1 THE JAK1 SH2 DOMAIN DOES NOT FULFILL A CLASSICAL SH2 FUNCTION IN JAK/STAT SIGNALING BUT PLAYS A STRUCTURAL ROLE FOR RECEPTOR INTERACTION AND UPREGULATION OF RECEPTOR SURFACE EXPRESSION Simone Radtke 1* , Serge Haan 1*# , Angela Jörissen 1* , Heike M. Hermanns 1 , Sandra Diefenbach 1 , Tanya Smyczek 1 , Hildegard Schmitz-VandeLeur 1 , Peter C. Heinrich 1 , Iris Behrmann 2* and Claude Haan 1,2*# 1 Institut für Biochemie, Uniklinik Aachen, Pauwelsstraße 30, 52074 Aachen, Germany; 2 Laboratoire de Biologie et Physiologie Intégrée, Fac. Sciences Techno. & Com., Université du Luxembourg, 162A, avenue de la Faïencerie, L-1511 Luxembourg * Authors contributed equally to this paper Running Title: Analysis of the Jak1 SH2 domain # Corresponding authors:: Dr. Claude Haan; Tel.: +352 466644 361; Fax: +352 466644 435; e-mail: [email protected]Dr. Serge Haan; Tel.: +49 241 8088414; Fax: +49 241 8082428; e-mail: [email protected]The presence of an SH2 domain sequence similarity in the sequence of Janus kinases (Jaks) has been discussed since the first descriptions of these enzymes. We performed an in depth study in order to determine the function of the Jak1 SH2 domain. We investigated the functionality of the Jak1 SH2 domain by stably reconstituting Jak1-defective human fibrosarcoma cells U4C with endogenous amounts of Jak1 in which the crucial arginine residue R466 within the SH2 domain has been replaced by lysine. This mutant still binds to the receptor subunits gp130 and OSMR. Moreover, the SH2 R466K mutant does not affect the subcellular distribution of Jak1 as assessed by cell fractionation and confocal microscopy of cells expressing endogenous levels of non-tagged or a YFP-tagged Jak1-R466K, respectively. Likewise, the signaling capacity of Jak1 was not affected by this point mutation. However, we found that the SH2 domain is structurally important for cytokine receptor binding and surface expression of the OSMR. The Janus family of protein tyrosine kinases comprises four mammalian members. Three, Jak1, Jak2 and Tyk2, are expressed in a wide variety of tissues, whereas Jak3 expression is restricted to cells of the haematopoietic system. Jak1 is membrane-localized by binding to cytokine receptors (1). It is involved in signal transduction of several cytokines including interferons (IFNα, IFNβ and IFNγ) as well as interleukin-6-type cytokines (IL-6 (interleukin-6), OSM (oncostatin M), IL-11 (interleukin-11), LIF (leukaemia inhibitory factor), CNTF (ciliary neurotrophic factor), CT-1 (cardiotrophin-1) and CLC (cardiotrophin-like cytokine)). IL-6-type cytokines signal either via homodimers of the signal transducing receptor subunit gp130 or via heterodimeric receptor complexes containing gp130 together with the LIFR or the OSMR [for a review see (2)]. All these signal transducing receptor subunits have been described to bind Jak1, Jak2 and Tyk2. Among them, Jak1 is essential for signal transduction as demonstrated for Jak1-deficient fibrosarcoma cells and for cells derived from Jak1 knock-out mice (3,4). Interestingly, the surface expression of the OSMR and other receptors (5-7) have been described to be dependent on Jak binding. The molecular mechanism of Jak activation upon cytokine stimulation is not understood. It is still under debate which functional domains exist in the Jaks and the interplay of these domains in kinase activation is not clear. Based on sequence similarities between the Jaks (molecular masses of 120-140 kDa) seven Jak homology (JH) regions have been defined (figure 2A) which match the more recently predicted domain structure only partially (8). The JH1 domain, a classical tyrosine kinase domain, is flanked by a non-functional kinase domain, the pseudokinase domain (JH2) that may play a regulatory role (9,10). The N-terminal half of the Jaks, domains JH3 to JH7, contains a predicted FERM domain (8) and a putative SH2 domain. The FERM domain is involved in binding to the cytokine receptors (11,12) and fixes the Jak permanently to the receptor, resulting in a complex which can be compared to a receptor tyrosine kinase (1,13,14). The presence of an SH2 domain sequence similarity (C-terminally to the FERM domain) has been discussed since the first JBC Papers in Press. Published on May 12, 2005 as Manuscript M500822200 Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on August 23, 2020 http://www.jbc.org/ Downloaded from
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THE JAK1 SH2 DOMAIN DOES NOT FULFILL A CLASSICAL SH2 FUNCTION IN JAK/STAT SIGNALING BUT PLAYS A STRUCTURAL ROLE FOR RECEPTOR INTERACTION AND UPREGULATION OF
Diefenbach1, Tanya Smyczek1, Hildegard Schmitz-VandeLeur1, Peter C. Heinrich1, Iris Behrmann2* and Claude Haan1,2*#
1Institut für Biochemie, Uniklinik Aachen, Pauwelsstraße 30, 52074 Aachen, Germany; 2Laboratoire de Biologie et Physiologie Intégrée, Fac. Sciences Techno. & Com., Université du Luxembourg, 162A, avenue de la Faïencerie, L-1511 Luxembourg * Authors contributed equally to this paper
Running Title: Analysis of the Jak1 SH2 domain # Corresponding authors:: Dr. Claude Haan; Tel.: +352 466644 361; Fax: +352 466644 435; e-mail: [email protected] Dr. Serge Haan; Tel.: +49 241 8088414; Fax: +49 241 8082428; e-mail: [email protected]
The presence of an SH2 domain sequence similarity in the sequence of Janus kinases (Jaks) has been discussed since the first descriptions of these enzymes. We performed an in depth study in order to determine the function of the Jak1 SH2 domain. We investigated the functionality of the Jak1 SH2 domain by stably reconstituting Jak1-defective human fibrosarcoma cells U4C with endogenous amounts of Jak1 in which the crucial arginine residue R466 within the SH2 domain has been replaced by lysine. This mutant still binds to the receptor subunits gp130 and OSMR. Moreover, the SH2 R466K mutant does not affect the subcellular distribution of Jak1 as assessed by cell fractionation and confocal microscopy of cells expressing endogenous levels of non-tagged or a YFP-tagged Jak1-R466K, respectively. Likewise, the signaling capacity of Jak1 was not affected by this point mutation. However, we found that the SH2 domain is structurally important for cytokine receptor binding and surface expression of the OSMR.
