds to both active and inactive JAK2. Previous results indicated that the SH2 domain of SH2-B is sufficient for SH2-B to bind to activated JAK2 (34). To examine whether tyrosyl phosphorylation of JAK2 and the SH2 domain of SH2-B are required for the interaction between JAK2 and SH2-B, 3T3-F442A cells were treated for 10 min with or without GH (500 ng/ml), a potent stimulator of the activity and tyrosyl phosphorylation of JAK2. Proteins in cell lysates were incubated with immobilized GST fusion protein containing SH2-B, SH2-B(R555E), which lacks a functional SH2 domain, or the C-terminal 167 amino acids of SH2-B (N504, amino acids 504 to 670), which contain the entire SH2 domain. The bound proteins were eluted and immunoblotted with JAK2. N504 bound to JAK2 from GH-treated but not control cells (Fig. 1A, lanes 7 and 8), consistent with our previous conclusion that the SH2 domain of SH2-B binds only to tyrosyl-phosphorylated JAK2 present in GH-treated but not control cells (34). Surprisingly, GST-SH2-B bound to JAK2 from both control and GH-treated cells, although it bound less JAK2 from control cells than from GH-treated cells (Fig. 1A, lanes 3 and 4). GST-SH2-B(R555E) bound equally well to JAK2 from GH-treated and control cells (Fig. 1A, lanes 5 and 6), while GST alone did not interact with JAK2 even from GH-treated cells (Fig. 1A, lanes 1 and 2). Reprobing the blots with PY revealed that the JAK2 that bound to GST-N504, GST-SH2-B, or GST-SH2-B(R555E) was tyrosyl phosphorylated in GH-treated cells but not in control cells (data not shown). Consistent with these observations, a small amount of endogenous JAK2 from 3T3-F442A cells coimmunoprecipitated with endogenous SH2-B even in the absence of GH (Fig. 1B). The amount of JAK2 coimmunoprecipitating with SH2-B was substantially increased by GH treatment (Fig. 1B, lane 2). These results indicate that full-length SH2-B can bind to non-tyrosyl-phosphorylated JAK2 and that at least one site of interaction of SH2-B with non-tyrosyl-phosphorylated JAK2 most likely lies outside the SH2 domain of SH2-B.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   The SH2 domain of SH2-B is not required for the interaction of SH2-B with non-tyrosyl-phosphorylated JAK2. 3T3-F442A cells were stimulated with 500 ng of human GH per ml for 10 min. (A) Cell lysates were incubated with GST or GST fusion protein containing SH2-B (GST-WT), SH2-B(R555E) (GST-R555E), or N504 (GST-N504), as indicated. The precipitated proteins were immunoblotted (IB) with JAK2. The amount of GST-SH2-B(R555E) used was three times that of GST-SH2-B. (B) Proteins in lysates of cells treated with (lane 2) or without (lane 1) human GH were immunoprecipitated (IP) with SH2-B and immunoblotted with JAK2 (top). The blot was reprobed with SH2-B (bottom).

View larger version (46K): [in this window] [in a new window]   FIG. 2.   SH2-B binds more strongly to wild-type, tyrosyl-phosphorylated JAK2 than to kinase-inactive, non-tyrosyl-phosphorylated JAK2(K882E). COS cells were cotransfected transiently with plasmid (5 µg) encoding either JAK2 (WT) or kinase-inactive JAK2(K882E) (K-E) and plasmid (5 µg) encoding either Myc-tagged SH2-B (WT) or SH2-B(R555E) (R555E). Proteins in cell lysates were immunoprecipitated (IP) with SH2-B and immunoblotted (IB) with JAK2 (top) or SH2-B (bottom).

