PDGF Stimulates Tyrosine Phosphorylation of JAK 1 Protein Tyrosine Similar to Other Receptor Tyrosine Kinases Stimulates Its Intrinsic Kinase in Human Mesangial Cells

Kidney International, I/of 49 (1996), pp. 19—25 LABORATORY INVESTIGATION

PDGF stimulates tyrosine phosphorylation of JAK1protein tyrosine kinase in human mesangial cells

GOUTAM GHOSH CHOUDHURY, FABI0 MARRA, HIDEYASU KIYOMOTO, and HANNA E. ABBOUD

Division of Nephrology, Department of Medicine, University of Texas Health Science Center at San Antonto, San Antonio, Texas, USA

PDGF stimulates tyrosine phosphorylation of JAK 1 protein tyrosine similar to other receptor tyrosine kinases stimulates its intrinsic kinase in human mesangial cells. Platelet-derived growth factor (PDGF) tyrosine kinase activity. Subsequent phosphorylation of several exerts multiple effects in glomerular mesangial cells, including transcription of genes that mediate its biological activity. We have partiallycytoplasmic SH2 domain-containing proteins on tyrosine residues characterized PDGF-mediated early mitogenic signal transduction path- results in their enzymatic activation [5]. These enzymes include ways that include activation of protein kinase Ca and phosphatidylinositol phospholipase Cyl (PLCy1), GTPase activating protein and the 3 kinase. However, the precise mechanism of PDGF-induced gene transcription is not yet clear. A family of cytoplasmic transcription factors tyrosine phosphatase SH-PTP 2 [6]. Tyrosine phosphorylated referred to as signal transducers and activators of transcription (STAT) PDGFR also binds cytoplasmic adaptor molecules such as Grb-2 has recently been identified. This group of transcription factors is activated and Nek followed by their translocation to the plasma membrane by different cytokines via tyrosine phosphorylation. We studied the effect [6—8]. Grb-2 exists in the cell as a complex with SOS which is a of PDGF on STATs in human mesangial cells. Using a gel retardation guanine nucleotide exchange factor for Ras. Thus activated assay, nuclear and cytoplasmic extracts from PDGF-stimulated mesangial cells contained protein factors that bind to a DNA sequence representing PDGFR stimulates Ras by recruiting Grb-21505complexto the the sis-inducible element (SIE) present in the c-fos gene promoter. These plasma membrane where Ras is located [6]. Activation of Ras protein factors also bind to the enhancer element present in interferon-y initiates a kinase cascade leading to stimulation of mitogen responsive genes, suggesting the involvement of STAT proteins. The activated protein kinase (MAPK) [6, 9]. MAPK then translocates addition of monoclonal antibody that recognizes STAT 1 results into the nucleus and phosphorylates transcription factors which "supershift" of the DNA-protein complex stimulated by PDGF indicating the presence of STAT 1. Immunoblotting experiments with a monoclonal stimulate gene expression induced by PDOF [9]. At least one STAT 1 antibody revealed the presence of STAT1a and STAT1J3 in other major pathway can be activated by a number of cytokines. mesangial cells. Since certain cytokines activate STATs via tyrosine A group of cytoplasmic proteins that participate in transcription phosphotylation mediated by JAK family of tyrosine kinases, we studied of genes induced by multiple cytokines such as interferons and the effect of PDGF on JAK kinases. Antiphosphotyrosine immunoblotting of JAK 1 immunoprecipitates from PDGF-stimulated mesangial cell lysate some interleukins have been identified. These proteins are re- showed increased tyrosine phosphorylation of this tyrosine kinase. In vitro ferred to as signal transducers and activators of transcription immune complex kinase assay of JAK 1 immunoprecipitates from PDGF- (STATs) [10]. To date, five members have been identified. stimulated mesangial cell lysate revealed activation of this tyrosine kinase. Activation of STATs by cytokine receptors which lack intrinsic Taken together, these data demonstrate that PDGF activates the transcription factor STAT 1 in mesangial cells. The data also provide the first tyrosine kinase activity utilize a group of recently identified evidence that PDGF stimulates tyrosine phosphorylation of JAK 1, the cytoplasmic tyrosine kinases. These are referred to as just another cytoplasmic tyrosine kinase stimulated by many other cytokines to activate kinases (JAKs) [10, 11]. Four members (JAK 1, JAK 2, JAK 3 and transcription via STATs. These observations indicate that JAK 1 is aTYK 2) have been identified. They are characterized by the downstream tyrosine kinase in PDGF receptor signaling and is a candidate presence of two tyrosine kinase domains of which the C-terminal for activation of STAT 1. one is catalytically active [11]. These kinases can associate with specific cytokine receptors [11—14]. These kinases also phosphor- ylate STATs on specific tyrosine residue, resulting in their trans- During glomerular injury, hypercellularity occurs because oflocation to the nucleus where they stimulate transcription of infiltration of inflammatory cells as well as proliferation ofcertain cytokine-inducible genes [10, 11]. intrinsic glomerular cells including mesangial cells. Inflammatory It has recently been shown that, similar to cytokines, polypep- cells and platelets secrete several cytokines and growth factors intide growth factors such as PDGF and epidermal growth factor the microenvironment of the mesangium. PDGF, a major cyto-(EGF) can also stimulate the STAT family of transcription factors kine secreted by these cells is the most potent mitogen for[15, 16]. EGF stimulation of A431 epidermal carcinoma cells mesangial cells [1—4]. Activation of PDGF receptor (PDGFR)results in the association of a member of STAT family, the STAT1a, with the EGF receptor (EGFR) [17]. EGF also stimulates tyrosine phosphorylation of JAKI [18]. In mesangial cells, the signal transduction pathways initiated by activated tyrosine Received for publication April 27, 1995 kinases have not been studied in detail [19, 20]. Specifically, the and in revised form August 18, 1995 role of newly identified JAK-STAT pathways in PDGF signal Accepted for publication August 21, 1995 transduction in mesangial cells have not been identified. © 1996 by the International Society of Nephrology In this study, we characterized activation of STAT by PDOF in