The Janus family of protein tyrosine kinases comprises four mammalian members. Three, Jak1, Jak2 and Tyk2, are expressed in a wide variety of tissues, whereas Jak3 expression is restricted to cells of the haematopoietic system. Jak1 is membrane-localized by binding to cytokine receptors (1). It is involved in signal transduction of several cytokines including interferons (IFNα, IFNβ and IFNγ) as well as interleukin-6-type cytokines (IL-6 (interleukin-6), OSM (oncostatin M), IL-11 (interleukin-11), LIF (leukaemia inhibitory factor), CNTF (ciliary neurotrophic
factor), CT-1 (cardiotrophin-1) and CLC (cardiotrophin-like cytokine)). IL-6-type cytokines signal either via homodimers of the signal transducing receptor subunit gp130 or via heterodimeric receptor complexes containing gp130 together with the LIFR or the OSMR [for a review see (2)]. All these signal transducing receptor subunits have been described to bind Jak1, Jak2 and Tyk2. Among them, Jak1 is essential for signal transduction as demonstrated for Jak1-deficient fibrosarcoma cells and for cells derived from Jak1 knock-out mice (3,4). Interestingly, the surface expression of the OSMR and other receptors (5-7) have been described to be dependent on Jak binding.
The molecular mechanism of Jak activation upon cytokine stimulation is not understood. It is still under debate which functional domains exist in the Jaks and the interplay of these domains in kinase activation is not clear. Based on sequence similarities between the Jaks (molecular masses of 120-140 kDa) seven Jak homology (JH) regions have been defined (figure 2A) which match the more recently predicted domain structure only partially (8). The JH1 domain, a classical tyrosine kinase domain, is flanked by a non-functional kinase domain, the pseudokinase domain (JH2) that may play a regulatory role (9,10). The N-terminal half of the Jaks, domains JH3 to JH7, contains a predicted FERM domain (8) and a putative SH2 domain. The FERM domain is involved in binding to the cytokine receptors (11,12) and fixes the Jak permanently to the receptor, resulting in a complex which can be compared to a receptor tyrosine kinase (1,13,14). The presence of an SH2 domain sequence similarity (C-terminally to the FERM domain) has been discussed since the first
JBC Papers in Press. Published on May 12, 2005 as Manuscript M500822200
Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
description of Jaks (15,16) and with the improvement of structure prediction tools the number of studies finding SH2 domain sequence similarities in Jaks have increased (17-19).
SH2 domains contain about 100 amino acids and consist of two α-helices and a central antiparallel β-sheet. They bind to specific motifs containing a tyrosine residue. Phosphorylation of this tyrosine is a prerequisite for SH2 domain binding. SH2 domains contain a conserved arginine at the βB5 position which contacts the phosphotyrosine in the motif recognized by the SH2 domain and makes a crucial contribution to the affinity. Non-functional SH2 domains can be generated by mutation of the crucial arginine residue to lysine (20-23).
In the present study we investigate the effects of an inactivating mutation of the Jak1 SH2 domain, Jak1-R466K, in stable transfectants. Whereas we did not find an effect of this mutant on IL-6-type cytokine or IFNγ/IFNα signaling, deletion mutants revealed that the SH2 domain is of structural importance for the previously described regulation of OSMR surface expression by Jaks.
EXPERIMENTAL PROCEDURES
Structural alignment of SH2 domains - The initial alignment of the SH2 domain sequences of Jak1, Jak2, Jak3 and Tyk2 from different species (h: human; m: mouse, r: rat; c: chicken; p: pig; q: rhesus macaque; z: zebrafish; y: carp and t: puffer fish) and of the Drosophila melanogaster Janus kinase homologue Hop with the sequences of the structurally explored SH2 domains of the human c-Src protein-tyrosine kinase, the human phosphatidylinositol 3-kinase p85 subunit, the bovine phospholipase C-γ, the human Bcr-abl protein-tyrosine kinase and the N-terminal and C-terminal SH2 domains of human SHP-2 protein-tyrosine phosphatase was performed by the use of the BLAST programme (24). Modifications were then introduced to meet structural requirements derived from the known SH2 structures. The sequential alignment of the known structures is based on the direct superposition of their backbone coordinates. Brookhaven data bank entry codes for the used structures are: 1hcs, 1a1b and 1shd for human c-Src (25-27); 1pic for human PI3-K p85 (28); 2pld for bovine PLC-γ1 (29); 2abl for human Bcr-abl (30) and 2SHP for human SHP2 (31). The Swiss-Prot/TrEMBL accession numbers for the sequences used are: P23458 (hJak1); P52332 (mJak1); Q9PWM9 (cJak1); O12990 (zJak1); O57612 (tJak1); Q9TTJ1 (pJak1); Q9N147
(qJak1); O35803 (rJak1); Q09178 (yJak1); Q62689 (rJak2); Q75R65 (cJak2); Q9PVI2 (tJak2); Q62120 (mJak2); O60674 (hJak2); O19064 (pJak2); O93596 (zJak2); P29597 (hTyk2); Q9PWD1 (tTyk2); Q9R117 (mTyk2); P52333 (hJak3); Q8BYU2 (mJak3); Q9PWD0 (tJak3); Q9PTN6 (yJak3); Q63272 (rJak3), Q24592 (Hop).For the alignment of reference SH2 domain sequences excluding the Janus kinases an initial alignment was performed with the MULTALIN (v5.4.1) software (32) using the Src SH2 sequence as template. After deletion of incomplete sequences and of sequences present more than once, the alignment of the remaining 420 SH2 domain sequences was corrected to meet the structural requirements derived from known SH2 structures.