The SH2 domain of SH2-B is not required for SH2-B to bind to kinase-inactive, non-tyrosyl-phosphorylated JAK2. To determine the region(s) of SH2-B responsible for the lower-affinity interaction with non-tyrosyl-phosphorylated JAK2, SH2-B was truncated at either the C or N terminus (Fig. 3A) and tagged with a Myc epitope at the N terminus. The SH2-B mutants were individually coexpressed with kinase-inactive JAK2(K882E) in COS cells and then immunoprecipitated with Myc. The immunoprecipitates were immunoblotted with JAK2. N504, which contains the entire SH2 domain and was shown previously to bind to tyrosyl-phosphorylated JAK2 (WT) (34), was unable to bind kinase-inactive, non-tyrosyl-phosphorylated JAK2(K882E) (Fig. 3B, lane 1, top). In contrast, two N-terminal fragments of SH2-B (C631 and C555 [Fig. 3A]) coimmunoprecipitated with JAK2(K882E) (Fig. 3B, lanes 2 and 3, top). Importantly, the C-terminally truncated SH2-B lacking most of its SH2 domain (C555) bound to kinase-inactive JAK2(K882E) to a similar extent as C631 that contains the entire SH2 domain (Fig. 3B, lanes 2 and 3, top). Reprobing the same blot with Myc revealed that similar amounts of N504, C631, and C555 were expressed and immunoprecipitated by Myc (Fig. 3B, bottom). These results indicate that an intact SH2 domain of SH2-B is neither able to bind to inactive, non-tyrosyl-phosphorylated JAK2 nor required for binding of SH2-B to inactive, non-tyrosyl-phosphorylated JAK2.

View larger version (39K): [in this window] [in a new window]   FIG. 3.   SH2-B binds to JAK2 via at least two sites. (A) Schematic representation of SH2-B and various mutants. (B) COS cells were cotransfected transiently with plasmid (10 µg) encoding kinase-inactive JAK2(K882E) (K-E) and plasmid (4 µg) encoding the indicated SH2-B mutant with a Myc tag at the N terminus. Proteins in cell lysates were immunoprecipitated (IP) with Myc and immunoblotted (IB) with JAK2 (top). The same blot was reprobed with Myc (bottom). (C) COS cells were cotransfected transiently with plasmid (5 µg) encoding C555 and plasmid (5 µg) encoding either JAK2 (WT) or kinase-inactive JAK2(K882E) (K-E). Proteins in cell lysates were immunoprecipitated with Myc and immunoblotted with JAK2 (top). The same blot was reprobed with PY (middle) or Myc (bottom). The positions of migration of JAK2 and SH2-B and SH2-B mutants are noted in panels B and C.

Multiple regions in SH2-B, including the PH domain, contribute to maximal binding of SH2-B to inactive JAK2. To identify amino acids in SH2-B responsible for the binding of SH2-B to inactive JAK2, SH2-B mutants with different deletions at the N or C terminus (Fig. 4A) were generated. The SH2-B mutants were tagged with a Myc epitope at their N termini and coexpressed individually with kinase-inactive JAK2(K882E) in COS cells. These truncated forms of SH2-B were then immunoprecipitated with Myc, and immunoprecipitated proteins were immunoblotted with JAK2. Removing the N-terminal 117 (N118) or 268 (N269) amino acids of SH2-B did not decrease their interaction with kinase-inactive JAK2(K882E) (Fig. 4B, lanes 3 and 4, top). However, when the PH domain of SH2-B was deleted (N397), the truncated SH2-B lost its ability to interact with JAK2(K882E) (Fig. 4B, lane 5, top), although this mutant SH2-B still retains the entire SH2 domain (Fig. 4A). Further truncation of SH2-B (N504) did not restore binding to JAK2(K882E). This finding is most consistent with maximal interaction between JAK2(K882E) and SH2-B requiring the PH domain. The alternative hypothesis, that truncation of SH2-B alters its three-dimensional structure to a point where it can no longer bind to JAK2 seems less likely, given that N504 is able to bind and activate JAK2 (WT).

View larger version (60K): [in this window] [in a new window]   FIG. 4.   SH2-B binds to JAK2 via multiple sites. (A) Schematic representation of SH2-B and various mutants. (B) COS cells were cotransfected transiently with plasmid (10 µg) encoding kinase-inactive JAK2(K882E) (K-E) and plasmid (4 µg) encoding the indicated SH2-B mutant containing a Myc tag at the N terminus or the Myc tag alone. Proteins in cell lysates were immunoprecipitated (IP) with Myc and immunoblotted (IB) with JAK2 (top). The blots were reprobed with Myc (bottom). Positions of migration of molecular weight standards (in thousands), JAK2, Myc-SH2-B, and Myc-SH2-B mutants are indicated. Lanes 1 to 6 were from one experiment; lanes 7 to 11 were from a separate experiment. Proteins of approximately 26 kDa detected by Myc in lanes 3 and 4 are believed to represent proteolytic products of Myc-N118 and Myc-N269, respectively.