19 20 Choudhuiy et a!: Tyrosine phosphoiylation of JAK 1 by PDGF

mesangial cells using two DNA sequences recognized by STATs(35 ms'i HEPES, pH 7.8, 0.5 mrvi EDTA, 0.5 mM DTF, 10% in gel mobility shift assay (GMSA) and a specific monoclonalglycerol) with 10 tg/m1 poly dl dC and 50,000 cpm of radiolabeled STAT 1 antibody. We also identified JAK 1 in these cells andoligonucleotide for 30 minutes at 25°C. For competition experi- provided the first evidence that this tyrosine kinase is phosphor-ments, 50-fold excess nonlabeled oligonucleotide was used. Some ylated on tyrosine in response to PDGF. These data suggest thatof the extracts were incubated on ice with STAT 1 or antiphos- JAK 1 is a candidate for PDGF-mediated activation of STAT 1.photyrosine antibody for 30 minutes before the binding reaction. Finally, the samples were separated on a native 5% polyacryl- Methods amide gel. After electrophoresis, the gel was dried and autoradio- Materials graphed. Tissue culture materials were purchased from Gibco (Grand Island, NY, USA). PDGF BB was obtained from Amgen (Thou- Immunoprecipitation, immunoblotting and immunokinase assay sand Oaks, CA, USA). Monoclonal STAT 1 antibody was pur- These were performed essentially as previously described [19, chased from Transduction Laboratories, Lexington, KY, USA).20, 24]. Briefly, the cells were lysed in RIPA buffer (20 mivi Antiphosphotyrosine and JAK 1 antibodies were obtained fromTris-HC1, pH 7.5, 150 mM NaC1, 5 mM EDTA, 1 mM Na3VO4, 1% UBI. Protein A sepharose CL 4B was from Pharmacia. ECLNP4O, 1 mivi PMSF and 0.1% aprotinin) at 4°C for 30 minutes. Western blotting chemicals were obtained from Amersham (Ar-Cleared cell lysate was prepared by centrifugation. The cell lysate lington Heights, IL, USA). Oligonucleotides were synthesized inwas incubated with the appropriate antibody on ice for 30 an Applied Biosystem DNAJRNA synthesizer. All other reagentsminutes. Protein A sepharose beads were added and the tubes were of analytical grade. were rotated at 4°C for two hours. The beads were washed twice with RIPA buffer, once with 50 mM Tris-HC1 pH 7.4, 1 mM Cell culture Na3VO4 and finally resuspended in 2 X sample buffer. Boiled Isolation and characterization of normal human glomerularimmunoprecipitates were separated on 7.5% SDS-polyacrylamide mesangial cells were described elsewhere [4]. Cells were grown ingel. The separated proteins were electrophoretically transferred Waymouths's medium with 17% fetal calf serum and were madeto polyvinyl membrane and blocked at 4°C overnight in 2% BSA quiescent by serum starvation for 48 hours in the same medium.prepared in PBS containing 0.1% tween 20 (PBS-T). The mem- In each experiment the cells were stimulated with 10 ng/ml PDGF.brane was then incubated with JAK 1 antibody (1:1000 dilution) Preparation of nuclear extracts. Nuclear extracts were preparedfor one hour at room temperature on a roller shaker. For STAT using a modified method described by Andrew and Faller [211.1 detection the antibody concentration was 1 sgIml. The mem- Mesangial cells in 150 mm dishes were washed twice with PBS and brane was washed four times with PBS-T and the protein was scraped in PBS using a rubber policeman. The cell pellet wasdetected by horseraddish peroxidase conjugated secondary anti- resuspended in buffer A [10 mivi HEPES pH 7.9, 1.5 mivi MgC12, body followed by developing with ECL reagent. Immune complex 10 mM KC1, 0.5 mrvi DTT, 2 mM Na3VO4, 0.5 mrvt phenylmethyl- kinase assay of JAK 1 immunoprecipitates was performed as we sulfonyl fluoride (PMSF), 0.1% aprotinin and 0.1% Triton X-1001described previously [19, 20]. Three microliters of JAK 1 antibody and gently homogenized. The lysate was centrifuged at 600 X g atwere used for immunoprecipitation. 