Cell culture and transfection - U4C cells (human fibrosarcoma cells kindly provided by Dr. I. M. Kerr, Cancer Research UK, London), COS-7 cells (simian kidney cells, ATCC CRL1619) were maintained in Dulbecco´s modified Eagle´s medium (Gibco). All media were supplemented with 10% fetal calf serum, 100 mg/l streptomycin, and 60 mg/l penicillin. G418 (400 µg/ml) was added to the medium of U4C-cells. The U4C-Jak1, U4C-Jak1-YFP, U4C-Jak1-R466K, U4C-Jak1-R466K-YFP, U4C-Jak1-K907E and U4C-Jak1-L80A/Y81A cells were cultured with 500 µg/ml hygromycin. Cells were grown at 37° C in a water-saturated atmosphere at 5% CO2.
Transient transfection of COS-7 cells was carried out using FuGENE (Roche, Mannheim, Germany) according to the manufacturer’s recommendations or using DEAE dextrane as described before (33). U4C cells were transfected using the Superfect reagent (Qiagen) according to the manufacturer’s recommendations. U4C cells stably expressing Jak1WT, Jak1R466K, Jak1-YFP, Jak1-R466K-YFP, Jak1-K907E and Jak1-L80A/Y81A were generated using the Flip-InTM T-RexTM System from Invitrogen according to the manufacturer’s recommendations. IFNα, IFNγ and OSM were obtained from Peprotech. Recombinant IL-6 and sIL-6R were prepared as described (34,35). Cells were stimulated with 20 ng/ml OSM, 1000 U/ml IFNγ or IFNα or with 200 ng/ml IL-6 in combination with 1 µg/ml sIL-6R.
Constructs - The pSVL- and pcDNA5/FRT/-constructs Jak1, Jak1-YFP, L80A/Y81A, L80A/Y81A-YFP were described previously (1,13). pSVL-Jak1-K907E was generated by restricting pBS-Jak1R907E with BspTI/BglII and inserting the fragment in the BspTI/BglII-digested pSVL-Jak1. Arginine 466
For generation of the Jak1 deletion constructs SH2∆, JH4∆ and FERM∆, DNA fragments were generated by standard PCR techniques inserting an in-frame HA-tag followed by a stop-codon and a SmaI site after the amino acid positions K558, E456 or C440, using pSVL-Jak1 as a template. PCR fragments and vector were digested by PstI/SmaI and the relevant fragments inserted. These constructs additionally contain an ApaI site upstream of the HA-tag and a NotI site directly following the stop codon which allows removal of the HA-tag. The constructs J3-SH2-HA and J2-SH2-HA were generated using pSVL-Jak2 and pSVL-Jak3 as templates. DNA-fragments encoding amino acids H377-L499 of Jak3 and amino acids H401-S523 of Jak2 were generated by PCR using the relevant primers, the sense primer additionally inserted an in-frame PstI site followed by DNA encoding amino acids T416-C440 of Jak1, the reverse primer contained an in-frame ApaI-site. The construct pSVL-Jak1-FERM∆ and the PCR fragments were digested by PstI/ApaI and the fragments were ligated into the isolated vector. All GFP-tagged Jak1 constructs were generated by digesting the relevant YFP- or HA-tagged parental constructs with ApaI/NotI, thus removing the tag and inserting a PCR-generated DNA fragment coding for eGFP flanked by the same restriction sites. The GFP-tagged JAK deletion constructs were subcloned into pcDNA3 (Invitrogen) by using the EcoRI/NotI restriction sites. The integrity of all constructs was verified by DNA sequencing using the ABI PRISM 310 Genetic Analyzer (PerkinElmer). The gp130 chimera β130 and the OSMR construct βOSMR∆1 (IL-5Rβ/OSMR∆1) were described previously (33,36). For generation of GFP-tagged OSMR, a BstEII site was introduced by PCR 3´ to the codon for amino acid C979 allowing in-frame insertion of cDNA for eGFP.
Cell fractionation - All fractionation and centrifugation steps were performed at 4°C using ice cold buffers. Cytoplasmic, membrane and
nuclear fractions were prepared using the protocol described in detail before (1).
Cell lysis, immunoprecipitation and Western blot analysis - All steps of cell lysis and immunoprecipitation were performed at 4°C using ice cold buffers. Cells were lysed on the dish with 500 µl of lysis buffer containing 1% Brij 96 or 1% Triton-X-100, 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 10 mM NaF, 1 mM sodium vanadate, 10 mM PMSF, 1 mM benzamidine, 5 µg/ml aprotinin, 3 µg/ml pepstatin, 5 µg/ml leupeptin and 1 mM EDTA. After incubation of the cleared lysates with antibodies, the immunoprecipitates were collected with protein-A-sepharose for 1 hour, washed several times and analyzed further by SDS-PAGE. The proteins were transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech) and probed with the respective antibodies. Anti-Jak1 (HR785, Santa Cruz) and anti-IL-5Rβ (S16, Santa Cruz Biotech) were used for immunoprecipitation. Anti-Jak1 (J24320, Transduction Laboratories and HR785, Santa Cruz), anti-HA (MMS-101R, Gibco), anti-GFP (Rockland), anti-Lamin-A/C (636, Santa Cruz Biotechnology), anti-Calnexin (610523, Transduction Laboratories), anti-phosphotyrosine (PY99, Santa Cruz Biotechnology), anti-STAT-3 (610189, Transduction Laboratories), anti-phospho-STAT-3 (Tyr705) (9131 from Cell signaling), anti-STAT-1 (S21120 from Transduction Laboratories), anti-phospho-STAT-1 (Tyr701) (9171 from Cell signaling) were used for detection. The horseradish peroxidase-conjugated secondary antibodies were purchased from Dako. Signals were detected using the ECL system (Amersham Pharmacia Biotech).