The SH2 domain of SH2-B is necessary and sufficient for its stimulatory effect on JAK2 and on JAK2-dependent phosphorylation of Stat5B on Tyr-699. We previously reported that SH2-B is a potent activator of JAK2 (30). To identify which region of SH2-B is involved in the activation of JAK2, we examined the stimulatory effect of different truncated SH2-B on JAK2. JAK2 was coexpressed with SH2-B mutants in COS cells, immunoprecipitated with JAK2, and subjected to an in vitro kinase assay by adding [-³²P]ATP in Mn²⁺-containing buffer. JAK2 autophosphorylation has been used previously to measure the kinase activity of JAK2. SH2-B strongly activated JAK2 (Fig. 5A, lanes 1 versus 2, top), consistent with our previous report (23). A similar stimulatory effect on JAK2 was observed for truncated SH2-B lacking N-terminal amino acids 1 to 268 (N269) or 1 to 503 (N504) (Fig. 5A, lanes 3 and 4, top) or C-terminally truncated SH2-B lacking amino acids 632 to 670 (C631) (Fig. 5A, lane 5, top). Since only the SH2 domain of SH2-B overlaps between N504 and C631 (Fig. 3A), these data suggest that the SH2 domain of SH2-B is sufficient to activate JAK2.

View larger version (38K): [in this window] [in a new window]   FIG. 5.   The SH2 domain of SH2-B is sufficient to stimulate JAK2. COS cells were cotransfected transiently with plasmid (2 µg) encoding JAK2 and plasmid (10 µg) encoding Myc alone (), Myc-SH2-B (WT), or the indicated Myc-SH2-B mutant. (A) JAK2 was immunoprecipitated (IP) with JAK2 and subjected to an in vitro kinase assay. Proteins in the JAK2 immunoprecipitates were then separated by SDS-PAGE, transferred onto nitrocellulose, and visualized by autoradiography (top). The same blot was immunoblotted (IB) with JAK2 (middle). Proteins in cell lysates were immunoblotted with Myc (bottom). Note that the bands comigrating with Myc-SH2-B in lane 3, Myc-N269 in lane 4, and Myc-N504 in lane 5 represent a small degree of spillover from the adjacent lane. The dark band migrating just below Myc-504 in lane 3 is believed to be a proteolytic product of Myc-N269 (see Fig. 4B, lane 4). (B) GST-SH2-B (10 µg of protein) was added to JAK2 immunoprecipitates and subjected to an in vitro kinase assay. After the assay, GST-SH2-B was purified using glutathione-agarose beads and visualized by autoradiography.

View larger version (23K): [in this window] [in a new window]   FIG. 6.   The SH2 domain of SH2-B is necessary and sufficient for the stimulatory effect of SH2-B on JAK2-mediated tyrosyl phosphorylation of Stat5B. COS cells were cotransfected transiently with plasmids encoding JAK2 (0.5 µg), Stat5B (5 µg) and either Myc alone, Myc-SH2-B (WT) (7 µg) or the indicated Myc-SH2-B mutant (7 µg). (A) Stat5B was immunoprecipitated (IP) with Stat5B and immunoblotted (IB) with PY (top). The same blot was reprobed with Stat5B (bottom). (B) Proteins in cell lysates (40 µg) were immunoblotted with antibody recognizing specifically Stat5 phosphorylated on Tyr-699 (pStat5; top), Stat5B (middle) or Myc (bottom).

A C-terminally truncated SH2-B lacking the SH2 domain acts as a dominant negative mutant to inhibit JAK2 and JAK2-mediated tyrosyl phosphorylation of Stat5B. Data in Fig. 5 and 6 raise the possibility that binding to JAK2 of regions in SH2-B other than the SH2 domain inhibits JAK2 activity. To test this hypothesis, we examined whether overexpression of C555, which lacks most of the SH2 domain, interferes with the activation of JAK2 by SH2-B or N504. JAK2 was coexpressed in COS cells with SH2-B or N504 in the presence or absence of C555, immunoprecipitated with JAK2, and subjected to an in vitro kinase assay. Both SH2-B (WT) and N504 stimulated JAK2 (Fig. 7A, lanes 2 and 4, top), consistent with Fig. 5. Overexpression of C555 significantly inhibited the activation of JAK2 induced by SH2-B (Fig. 7A, lanes 3 versus 2, top). When the amount of ³²P incorporated into JAK2 was quantified by scanning and normalized to levels of JAK2 immunoprecipitated as judged by JAK2 immunoblotting, C555 was estimated to inhibit SH2-B by greater than 55% (n = 3). Surprisingly, overexpression of C555 almost abolished the activation of JAK2 by N504 (Fig. 7A, lanes 1, 4, and 5, top). Overexpression of C555 did not change significantly the level of expression of JAK2 (Fig. 7A, middle), SH2-B, or N504 (Fig. 7A, bottom). These results strongly support the conclusion that amino acids 1 to 555 of SH2-B bind and inhibit JAK2.