4°C. The pellet was resuspended in the same buffer and centri- Unless otherwise indicated, each observation was confirmed at fuged again at 600 X g. The final pellet was then resuspended inleast twice. buffer B (20 mivi HEPES pH 7.9, 1.5 mM MgCl2, 420 mivi NaC1, 0.2 mM EDTA, 0.5 ms'i PMSF, 0.1% aprotinin and 2 mrvt Na3VO4) Results and incubated at 4°C for 30 minutes. The mixture was centrifuged To identify factors which bind DNA elements similar to STATs at 10,000 X g and the supernatant was collected as the nuclearin mesangial cells, we performed gel mobility shift assay using a extract. The supernatant from the first low speed spin washigh affinity variant of sis-inducible element (m67) present in the centrifuged at 10,000 X g at 4°C and collected as the cytosolicfos promoter as the labeled probe [22]. Nuclear extracts from fraction. After determining protein concentration, aliquots werenon-treated and PDGF-treated human mesangial cells were used. stored at —80°C. The data in Figure 1 show that PDGF stimulates binding of nuclear as well as cytoplasmic factors to this element in mesangial Gel mobility shift assay (GMSA) cells (compare lanes 2 and 4 with lanes 1 and 3). To test the Elements that bind STATs are present in several cytokinespecificity of binding, competition experiments were performed inducible genes including PDGF-induced c-fos promoter [22]. Thewith extracts from PDGF-stimulated mesangial cells in the pres- sequence present in the c-fos gene is called the sis-inducibleence of excess nonlabeled oligonucleotide. The data show com- element (SIE). A variant of STE has also been reported to interactplete disappearance of the protein-DNA complex in the presence with protein factors even with higher affinity than the cytokineof the competitor, indicating that this DNA protein interaction is inducible gene elements [10, 11, 22]. For this study, we thereforehighly specific (Fig. 2, compare lane 3 with 2). To confirm that one chose SIE and an IFNy responsive Ly-6E gene element. Doubleof STAT proteins is involved in this interaction, we used labeled stranded oligonucleotides corresponding to high affinity SIE,GAS as a probe in the GMSA. Similar to m67, GAS also binds to called m67 (CAGTTCCCGTCAATC) [22J, and the interferon-yprotein(s) in extracts from PDGF-treated cells (Fig. 2, compare activating sequence (GAS) from the Ly-6E gene (ATA]ITCCT-lane 5 with 4). Effective competition using excess nonlabeled GAS GTAAGTG) [23] were used. The DNA was labeled with T4oligonucleotide indicate the specificity of this interaction (com- polynucleotide kinase and y32P-ATP. Eight to 15 g of nuclear,pare lane 6 with 5). These data indicate that PDGF activates cytoplasmic or total extracts were incubated in a binding buffertranscription factor(s) similar to STATs in human mesangial cells. Choudhuiy et al: Tyrosine phosphoiylation of JAK 1 by PDGF 21