Fluorescence microscopy - Confocal imaging in living cells was carried out on a Zeiss LSM 510 confocal microscope using a water corrected Plan-Apochromat (63x) objective. The LSM 510 is equipped with an argon-ion laser (λ = 458 nm, λ = 488 nm and λ = 514 nm) and a helium/neon laser (λ = 543 nm) (Zeiss, Jena, Germany). The laser was modulated by an acusto-optical modulator. The beam path of YFP (enhanced yellow fluorescent protein) contained excitation at 514 nm, a main dichroic mirror of 514 nm and a longpass filter of >530 nm. Transfected cells were grown on 18 mm glass cover slips. 48 h after transfection the cover slips were placed in a perfusion chamber that allowed a constant flow with DMEM supplemented with 10% FCS, 100 mg/l streptomycin, and 60 mg/l penicillin. To maintain 37°C during all image acquisition, the perfusion chamber and the
objective were thermostatized. For qualitative cell imaging, a laser power of 35% of 25 mW was used. The transmission and detector gains were set to achieve best signals.
Flow cytometry - Cells were resuspended in cold PBS supplemented with 5 % FCS and 0.1 % sodium azide (PBS/azide). 5 x 105 to 1 x 106 cells in 100 µl PBS/azide were incubated with 1 µg/ml of monoclonal anti-MHC-I (W6/32, Sigma), anti-OSMR (ANA2, Santa Cruz) or anti-IL5Rβ (S16, Santa Cruz) for 30 min at 4°C. Cells were then washed with cold PBS/azide. To visualize the bound antibodies, the cells were subsequently incubated in darkness with a 1/100 dilution of a R-phycoerythrin-conjugated anti-mouse IgG-Fab (Dianova, Hamburg) for 30 min at 4°C. Cells were again washed with cold PBS-azide and then 104 cells per sample were analyzed by flow cytometry using a FACScalibur (Beckton Dickinson) equipped with a 488 nm argon laser.
RESULTS
The members of the Janus kinase family contain a divergent SH2 domain.
In order to determine whether the potential SH2 domain of Janus kinases possesses the features required for proper recognition of phosphotyrosine motifs, we performed a structural alignment of 25 SH2 sequences of Janus kinases with the sequences of structurally explored SH2 domains of other proteins (Figure 1). To better evaluate the variability of the different amino acid positions, an additional alignment of 420 SH2 domain sequences excluding the Janus kinases was generated (data not shown). Residues conserved in SH2 domains to at least 30% or 90% are indicated in figure 1 in blue and red, respectively. We found that critical residues involved in the hydrophobic core of the domain (e.g. positions αA9, βB2, βB3, βB4, βC3, βC5, βD7, αB2 and αB5) are strictly conserved in the 420 SH2 domains (hydrophobic amino acid side chains in 100% of the 420 SH2 sequences) as well as in all the Jaks (highlighted by an h in figure 1). Furthermore, secondary structure prediction analysis of the Janus kinase family members revealed the typical secondary structure pattern found in SH2 domains (data not shown). For functionality, SH2 domains depend on the arginine residue at position βB5, which contacts the phosphate group of a binding phosphotyrosine motif. Accordingly, this residue was present in 419 sequences (99.8%) in our alignment of 420 reference SH2 domain sequences, highlighting the strict requirement for this amino acid at this position. Strikingly, this
arginine is not equally well conserved in all the Jaks. Whereas this residue is present in all the sequences of Jak1 and Jak3, it is exchanged to glutamine in one Jak2 sequence (puffer fish) and none of the Tyk2 sequences contains an arginine at this position (histidine in humans, glutamine in mouse and cysteine in puffer fish). Thus, considering all the Jaks, there is a striking discrepancy of conservation between structural (conserved in all) and functional residues (conserved in only some Jaks). While this finding precludes a general requirement of the SH2 domain in Janus kinases, the fact that the crucial arginine is conserved in all Jak1 sequences may suggest that the SH2 domain may play a role in Jak1-mediated signal transduction. We therefore decided to generate a non-functional SH2 mutant of Jak1. Mutation of R466 to lysine has no effect on Jak1/cytokine-receptor binding and on surface expression of the OSM receptor.
To investigate the effects of a non-functional SH2 domain within Jak1, we introduced an arginine to lysine mutation at position βB5 (amino acid 466) of Jak1 (Figure 2A). First, we checked whether the mutation of this residue would affect Jak1/receptor binding. Due to low endogenous protein levels and lack of sufficiently efficient antibodies against gp130 or OSMR the Jak/receptor binding was investigated by transiently expressing Jak1 constructs and a chimeric IL-5Rβ/gp130-construct in COS-7 cells as described before (11). To monitor Jak1 association, the chimeric receptors were immunoprecipitated with an IL-5Rβ-specific antibody and subsequently analyzed by Western blotting (Figure 2B). Co-precipitated Jak1 was detected using a Jak1-specific antibody. As described previously (11), the IL-5Rβ/gp130-constructs efficiently bound wild-type Jak1 whereas association of the non-binding mutant Jak1-L80A/Y81A was greatly impaired. The single amino acid exchange of the phosphotyrosine binding residue in the Jak1 SH2 domain arginine 466 to lysine did not affect Jak association to the gp130 cytoplasmic part (Figure 2B). The same results were obtained using an IL-5Rβ/OSMR-construct (data not shown).