View larger version (62K): [in this window] [in a new window]   FIG. 7.   Amino acids 1 to 555 of SH2-B inhibit JAK2. (A) COS cells were cotransfected transiently with plasmids (2 µg) encoding JAK2 and either Myc-SH2-B, Myc-N504, or Myc alone in the presence of plasmids (10 µg) encoding Myc-C555 or Myc alone. JAK2 was immunoprecipitated (IP) with JAK2, subjected to an in vitro kinase assay, and visualized by autoradiography (top) or immunoblotting (IB) with JAK2 (middle) as described for Fig. 5A. Proteins (30 µg) in cell lysates were immunoblotted with Myc (bottom). (B) COS cells were cotransfected transiently with plasmids (2 µg) encoding JAK2 and/or Myc-SH2-B or Myc alone in the presence or absence of plasmids (9 µg) encoding Myc-C555, Myc-PH, Myc-C266, Myc-C410, or Myc alone as indicated. JAK2 was immunoprecipitated with JAK2, subjected to an in vitro kinase assay, and visualized by autoradiography (top) or immunoblotting with JAK2 (middle panel). Proteins (30 µg) in cell lysates were immunoblotted with Myc (bottom). The migration of molecular weight standards (in thousands), JAK2, SH2-B and SH2-B mutants is indicated.

View larger version (53K): [in this window] [in a new window]   FIG. 8.   C-terminally truncated SH2-B without a functional SH2 domain (C555) inhibits phosphorylation of Stat5B on Tyr-699 by JAK2. COS cells were cotransfected transiently with plasmids encoding JAK2 (0.4 µg), Stat5B (5 µg), SH2-B (2 µg), or N504 (2 µg) in the presence or absence of C555 (10 µg) as indicated. (A) Stat5B was immunoprecipitated (IP) with Stat5B and immunoblotted (IB) with PY (top). The same blot was reprobed with Stat5B (bottom). (B) Proteins in cell lysates (40 µg) were immunoblotted with antibody recognizing specifically Stat5 phosphorylated on Tyr-699 (pStat5; top). The same blot was reprobed with Stat5B (bottom).

C555 inhibits GH-stimulated nuclear accumulation of Stat5B. To determine whether C555 acts as a dominant negative mutant to inhibit ligand-stimulated activation of Stat5B mediated by endogenous JAK2, GFP-tagged Stat5B was transiently coexpressed in 3T3-F442A cells with C555 and visualized by confocal microscopy. A GFP tag has been used successfully to monitor the migration of Stat5B in response to GH (12). In control cells, GH stimulated activation and accumulation of GFP-Stat5B in the nucleus (Fig. 9), as reported previously (12). In contrast, overexpression of C555 dramatically inhibited GH-stimulated accumulation of GFP-Stat5B in the nucleus (Fig. 9). These data indicate that C555 inhibits ligand-stimulated activation of Stat5B by endogenous receptor and JAK2.

View larger version (24K): [in this window] [in a new window]   FIG. 9.   C-terminally truncated SH2-B without a functional SH2 domain (C555) acts as a dominant negative to inhibit GH-stimulated nuclear migration of Stat5B. 3T3-F442A cells were cotransfected with plasmids encoding GFP-Stat5B (1 µg) and plasmid (2.5 µg) encoding either Myc alone (vector) or Myc-C555. Cells were then treated with or without 500 ng of GH per ml for 40 min. (A) Representative confocal images of cells. Scale bar represents 20 µm. (B) Nucleus-to-cytosol GFP fluorescence intensity ratios. Bars represent the mean ± SEM ratio of 20 cells per condition. *, significantly different from GH-treated cells transfected with empty vector (P < 0.05).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We originally identified SH2-B as a JAK2-interacting protein in a yeast two-hybrid assay (34). In the present study, we provide evidence that SH2-B interacts with JAK2 via at least two sites: the SH2 domain of SH2-B, which presumably binds to one or more phosphorylated tyrosines within activated JAK2, and a site(s) within or encompassing amino acids 1 to 555 of SH2-B which can bind to non-tyrosyl-phosphorylated, inactive JAK2. We showed previously that the SH2 domain of SH2-B alone is sufficient to bind to tyrosyl-phosphorylated JAK2 (34). Here we extend this earlier observation and establish that the SH2 domain of SH2-B is the primary binding site between SH2-B and tyrosyl-phosphorylated, active JAK2 in cells. In support of this, we showed that GST-N504, which contains only the SH2 domain and C-terminal 49 amino acids of SH2-B, binds only to tyrosyl-phosphorylated JAK2. SH2-B coimmunoprecipitates with tyrosyl-phosphorylated JAK2 to a much greater extent than with non-tyrosyl-phosphorylated JAK2(K882E). Finally, when the critical Arg-555 within the SH2 domain of SH2-B is replaced by Glu, the binding of the mutant SH2-B to JAK2 is reduced dramatically.

