rel Is rapidly tyrosine-phosphorylated following granulocyte-colony stimulating factor treatment of human neutrophils.

Stimulation of neutrophils with granulocyte-colony stimulating factor (G-CSF) results in an enhanced respiratory burst, prolonged survival, and increased tumor cell killing. The effects of G-CSF are mediated by binding to specific, high affinity receptors. G-CSF receptors lack intrinsic tyrosine kinase activity, but activation of the receptor results in the rapid induction of tyrosine kinase activity. Antiphosphotyrosine immunoblots of whole cell lysates prepared from neutrophils show that the G-CSF rapidly induces prominent tyrosine phosphorylation of a protein of a relative molecular mass of 80 kDa. Using monospecific antibodies, the 80-kDa tyrosine-phosphorylated protein has been shown to be p80c-rel, a proto-oncogene belonging to a family of transcriptional regulators which include NF-kB. The induction of tyrosine phosphorylation of p80c-rel was unique to G-CSF in that granulocyte-macrophage colony stimulating factor which also stimulates neutrophils and induces tyrosine phosphorylation does not result in tyrosine phosphorylation of p80c-rel. The consequences of p80c-rel tyrosine phosphorylation are not yet known; however, tyrosine-phosphorylated p80c-rel is capable of binding to DNA, and G-CSF stimulation results in an increase in the amount of p80c-rel which binds to DNA. These results demonstrate that one of the first biochemical events which occurs in neutrophils following G-CSF stimulation, activation of a tyrosine kinase, leads directly to the tyrosine phosphorylation of p80c-rel. Thus, the tyrosine kinase activated by G-CSF appears to directly transduce a signal to a protein which functions as a transcriptional regulator.

Stimulation of neutrophils with granulocyte-colony stimulating factor (G-CSF) results in an enhanced respiratory burst, prolonged survival, and increased tumor cell killing. The effects of G-CSF are mediated by binding to specific, high affinity receptors. G-CSF receptors lack intrinsic tyrosine kinase activity, but activation of the receptor results in the rapid induction of tyrosine kinase activity. Antiphosphotyrosine immunoblots of whole cell lysates prepared from neutrophils show that the G-CSF rapidly induces prominent tyrosine phosphorylation of a protein of a relative molecular mass of 80 kDa. Using monospecific antibodies, the 80-kDa tyrosine-phosphorylated protein has been shown to be p8OC-", a proto-oncogene belonging to a family of transcriptional regulators which include NF-kB. The induction of tyrosine phosphorylation of p8OC"' was unique to G-CSF in that granulocyte-macrophage colony stimulating factor which also stimulates neutrophils and induces tyrosine phosphorylation does not result in tyrosine phosphorylation of ~8 0~-~' .
The consequences of p80c'm' tyrosine phosphorylation are not yet known; however, tyrosine-phosphorylated p8OC-=' is capable of binding to DNA, and G-CSF stimulation results in an increase in the amount of ~8 0~-~' which binds to DNA. These results demonstrate that one of the first biochemical events which occurs in neutrophils following G-CSF stimulation, activation of a tyrosine kinase, leads directly to the tyrosine phosphorylation of ~8 0~-~' .
Thus, the tyrosine kinase activated by G-CSF appears to directly transduce a signal to a protein which functions as a transcriptional regulator.
Granulocyte-colony stimulating factor (G-CSF)I has diverse effects on the proliferation, differentiation, and function of mature neutrophils and their precursors (1-3). These biologic ef-CA01422 (to B. J. D.), CA36167 and CA34183 (to J. D. G.), and * This work was supported by National Institutes of Health Grants CA32028 and CA40512 (to B. R. F.) and by Sandoz Pharmaceuticals (to J. D. G. and B. J. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18   The abbreviations used are: G-CSF, granulocyte-colony stimulating factor; GM, granulocyte-macrophage; DNAP, DNA affinity precipitation.
fects are mediated through binding of G-CSF to a specific, high affinity cell surface receptor. The human G-CSF receptor is a glycoprotein of relative molecular mass 100-130 kDa (4). Homodimerization of the receptor is believed to be necessary to create a high affinity binding site for G-CSF (5). However, post-receptor signal transduction mechanisms remain largely unknown. Although the G-CSF receptor does not possess intrinsic tyrosine kinase activity, one of the earliest events following G-CSF binding to its receptor is the induction of tyrosine phosphorylation of at least one and possibly several intracellular proteins (6). Identification of the proteins which become tyrosine-phosphorylated following stimdation of neutrophils with G-CSF is likely to be important in understanding the signal transduction pathways required for neutrophil function. In this study, the major protein which is inducibly tyrosine-phosphorylated following treatment of neutrophils with G-CSF has been identified as p8OC-"'. The ~8 0~"~' protein is encoded for by a protooncogene (7, 8) which belongs to a family of transcriptional regulatory proteins which include v-rel, the oncogene product of the avian reticuloendotheliosis virus strain T (rev-T) (91, the maternal effects gene dorsal of Drosophila (10)(11)(12), and the ubiquitous factor NF-kB (13)(14)(15). These results demonstrate that one of the first biochemical events which occurs in neutrophils following G-CSF stimulation, activation of a tyrosine kinase, leads directly and rapidly to the tyrosine phosphorylation of ~80""'. Thus, the tyrosine kinase activated by G-CSF appears to directly transduce a signal to a protein which functions as a transcriptional regulator.