Since the Jak1 N-terminus comprising the FERM and the SH2 domains is involved in cytokine receptor binding and since Jak association to the OSMR has recently been shown to promote an upregulation of the surface expression of this receptor (6) we hypothesized that the SH2 domain might play a role in the latter process. Upon coexpression of a GFP-tagged
OSMR with different Jak mutants in COS-7 cells, both Jak1 and Jak1-R466K efficiently upregulated the OSM receptor while Jak1-L80A/Y81A, the non-receptor-binding mutant, failed to do so (Figure 2C). Thus, the non-functional Jak1 SH2 domain mutant, Jak1-R466K, does not only have the ability to bind to cytokine receptors like wild-type Jak1 but also retains the ability to promote surface expression of the OSMR. Mutation of arginine 466 to lysine has no effect on the subcellular distribution of Jak1.
Jak membrane localization was recently shown to be dependent on cytokine receptor binding (1). To examine whether a low level constitutive phosphotyrosine/SH2 domain interaction could have an effect on Jak localization, we investigated the localisation of Jak1, Jak1-R466K and Jak1-L80A/Y81A in stably transfected U4C-cells by using cell fractionation techniques (Figure 3A). Jak1-L80A/Y81A was used as control since it is known to be localised to the cytoplasm (1). The cells were fractionated and Jak1 was detected by Western blot analysis. The blots were counterstained with antibodies against marker proteins of the different subcellular fractions. Calnexin was used as membrane fraction marker and lamin A and C as nuclear marker proteins. Cytoplasmic proteins have been defined as those soluble after 1h ultracentrifugation at 100,000 g. A predominant membrane localisation was found for Jak1 and Jak1-R466K while the non-receptor-binding control Jak1-L80A/Y81A was mainly found in the cytoplasm. Thus, the localization of the non-functional SH2 mutant Jak1-R466K is identical to the localization of wild-type Jak1. As a second independent assay to show Jak localization, yellow fluorescent fusion proteins of Jak1 and Jak1-R466K were generated (Figure 3B) and stably expressed in U4C cells. Importantly, the YFP-tag does not interfere with Jak1 signaling capacity as previously demonstrated (1). In order to compare the subcellular localisation of YFP-Jak1 and YFP-Jak1-R466K in living cells, the cells were seeded onto coverslips and analyzed with a confocal laser scanning microscope. Figure 3B shows microscopic images of living U4C-Jak1-YFP- and U4C-Jak1-R466K-YFP-cells expressing the YFP-Jak1 fusion proteins. Fluorescence of both Jak1-YFP and Jak1-R466K-YFP is concentrated at the plasma membrane, as described previously for Jak1-YFP (1).
Taken together, the results from the subcellular fractionation experiments and the localisation studies using YFP-fusion proteins show that the localisation of Jak1 does not require
a functionally intact SH2 domain. Mutation of arginine 466 to lysine does not affect signal transduction through the Jak/STAT pathway.
We next compared the signal transducing capacity of Jak1 and Jak1-R466K in the stably reconstituted JAK1-deficient U4C cells. Cells expressing a kinase-negative Jak1 mutant Jak1-K907E were used as negative control. The cells were stimulated with different cytokines signaling via Jak1 (IL-6, OSM, IFNγ and IFNα) and total cellular lysates were prepared. Part of each lysate was used for an immunoprecipitation with a Jak1 antibody. The immunoprecipitates and the lysates were then subjected to SDS-PAGE and Western blotting (Figure 4A). Jak1-R466K does not show any differences in signal transduction compared to Jak1. Jak1 phosphorylation as well as STAT-1 and STAT-3 tyrosine phosphorylation after cytokine stimulation were identical in U4C-Jak1 and U4C-Jak1-R466K cells, whereas these signaling events were impaired in U4C-Jak1-K907E cells. Single clones and pools of stable U4C transfectants always showed identical results (data not shown).
To examine possible changes in STAT activation kinetics, the U4C-Jak1, U4C-Jak1-R466K and U4C-Jak1-K907E cells were stimulated with OSM or IFNγ for different times, total cellular lysates were prepared and analyzed by Western blotting (Figure 4B). The extent and the kinetics of tyrosine phosphorylation of STAT-1 and STAT-3 after cytokine stimulation were identical in U4C-Jak1 and U4C-Jak1-R466K cells, whereas the negative control U4C-Jak1-K907E-cells show disturbed signaling.
To monitor MHC-I gene expression upon IFNα or IFNγ stimulation, U4C-Jak1 and U4C-Jak1-R466K cells were stimulated for 3 days and then analyzed using flow cytometry (Figure 4C). The upregulation of MHC-I surface expression in U4C-Jak1-R466K in response to both cytokines was identical to the one found in U4C-Jak1 cells. U4C-Jak1-K907E control cells did not show upregulation of MHC surface expression (data not shown). The SH2 domain of Jak1 is structurally important for supporting OSMR binding and surface expression.
We generated a number of Jak1 constructs to test whether the SH2 domain is structurally needed for proper Jak1 binding to the OSMR and consequent receptor surface expression. All Jak1 constructs used in this assay are C-terminally tagged with GFP so that their overall expression can be easily measured by Western blot or FACS
analysis (Figure 5A). The construct Jak1-SH2∆ comprises the N-terminus of Jak1 and has intact FERM and SH2 domains (residues 1-558). The construct JH4∆ has an intact FERM domain but only part of the SH2 domain (residues 1-456). This deletion disrupts the structural integrity of the SH2 domain. The FERM∆ construct represents the isolated FERM domain (residues 1-440). To test whether SH2 domains of other Jaks could substitute for the Jak1 SH2 domain we generated constructs where the SH2 domains of Jak2 or Jak3 were fused to the FERM∆ construct (Figure 5A, J2-SH2-GFP and J3-SH2 GFP).