Probe m67 I GAS 0 + — PDGF a- PDGF — + + + —— —.. a aSs a

Free probe —* 1 2 3 4 5 Fig. 1. Binding of nuclear and cytoplasmic protein factors from control and PDGF-stimulated mesangial cells to sis-inducihie element (SIE). Quiescent mesangial cells were stimulated with PDGF for 30 minutes at 37°C. 1 2 3 4 5 6 Nuclear and cytoplasmic extracts were used in gel mobility shift assay (GMSA) with labeled m67 oligonucleotide as the SIE probe, as described Fig. 2. Binding of protein factors from control and PDGF-stimulated me- in Methods. Arrow indicates the DNA-protein complexes formed when sangial cells to m67 and GAS elements. Mesangial cell extracts were used in extracts from PDGF-stimulated cells are used. Lane 5 shows the mobility GMSA with labeled m67 or GAS as described in Methods. Solid arrow of the free oligonucleotide probe. indicates the position of PDGF-stimulated DNA-protein complex. Lanes 3 and 6 demonstrate specificity of binding using 50-fold excess nonlabeled m67 and GAS as competitors, respectively.

To identify STAT family member in mesangial cells, we performed immunoblot experiments using a STAT 1 specific mono-associated with GAS element was shifted by STAT 1 antibody clonal antibody. The data demonstrate that human mesangial cells(Fig. 3B, compare lane 6 with 5). These data clearly indicate that express STAT 1 as a doublet, STAT1a and STATI f3(Fig.3A).PDGF activates STAT 1 in human mesangial cells. It has been We also studied the effect of the same STAT 1 antibody onshown that STAT 1 is tyrosine phosphorylated in response to migration of DNA-protein complex in GMSA. Extracts fromgrowth factors and cytokines and this tyrosine phosphorylation is PDGF-stimulated mesangial cells were used in GMSA in therequired for its translocation to the nucleus as well as for DNA presence and absence of STAT 1 antibody. The band representingbinding [10, 11]. Our attempts to detect direct tyrosine phosphor- binding of protein(s) to the m67 oligonucleotide was shifted in theylation of STAT 1 protein in mesangial cells using antiphospho- presence of the antibody (Fig. 3B, compare lane 3 with 2,tyrosine immunoblot analysis of STAT 1 immunoprecipitates indicated by dark arrow). Similarly, all the protein/DNA complexwere unsuccessful (see Discussion). However, we tested for the 22 Choudhuiy et al: Tyrosine phospho,ylation of JAK 1 by PDGF

A B

Probe m67 GAS PDGFS — + +• — + + Fig. 3. Binding activity in extracts from PDGF- treated mesangial cells is signal transducer and activator of trascription I (STAT 1). (A) STAT I protein expression in mesangial cells. Total cell 97— lysate was analyzed by immunoblotting using a monoclonal STAT 1 antibody as described in Methods. Lane 1, 15 g and lane 2, 25 jsg of cell lysate, respectively. Molecular weight markers are shown in the left margin. The -1 doublet in lane 2 indicated by arrow represents STAT1a and STAT1I3, respectively. (B) "Supershift" analysis of DNA-STAT 1 complex. 68 — Extracts from control or PDGF-treated mesangial cells were used in GMSA with labeled m67 or GAS probes. Extracts in lanes 3 and 6 were preincubated with STAT 1 antibody before incubation with labeled probes as described in Methods. Open arrow indicates the DNA-protein complex stimulated by PDGF. Dark arrow indicates the "supershifted" band in lanes 3 and 6 in the presence of STAT 1 12 123 456 antibody.