MATERIALS AND METHODS
Reagents-Highly purified recombinant human G-CSF and GM-CSF were gifts of Dr. Steven Clark and Gordon Wong, Genetics Institute, Cambridge, MA. Endotoxin, tumor necrosis factor, platelet activating factor, C5a, met-Leu-Phe, and phorbol ester were purchased from Sigma.
Antisera-The antiphosphotyrosine antibody 4G10 was generated using phosphotyramine as the immunogen and was used as described (16). The rel antiserum is a polyclonal rabbit antiserum prepared against a synthetic peptide corresponding to the unique carboxyl-terminal 16 amino acids of human ~80"" (17, 18).
Preparation of Human Neutrophils-Normal peripheral blood was obtained from healthy adult volunteers and fractionated on a Ficoll-Hypaque density step gradient (1.077 g/ml). The pellet was washed twice with Hanks' balanced salt solution (Life Technologies, Inc.). Red blood cells were lysed by incubation for 30 min at 4 "C in ammonium chloride (8.29 g/liter, pH 7.27). The neutrophil pellet was resuspended in RPMI 1640 (Life Technologies, Inc.) containing 0.5% bovine serum albumin, Fraction V (Sigma) at 5 x lo6 cells/ml.

Stimulation with Factors and Cell Lysis-Neutrophils were
pretreated with 1 m~ diisopropyl fluorophosphate (Sigma) for 30 min at 4 "C to inhibit proteases before factor stimulation. Cells were then
Insoluble material was removed by centrifugation at 4 "C for 10 min a t 10,000 x g. Alternatively, cells were lysed in DNA affinity precipitation buffer as described below. It should be noted that we have compared lysates prepared by boiling in SDS to lysates made in NP40 with and without EDTA and have found no significant differences in tyrosinephosphorylated proteins.
Zmmunoprecipitations, Gel Electrophoresis, and Zmmunoblotting-Whole cell lysates (5 x lo6 cells) were mixed 1:l with 2 x SDS buffer (19) with 2-mercaptoethanol (Life Technologies, Inc.) and heated at 95 "C for 3 min prior to one-dimensional SDS-polyacrylamide gel electrophoresis with 7.5% polyacrylamide. For immunoprecipitations, lysates containing 1 x lo7 cells were incubated with 20 pl of 4G10 cross-linked to Protein A-Sepharose (Pharmacia LKB Biotechnolgy Inc.) at 2 mg/ml as described elsewhere (20) or 5 p1 of polyclonal ~80"" antiserum at 4 "C for 4 h. Anti-re1 immune complexes were collected by incubation with 25 p1 of Protein A-Sepharose for an additional 30 min a t 4 "C. Immune complexes were washed once with lysis buffer, twice with 0.5 M LiCI, 20 mM Tris-HC1, pH 8.0, and once with phosphate-buffered saline. Washed immunoprecipitates were resuspended in an equal volume of water, then mixed with sample buffer and treated as described above prior to gel electrophoresis. After electrophoresis, proteins were electrophoretically transfemed to 0.2-pm nitrocellulose (Schleicher & Schuell) in a buffer containing 25 mM Tris, 192 m~ glycine, and 20% methanol a t 0.4 A for 4 h a t 4 "C. Residual binding sites were blocked by incubation in TBS (10 m~ Tris, pH 8.0, 150 m~ NaCI) containing 2% gelatin for 60 min a t 25 "C. The blots were incubated overnight a t room temperature with antiphosphotyrosine antibody (1.5 pg/ml) or for 2 h with re1 antiserum diluted 1:lOOO in TBST (TBS with 0.05% Tween 20). Antibody reactions were detected as previously described (16,18). Prestained molecular weight markers were run on each gel and were obtained from Life Technologies, Inc. DNA Affinity Precipitation (DNAP) Assay-Human neutrophils (2 x 109 either untreated or stimulated with G-CSF were lysed twice in 600 pl of DNAP buffer as described elsewhere (21). Dithiothreitol (final concentration 1 m~) , phenylmethylsulfonyl fluoride (1 m~) , leupeptin (10 mdml), and phenyl arsine oxide (37 p~) were added immediately before use. The final potassium chloride (KCI) concentration was 250 mM for the extraction and 50 mM for the binding assay. The final sodium fluoride (NaF) concentration was 50 mM. A 40-fold excess of poly(d1-dC) (Pharmacia) was used per DNAP assay. The binding reaction was performed with 100 pmol of biotinylated oligonucleotide. The oligonucleotides were synthesized by ISIS Pharmaceuticals, San Diego, CA. The oligonucleotides were synthesized with a 3' end biotin group and had a phosphothiorate backbone rendering them nuclease insensitive. They were otherwise identical to previously reported HIV-1 kB wild type and mutant oligonucleotides (21). The biotinylated oligonucleotide-protein complexes recovered by binding to streptavidin-agarose beads were washed four times with 400 pl of DNAP buffer containing 50 m~ NaF and 75 m~ KCI. A DNaseRNase digest was performed for 2 min on ice (21). The complexes were heated for 3 min at 95 "C in Laemmli sample buffer (50 pl/pellet) (19) and were subjected to polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred to nitrocellulose and detected by immunoblotting as described above.