We also provide substantial evidence showing that in addition to the SH2 domain, SH2-B has another site(s) that binds to JAK2; binding via this additional site(s) is of lower affinity and does not require tyrosyl phosphorylation of JAK2. First, we show that GST-SH2-B is able to bind to non-tyrosyl-phosphorylated JAK2, although to a lesser extent than to tyrosyl-phosphorylated JAK2. Second, GST-SH2-B(R555E) with a defective SH2 domain binds similarly to tyrosyl-phosphorylated and non-tyrosyl-phosphorylated JAK2, at a level substantially reduced compared to binding of GST-SH2-B to tyrosyl-phosphorylated JAK2. Third, when coexpressed with kinase-inactive JAK2(K882E), both SH2-B and SH2-B(R555E) coimmunoprecipitate with non-tyrosyl-phosphorylated JAK2(K882E). Similarly, C-terminally truncated SH2-B (C555), which lacks most of the SH2 domain, coimmunoprecipitates similarly with wild-type, tyrosyl-phosphorylated JAK2 and kinase-inactive, non-tyrosyl-phosphorylated JAK2(K882E). These results clearly demonstrate that in addition to the SH2 domain, SH2-B has one or more additional sites residing within the N-terminal 555 amino acids of SH2-B that bind to JAK2 independent of tyrosyl phosphorylation of JAK2.

The N-terminal part of SH2-B that contains the additional JAK2 binding site(s) contains a PH domain, multiple proline-rich motifs, and numerous potential phosphorylation sites. Experiments using N-terminal deletion mutants of SH2-B indicated that the PH domain is necessary for binding inactive JAK2 whereas amino acids 1 to 269 are not. They also indicated that amino acids 410 to 638 are not sufficient for binding to inactive JAK2. Experiments using C-terminal deletion mutants of SH2-B and the PH domain alone revealed that the PH domain of SH2-B, the first 269 amino acids of SH2-B, and the first 410 amino acids can bind to inactive, non-tyrosyl-phosphorylated JAK2. However, all three bind significantly less well than amino acids 1 to 555. Taken together, these data with both sets of mutants indicate that both the PH domain and amino acids 410 to 555 are required for maximal binding of SH2-B to inactive JAK2. PH domains are widely believed to mediate the interaction of signaling molecules containing a PH domain with phospholipids on the plasma membrane and recruit these signaling molecules to the plasma membrane (8, 10, 17). If the interaction of the PH domain of SH2-B with JAK2 observed in this study turns out to be a direct interaction, rather than one mediated via phospholipids, it would represent one of a few examples of PH domain-mediated protein-protein interactions identified to date (3, 16, 28, 42).

We have previously shown that SH2-B is a potent activator of JAK2 (30). In this study, we demonstrate that the SH2 domain of SH2-B is not only required but also sufficient for the stimulatory action of SH2-B on JAK2. Coexpression of SH2-B with JAK2 stimulates the kinase activity of JAK2, as assessed by an in vitro kinase assay of JAK2 autophosphorylation and JAK2 phosphorylation of GST-SH2-B. It also enhances dramatically JAK2-mediated phosphorylation of Stat5B on Tyr-699, a physiological cytokine-induced phosphorylation site in cells. When the critical Arg-555 within the SH2 domain of SH2-B is replaced with Glu, or the SH2 domain is deleted (C555), the resultant SH2-B mutants are unable to activate JAK2 and stimulate JAK2-mediated tyrosyl phosphorylation of Stat5B on Tyr-699. In contrast, two other truncated SH2-B mutants, N504 and C631, both of which contain the entire SH2 domain, are fully capable of activating JAK2 and enhancing JAK2-mediated phosphorylation of Stat5B on Tyr-699. The only region shared by N504 and C631 is the SH2 domain. Therefore, the SH2 domain of SH2-B appears to be necessary and sufficient for the stimulatory action of SH2-B on JAK2 kinase activity. Because the SH2 domain is conserved in all three isoforms of known SH2-B (, , and ), we believe that all three isoforms of SH2-B are able to stimulate JAK2 activity.