We next studied the binding of the different Jak1 constructs to an IL-5Rβ/OSMR∆1 chimera which we previously used to monitor Jak1 receptor association (Figure 5B) (6). The chimeric receptor was precipitated with an IL-5Rβ antibody and co-precipitated Jak1 was detected with an antibody recognizing the GFP moiety of wild-type Jak1 and the Jak mutants. As can be seen in figure 5B (left panels), wild-type Jak1 and Jak1-SH2∆ can be efficiently precipitated with the chimeric OSMR, whereas the shorter Jak1 proteins Jak1-JH4∆ and Jak1-FERM∆ show a decreased binding. We also found that the construct containing the Jak2 SH2 domain fused to the Jak1 FERM domain (J2-SH2) efficiently bound to the IL-5Rβ/OSMR whereas the fusion construct encompassing the Jak3 SH2 domain (J3-SH2) did not bind (Figure 5B, right panels). Similarly, a chimera in which not only the SH2 domain of Jak3 but the whole C-terminus of Jak3 is present, is also deficient in receptor upregulation (suppl. Fig. 1). As described before (6), a glycosylated IL-5Rβ/OSMR∆1 band of lower mobility was detected for all the constructs that bound to the IL-5Rβ/OSMR∆1 (Fig. 5B, upper panel). This band has been shown to represent the mature form of the IL-5Rβ/OSMR∆1 (6). In addition, we monitored the surface expression of the chimeric construct by FACS analysis. We found that all the constructs that bind to the IL-5Rβ/OSMR∆1 also lead to the upregulation of the OSMR chimera (Figure 5B, bar diagrams). Similar results were obtained using a GFP-tagged OSMR together with HA-tagged Jak1 constructs (data not shown). Taken together, the upregulation of OSMR surface expression stictly correlates with the binding of the different Jak constructs.
To investigate whether the Jak1 mutants would similarly upregulate the endogenous OSMR we reconstituted Jak1 deficient U4C cells with different Jak1-GFP constructs (Figure 5C). The Jak1-SH2∆ showed a significant upregulation of endogenous OSMR if compared to the control
cells. The FERM∆ construct lacking the SH2 domain showed impaired receptor upregulation. This defective upregulation could be rescued by fusing the Jak2 SH2 domain to the Jak1 FERM domain (J2-SH2) but not by the Jak3 SH2 domain fusion (J3-SH2). This correlates with the effects shown in figures 5B. To exclude any non-specific effects we also generated a control construct, SH2∆-L80A/Y81A, in which two residues in the FERM domain that participate in receptor binding, L80 and Y81 are mutated. As shown in suppl. Fig. 2, the upregulation of the endogenous OSMR surface expression observed using the SH2∆ construct is lost if these residues are mutated (SH2∆-L80A/Y81A).
DISCUSSION
We set out to explore whether the predicted SH2 domain of Jak1 was of functional relevance for Jak1-mediated signal transduction. Our initial sequence alignment of 420 SH2 domains demonstrated that in case of Jak1, all amino acids needed for proper SH2 structure (e.g. αA9, βB2, βB3, βB4, βC3, βC5, βD7, αB2 and αB5) and function (e.g. the arginine residue at position at βB5) are well conserved in all available sequences (Figure 1).
We therefore introduced an arginine to lysine point mutation (R466K) into Jak1 which impairs the function of the SH2 domain but does not interfere with the structural integrity of the domain. This exchange is a common loss of function mutation used in SH2 domain studies (20-23). It is commonly accepted that the lesser length of a lysine causes a disruption of the binding to the phosphate oxygens of the phosphotyrosine residue (20). In addition, the positively charged amino group from the lysine cannot mimic the binding of the terminal guanidinium nitrogens of the arginine to the phosphate oxygens.
We show that mutation of R466 to lysine has no effect on Jak1/cytokine-receptor binding as well as on its subcellular distribution: Subcellular fractionation experiments and confocal microscopy with YFP-tagged Jak1 and Jak1-R466K showed that the mutant is localized to membranes as is the wild-type. Thus, there does not seem to be any basal phosphotyrosine/SH2 interaction involved in the localization of Jak1.
Next, we investigated the effect of the Jak1 SH2 domain knock-out R466K on signal transduction in the context of different cytokine receptor complexes. The IL-6- and OSM-receptor complexes represent a setting of cytokine receptors using predominantly Jak1 (3,4) but can promiscuitely recruit Jak2 and Tyk2. In the IFN-γ
receptor, the Jak1 SH2 knock-out mutant is paired with a Jak2 containing an intact SH2 domain. Finally, in the case of the IFNα receptor complex, the Jak1 SH2 mutant is paired with Tyk2, which is naturally defective in SH2 function (Figure 1). STAT factor activation kinetics and Jak1 activation itself was unchanged in stable Jak1-R466K transfectants upon stimulation with any of these cytokines. Since even IFNα stimulation in U4C-Jak1-R466K-cells shows unaltered Jak and STAT activation as well as an efficient upregulation of MHC class I in comparison to Jak1-WT, it is clear that there is no need for any functional Jak SH2 domain in IFNα signaling. Thus, we conclude that in case of the tested cytokines, the SH2 domain of Jak1 does not contribute to signaling via the Jak/STAT pathway. As in Jak1, the crucial arginine is conserved in all available Jak3 sequences. Interestingly, it was reported recently that this arginine is mutated to histidine in a SCID patient. Ectopic expression of this mutant in HeLa cells revealed an altered subcellular localisation (37). It is an intriguing thought that a functional SH2 domain may be required for Jak3 signaling.