presence of tyrosine phosphorylated STAT 1 in the DNA-protein PDGF — + + complex. Using m67 as a probe, gel mobility shift analysis was AntiPTyrAb — — + performed using PDGF-stimulated mesangial cell lysate preincubated with or without a monoclonal antiphosphotyrosine antibody. The data show inhibition of DNA binding in the presence of the antiphosphotyrosine antibody (Fig. 4). These results suggest that STAT 1 present in DNA-protein complex is tyrosine phosphorylated since incubation of mesangial cell extract with antiphosphotyrosine antibody neutralizes the DNA binding activity of STAT 1. Preincubation of PDGF-stimulated mesangial cell extract with another mouse monoclonal antibody (which recognizes phosphatidylinositol 3 kinase) did not inhibit the formation of DNA-protein complex (data not shown) suggesting that the inhibition by antiphosphotyrosine antibody is specific. It has recently been shown that JAK family of tyrosine kinases is involved in various cytokine-mediated activation of STAT 1 [10, 11]. Specifically JAK 1 is tyrosine phosphotylated and involved in 1 23 signal transduction of hormones such as prolactin and cytokinesFig.4. Effect of antiphosphotyrosine antibody on the DNA-protein complex formation from PDGF-stimulated mesangial cell extract. Gel mobility shift including interferons, IL-2, IL-4, IL-6, IL-7 and IL-9 [10, 11, 25].analysis was performed using m67 as probe from control (lane 1) and We first determined if mesangial cells express JAK 1 protein.PDGF-stimulated (lane 2) cell extracts. Extracts in lane 3 was preincu- Since the abundance of this protein is low, we first immunopre-bated with antiphosphotyrosine antibody before incubation with labeled cipitated JAK 1 protein and the immunoprecipitates were thenprobe. The arrow indicates the protein-DNA complex stimulated by PDGF. analyzed by immunoblotting using the same JAK 1 antibody. The data show the presence of 130 kD JAK 1 in human mesangial cells (Fig. 5). Immunoprecipitation of mesangial cell lysate with nonimmune serum did not show any cross reactivity with JAK 1 (Fig.phosphorylated in control quiescent cells in the absence of PDGF 5, lane 1). To determine if this kinase is tyrosine phosphorylated(Fig. 6, lane 1). However, treatment of cells with PDGF resulted in response to PDGF in mesangial cells, we immunoprecipitatedin a further increase in tyrosirie phosphorylation of JAK 1 JAK 1 protein from PDGF-stimulated cell lysate and the immu-(compare lanes 2 and 3 with lane 1). To determine if the increase noprecipitates were analyzed by immunoblotting using an an-in tyrosine phosphorylation of JAK 1 is associated with increased tiphosphotyrosine antibody. The data show that JAK 1 is tyrosinetyrosine kinase activity, the enzyme was immunoprecipitated from Choudhuiy et al: Tyrosine phosphotylation of JAK I by PDGF 23 KD PDGF, minutes 200 — 0 15 30

200 —

—JAK1

97 — 97 —

68 — *- IgO 68 —

1 2

Fig. 5.Expressionof JAK 1 in human mesangial cells. Equal amounts of mesangial cell lysates were immunoprecipitated with either nonimmune 1 2 3 serum (lane 1) or anti-JAK 1 antibody (lane 2). The precipitated proteins were analyzed by immunoblotting using JAK 1 antibody. Molecular weight markers are shown in left margin. Arrow indicates the 130 kD JAK 1 Fig. 6. Tyrosine phospho..ylation of JAK I in PDGF-stimulated mesangial tyrosine kinase. cells. Quiescent mesangial cells were stimulated with PDGF for 15 and 30 minutes respectively. Equal amounts of cell lysate were immunoprecipitated with anti-JAK 1 antibody and analyzed by antiphosphotyrosine immunoblotting. Molecular weight markers are shown in the left margin. control and PDGF-treatcd mesangial cell lysate and its tyrosineRepresentative of three independent experiments is shown. kinase activity was measured in vitro. The data in Figure 7 show that PDGF stimulates tyrosine kinase activity of JAK 1 as revealed by the presence of autophosphorylated JAK1proteining sis-inducible element present in the fos promoter as a probe, (indicated by solid arrow). A slower migrating band was alsorevealed the presence of protein factor(s) which bind to this observed 30 minutes after PDGF treatment (indicated by openelement in a specific manner (Figs. I and 2). These proteins also arrow). This band may represent hyperphosphorylation of JAK 1bind to GAS element suggesting that these are members of the protein. These data indicate that the cytoplasmic tyrosine kinaseSTAT family (Fig. 2). Use of a specific STAT I antibody in GMSA JAK1is a target protein in PDGF-mediated signal transductionconfirmed that STAT 1 represent the major protein binding in mesangial cells. activity present in the DNA-protein complex in extracts of mesangial cells treated with PDGF (Fig. 3B). We also identified the Discussion presence of STAT 1 proteins in human mesangial cell by immu- The JAK/STAT pathways involved in transcriptional activationnoblotting (Fig. 3A). However, we were unable to detect tyrosine of certain hormones, growth factors and cytokine-inducible genesphosphorylated STAT 1 protein by antiphosphotyrsine immuno- have not yet been characterized in primary human cells such asblot analysis. This may be due to the fact that PDGF stimulates mesangial cells. This study aimed at characterizing protein factorsrather weak tyrosine phosphorylation of STAT 1 in mesangial that are involved in transcriptional activation of genes induced bycells. It is known that only one tyrosine residue (Tyr 701) in the PDGF. Gel retardation assay, using an oligonucleotide represent-STAT 1 molecule is phosphorylated in response to agonists [10, 24 Choudhuiy et al: Tyrosine phosphotylation of JAK I by PDGF