The mechanism by which SH2-B activates JAK2 is unclear. One possibility is that interaction of SH2-B via its SH2 domain with JAK2 causes a change in conformation of JAK2 that stabilizes JAK2 in an active state. Alternatively, the binding of the SH2 domain of SH2-B to a critical phosphorylated tyrosine in JAK2 protects this tyrosine from being dephosphorylated by tyrosine phosphatase(s). Dephosphorylation of this critical tyrosine may lead to inactivation of JAK2. A third possibility is that SH2-B prevents the binding of SOCS-1/JAB or another inhibitory protein to JAK2. SOCS-1/JAB, a member of the SOCS family, has been shown to bind via its SH2 domain to a phosphotyrosine within the activation loop in the kinase domain of JAK2 and inhibit JAK2 (24, 44). The finding that SH2-B has multiple binding sites for JAK2 raises the possibility that SH2-B activates JAK2 by causing dimerization of JAK2 (i.e., one site in SH2-B binds to one JAK2, and a second site in the same SH2-B binds to a second JAK2). However, this latter explanation does not explain why N504, which lacks the site that binds to inactive JAK2, is a potent activator of JAK2.

An interesting finding of this study is that C555, a C-terminally truncated SH2-B that contains the PH domain and several proline-rich domains but lacks most of the SH2 domain, inhibits the kinase activity of JAK2, JAK2-mediated tyrosyl phosphorylation of Stat5B, and JAK2-mediated movement of Stat5B to the nucleus in response to GH. One can envision this dominant negative effect of C555 as a consequence of C555 competing with endogenous SH2-B for binding to the low-affinity site of interaction in JAK2, thereby decreasing the ability of endogenous SH2-B to bind via its SH2 domain to JAK2 and preventing activation of JAK2. Because C555 inhibits the activation of JAK2 not only by SH2-B but also by N504, an N-terminally truncated SH2-B that shares only 51 amino acids with C555, one can also envision binding of the low-affinity site of SH2-B to JAK2 actually inhibiting JAK2. Consistent with this, the PH domain alone inhibits the activity of overexpressed JAK2, in the absence of overexpressed SH2-B.

Based on our data, we propose the following model by which SH2-B regulates JAK2. In the basal state, SH2-B binds via a site(s) in its N-terminal 555 amino acids to non-tyrosyl-phosphorylated JAK2 with a relatively low affinity and inhibits spontaneous, abnormal activation of JAK2. This interaction also increases the subcellular local concentration of SH2-B around JAK2. When JAK2 is partially activated and tyrosyl phosphorylated in response to hormones and cytokines, the SH2 domain of SH2-B is then able to bind rapidly with high affinity to phosphorylated tyrosine(s) in JAK2, thereby rapidly and robustly enhancing JAK2 activity. SH2-B is phosphorylated on tyrosines as well as on serines and threonines in response to a variety of growth factors, cytokines, and hormones (31-34). It is appealing to hypothesize that differential phosphorylation of SH2-B may affect its ability to bind and/or activate JAK2.

In summary, we show that SH2-B binds to JAK2 via at least two sites. One or more sites within the first 555 amino acids of SH2-B binds to JAK2 independent of tyrosyl phosphorylation of JAK2 and inhibits JAK2. The SH2 domain of SH2-B binds only to activated JAK2, presumably to phosphorylated tyrosine(s). This latter interaction is necessary and sufficient for SH2-B to stimulate JAK2. The inhibition of JAK2 mediated by the low-affinity interaction of JAK2 with SH2-B or its related molecules may serve as a checkpoint to prevent abnormal activation of JAK2 in the absence of ligand. In addition, the accumulation of SH2-B around JAK2 due to this low-affinity interaction of SH2-B with inactive JAK2 may increase the efficiency with which the SH2 domain is able to bind and activate JAK2. This allows for a more rapid and robust response of cells to hormones and cytokines that utilize JAK2 in their signaling.