We demonstrated in a previous study that Jak1 is important for efficient surface expression of the OSMR. In particular, we could show that this effect is mediated by binding of Jak1 to the receptor but that Jak1 kinase activity is not required since a kinase negative mutant (K907E of Jak1) was as efficient as wild type Jak1 in mediating OSMR surface expression. Further data suggested that the OSMR contains a negative regulatory signal in its membrane proximal region that may be masked by Jak1 upon its binding to the receptor (6).
Here we show that a truncated Jak1 encompassing its N-terminal region can bind to the OSMR and upregulate its surface expression as efficiently as full length Jak1 demonstrating that the C-terminal part of the enzyme does not structurally contribute to receptor association and upregulation. However, we found the SH2 domain of Jak1 to be structurally important for the binding to the OSMR and consequently for efficient OSMR surface expression. Truncated constructs lacking the full SH2 domain or lacking the major part of the SH2 domain show a clear reduction in their abiliy to promote receptor surface expression. This finding is in accordance with data published for the EpoR and the IFNARI. In both cases, only constructs (Jak2 in case of the EpoR and Tyk2 in case of the IFNAR1 chain) comprising an intact SH2 domain are also able to support efficient surface expression of the bound receptors (5,7). In these cases, however, deletion of the SH2 domain
did not affect binding to the receptors. This is clearly different in case of the OSMR: partial or full deletion of the SH2 domain leads to constructs that bind to a much lesser extent to the chimeric OSMR than the construct containing the intact SH2 domain (Figure 5A). This implies that in case of the OSMR/Jak1 interaction, the FERM domain by itself is not sufficient for high affinity receptor binding. In case of gp130, it was shown by the use of chimeric constructs of Jak1 and Jak3 that the FERM domain is sufficient for binding (12). We also observed that the FERM∆ construct of Jak 1 binds to gp130 ((11) and data not shown). Thus, the structural requirements for receptor interaction might vary between different receptor systems. Interstingly, we found that in case of the binding of Jak1 to the OSMR, the SH2 domain of Jak2, but not the SH2 domain of Jak3, is able to compensate for a missing Jak1 SH2 domain.
The role of the SH2 domain may be to anchor the FERM domain in a binding-competent state which would imply that it is very unlikely that the FERM and SH2 domains exist as independent entities. We rather hypothesize that there are some structural interactions between these domains which could explain our finding that the SH2 domain of Jak2 but not Jak3 can substitute for the SH2 domain of Jak1 in OSMR upregulation. Further support for the structural role of the Jak SH2 domain can be deduced from the alignment shown in figure 1. Interestingly, the extremely well conserved tryptophan and tyrosine residues (conserved to greater 90 % in SH2 domains) in the βA strand can not be found in any of the Jak sequences. The tryptophan residue normally anchors the N-terminal tail at the back of the SH2 domain and directs it away from the phosphotyrosine recognition site. As the C-terminal end of SH2 domains is also directed towards the back of the domain, interference of N- and C-terminal domains with SH2-binding partners is prevented. The absence of the well conserved tryptophan indicates that, in case of the Jaks, the domain preceding the SH2 domain, namely the FERM domain, could be positioned not behind, but aside the SH2 domain. Thus, the SH2 domain could more function as a spacer between its neighbouring domains and as such be important for the conformation of the molecule. Nevertheless, it cannot be excluded that the SH2 domain in Jaks still may be an interaction domain. In theory, it could bind non-classical motifs, like non-phosphorylated peptides or bind proteins by a totally different mechanism. Our attempts to pull down interaction partners with isolated SH2 domains of Jaks have so far yielded
no results (data not shown). Unfortunately, the structure of Janus kinases has not been successfully explored so far as it may give valuable information about the function of the different domains.
Taken together, the present in depth study on the SH2 domain of Jaks - indicating that the SH2 domain does not fulfill a classical SH2 function - adds to the mystery of these kinases that already harbour a kinase domain with a non-classical function.
FOOTNOTES Abbreviations used: FERM, four-point-one, ezrin, radixin, moesin; GFP, green fluorescent protein; IFN, interferon; IL, interleukin; Jak, Janus kinase; JH, Jak homology; OSM, oncostatin M; R, receptor; SH2, Src homology 2; STAT, signal transducer and activator of transcription; Tyk, tyrosine kinase; YFP, yellow fluorescent protein We thank Dr. Ian M. Kerr (London) for the kind gift of the U4C cells and Dr. Catharien M. Hilkens (Newcastle) for providing the construct encoding the chimeric protein Jak1/3. This work was supported by grants from the Fonds der Chemischen Industrie and the Deutsche Forschungsgemeinschaft (SFB 542; HA 3433/1-1). REFERENCES 1. Behrmann, I., Smyczek, T., Heinrich, P. C., Schmitz-Van De Leur, H., Komyod, W.,