PDGF,minutes 0 15 30 KD 200 —

Fig. 7. Activation of JAK I ryrosine kinase activity in response to PDGF in mesangial cells. 4- Quiescent mesangial cells were stimulated with PDGF and the cell lysate was —JAK1 immunoprecipitated with JAK 1 antibody as described in Figure 6. The immunebeads were assayed for tyrosine kinase activity in vitro in the presence of y32P-ATP as described in Methods. The reaction products were analyzed on SDS gel, dried and autoradiographed. The 97 — molecular weight markers are shown in left margin. Bold arrow indicates the phosphorylated JAK 1 protein and the open arrow indicates the hyperphosphorylated JAK 1. Note that there are two other phosphorylated proteins present only in PDGF-stimulated lanes, the identity of which are not known yet.

11, 26]. However, functional analysis using antiphosphotyrosinekD JAK 1 protein in human mesangial cells (Fig. 5) and demon- antibody suggested that STAT 1 obtained from PDGF-stimulatedstrate that this tyrosine kinase is tyrosine phosphorylated in these mesangial cell lysate in protein-DNA complex is tyrosine phos-cells in the basal state (Fig. 6, lane 1). It has recently been shown phorylated (Fig. 4). that JAK 1 protein is tyrosine phosphorylated in other cell types Growth factor and cytokine receptors utilize protein tyrosinein the absence of exogenous agonists [13]. Our data provide the kinases that are present either as an intrinsic domain of thefirst evidence that activation of PDGFR results in increased receptor and/or as cytoplasmic protein(s) that has the capability totyrosine phosphorylation of JAK 1 (Fig. 6). In vitro kinase assay interact with the receptor. Upon activation of the PDGFR, itsof JAK I immunoprecipitates from PDGF-stimulated mesangial intrinsic tyrosine kinase activity is necessary for the full spectrumcell lysate also showed increased tyrosine kinase activity of its biological activities [51. Activation of PDGFR also stimu-(Fig. 7). Taken together, these data indicate that JAK 1 is a lates src family of tyrosine kinases [271. This family of kinases can downstream tyrosine kinase in PDGFR-mediated signal transduc- physically associate with the juxtamembrane region of thetion. Whether JAK I is the candidate tyrosine kinase to stimulate PDGFR, an association which is necessary for the mitogenictranscription via STAT 1 in response to PDGF is not established. signal induced by PDGF. These data suggest that despite theHowever, it is known that this is one of the kinases that stimulate requirement of intrinsic tyrosine kinase activity that is associatedSTAT 1-mediated transcription in response to several cytokines with PDGFR, cytoplasmic tyrosine kinases are also involved in[10, 11, 25]. More work is needed to identify the direct mechanism PDGF-mediated signaling. involved in the activation of STAT I in PDGF-induced signaling. Recently a unique family of tyrosine kinases has been impli- cated in the transcriptional regulation of cytokine-induced gene Acknowledgments expression. This family consists of four members, three JAKs and This study was supported in part by VA Medical Research Service and TYK 2 [10, 11]. Activation of certain cytokine receptors stimulateNIH grants DK 43988 and DK 33665 to HEA and a grant in aid from the these kinases and result in the activation of protein factors thatAHA-Texas affiliate to GGC. We thank Sergio Garcia for help with the eventually result in gene transcription. Very little is knowncell culture. concerning the role of these kinases in growth factor receptor tyrosine kinase-mediated signal transduction. It has recently been Reprint requests to Goutam Ghosh Choudhu,y, Ph.D., Division of Ne- phrology, Department of Medicine, University of Texas Health Science shown that EGF stimulates tyrosine phosphorylation of JAK I inCenter, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7882, USA. A431 epidermal carcinoma cells suggesting its involvement in EGF signaling in these cells [18]. Also EGF stimulates STAT ito Appendix transcribe genes containing SIE [15]. Whether JAK 1 directly Abbreviations are: STAT, signal transducer and activator of transcrip- stimulates this family of transcription factors in EGF-stimulated tion; JAK, Janus kinase; GMSA, gel mobility shift assay; SIE, sis-inducible cells is not yet clear. In the present study, we have identified 130element; GAS, gamma interferon activating sequence. Choudhuiy et al: Tyrosine phosphoy1ation of JAK I by PDGF 25

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