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FIGURE LEGENDS Figure 1: SH2 domain alignment of Jak1, Jak2, Jak3 and Tyk2 with other SH2 domains. The sequences the Janus kinase SH2 domains of different origin (h: human; m: mouse, r: rat; c: chicken; p: pig; q: macaque; z: zebrafish; y: carp and t: puffer fish) were aligned with the SH2- domain of human phosphatidylinositol 3-kinase p85 subunit (P85αC) (28), the bovine phospholipase C-γ (PLCγ) (29), the human Bcr-abl protein-tyrosine kinase (Abl) (30) as well as with the N-terminal and C-terminal SH2 domains of murine SHP2 protein-tyrosine phosphatase (SHP2N, SHP2C) (31). Secondary structure characteristics are given on top following the common nomenclature (38). Residues that are highly conserved among the Janus kinase sequences are highlighted in green. Blue and red characters indicate residues conserved in SH2 domains to at least 30% or 90%, respectively (based on our alignment of 420 SH2 domain sequences). Amino acid positions critical for the build-up of the hydrophobic core of the domain where a hydrophobic amino acid side chain is present in 100% of the sequences are indicated by h. The arginine residue at position βB5 (*) that is crucial for the function of SH2 domains is highlighted in yellow. Figure 2: The R466K mutation does not affect receptor binding and OSMR surface expression. (A) Schematic representation of the Jak1 mutants. (B) Transiently transfected COS-7 cells expressing IL-5Rβ/gp130 and Jak1 mutants were lysed and subjected to immunoprecipitation using a Jak1-antiserum (provided by A. Ziemiecki). The immunoprecipitates and lysates were analysed by Western blotting using an IL-5Rβ (N20) antibody and a Jak1 polyclonal antiserum. The signals were detected using the ECL system. IP: Immunoprecipitation, WB: Western Blot. (C) COS-7 cells were transfected with OSMR-GFP together with Jak1-WT, Jak1-R466K or Jak1-L80A/Y81A. For FACS analysis cells were stained with an antibody recognizing the OSMR and surface expression of cells expressing similar amounts of the GFP-tagged OSMR were analysed. The means and standard deviations obtained from three independent experiments are depicted. The mean fluorescence of cells expressing OSMR-GFP and Jak1-L80A/Y81A was set to 100%. Figure 3: Jak1-YFP and Jak1-R466K-YFP are membrane-localised. (A) U4C, U4C-Jak1, U4C-Jak1-R466K and U4C-Jak1-L80A/Y81A cells were fractionated as described in Materials and Methods and Jak1 was detected by Western blot analysis. The blots were counterstained with antibodies against marker proteins of the different subcellular fractions. Calnexin was used as membrane fraction marker and lamin A and C as nuclear marker proteins. (B) The localization of the YFP-tagged proteins in living U4C cells stably expressing Jak1-YFP or the mutant Jak1-R466K-YFP was monitored by confocal microscopy. Figure 4: Mutation of R466 to lysine does not affect signal transduction through the Jak/STAT pathway upon cytokine stimulation. (A) Stably transfected U4C-Jak1, U4C-Jak1-R466K and U4C-Jak1-K907E (kinase-dead mutant) cells were stimulated with IL-6, OSM, IFNγ or IFNα and total cellular lysates were prepared. Part of the lysates was subjected to an immunoprecipitation with a Jak1 antibody. Lysates and immunoprecipitates were resolved by SDS-PAGE and transferred to membranes by Western blot. The blot of the immunoprecipitates was detected using a phosphotyrosine antibody and reprobed using a Jak1 antibody. The lysate-blots were detected with phospho-specific STAT1 and STAT3 antibodies and reprobed with STAT antibodies. (B) U4C-Jak1, U4C-Jak1-R466K and U4C-Jak1-K907E cells were stimulated with OSM or IFNγ for different periods of time and total cellular lysates were prepared. Lysates were resolved by SDS-PAGE and transferred to membranes by Western blot. The blots were detected with phospho-specific STAT1 and STAT3 antibodies and reprobed with STAT antibodies. (C) U4C cells stably expressing wild-type Jak1 or Jak1-R466K were stimulated for 3 days with IFNγ or IFNα and MHC-I expression was monitored by FACS analysis using an MHC-I-specific antibody. Histograms from unstimulated cells are shown in grey, those from cells treated with IFNα or IFNγ are depicted as solid or broken lines, respectively.
Figure 5: The SH2 domain of Jak1 is structurally important for supporting OSMR binding and surface expression. (A) Schematic representation of the Jak1 constructs used to study the binding of Jak1 to the OSMR. (B) COS-7 cells were transfected with the chimeric receptor IL-5Rβ/OSMR∆1 together with the indicated GFP-tagged Jak1 constructs in a pSVL vector. Cells were stained with antibodies against IL-5Rβ and secondary antibody. The surface expression of chimeric receptors in cells displaying similar GFP-fluorescence was analyzed by FACS analysis. The values obtained for cells expressing the non-binding Jak1 mutant L80A/Y81A were set to 100%. Mean values and standard deviation obtained from at least 4 independent experiments are depicted (bar diagram). For Western blot analysis, cells were lysed and IL-5Rβ/OSMR∆1 was precipitated using an antibody recognizing IL-5Rβ. Using a GFP antibody, co-precipitation of the GFP-tagged Jak constructs and expression levels in whole cell lysates were monitored. (C) Jak1 deficient U4C cells were transfected with Jak1-GFP constructs (in a pcDNA3 vector) or with empty vector. Surface expression of the endogenous OSMR was monitored by FACS analysis using a monoclonal antibody against the OSMR. The values obtained for mock transfected cells were set to 100%. Mean values and standard deviation obtained from 3 independent experiments are depicted.
The FERM domain of Jak1 is not sufficient to upregulate OSMR surface expression.COS-7 cells were transfected with the construct encoding IL-5Rβ/OSMR and expression plasmids for either Jak3, Jak1 or the chimeric protein Jak1/3 as indicated. 48 h post transfection, cells were harvested and FACS analysis was carried out using monoclonal IL-5Rβ and secondary PE-conjugated antibodies. Values were normalised to fluorescence intensities of cells expressing a non-binding Jak1. Mean values and standard deviations of at least three independent experiments are depicted.
Mutation of L80 and Y81 to alanine abrogates SH2∆ mediated upregulation of the surface expression of endogenous OSMR.Jak1 deficient U4C fibrosarcoma cells were transfected with the GFP-tagged constructs encoding either Jak1-L80A/Y81A, SH2∆ or SH2∆-L80A/Y81A as indicated. Surface expression of the endogenous OSMR was monitored by FACS analysis using a monoclonal antibody against the OSMR. The mean fluorescence values obtained for Jak1-L80A/Y81A-GFP transfected cells was set to 100%. Mean values and standard deviations derived from three independent experiments are depicted.
signaling but plays a structural role for receptor interaction and upregulation of The JAK1 SH2 domain does not fulfill a classical SH2 function in JAK/STAT
published online May 12, 2005J. Biol. Chem.
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