RESULTS
G-CSF Znduces Tyrosine Phosphorylation of a n 80-kDa Protein in Human Neutrophils-Human neutrophils stimulated with G-CSF were evaluated by immunoblotting with an antiphosphotyrosine antibody in order to determine whether G-CSF receptor stimulation results in the tyrosine phosphorylation of any novel intracellular proteins. As can be seen (Fig. l), the pattern of tyrosine phosphorylated proteins seen following G-CSF stimulation of neutrophils was relatively simple. The major change was the inducible tyrosine phosphorylation of a protein of relative molecular mass of 80 kDa. Tyrosine phosphorylation of this 80-kDa protein could be seen as early as 5 min following G-CSF treatment of neutrophils and remained relatively constant when examined in a 1-h time course (Fig. 2). Tyrosine Phosphorylation of the 80-kDa Protein Is Specific to Stimulation of Neutrophils with G-CSF-Signal transduction through receptor linked tyrosine kinases is a relatively common phenomenon. Therefore, the pattern of tyrosine-phosphorylated proteins obtained following G-CSF stimulation of neutrophils was compared to that obtained following treatment with a variety of other factors known to stimulate neutrophil function. Treatment of neutrophils with GM-CSF resulted in tyrosine phosphorylation of proteins of 130,93,70, and 42 kDa as previously described (16,22). Similarly, phorbol ester stimulation resulted in the tyrosine phosphorylation of a 42-kDa protein which has been shown to be MAP-2 kinase (23). Other factors for which neutrophils contain receptors, such as endotoxin, C5a, tissue necrosis factor, and met-Leu-Phe failed to result in any discernible changes in tyrosine phosphorylation at the time point chosen (Fig. 3). Thus, tyrosine phosphorylation of the 80-kDa protein following G-CSF stimulation appears to be specific to the G-CSF signal transduction pathway.
The 80-kDa Tyrosine-phosphorylated Protein Is p8@-"' -Previous reports have shown ~80""' to be inducibly tyrosinephosphorylated in T cells following stimulation with phytohemagglutinin and phorbol 12-myristate 13-acetate (18). Given this finding, a rel-specific antiserum was employed to determine whether the 80-kDa tyrosine-phosphorylated protein seen following stimulation of neutrophils with G-CSF was p8OC-"'. In unstimulated neutrophils, no detectable tyrosine phosphorylation of p8OC-"' could be detected. However, G-CSF treatment of neutrophils resulted in the presence of an 80-kDa protein which can be detected in anti-re1 immunoprecipitates followed by antiphosphotyrosine immunoblotting or by antiphosphotyrosine immunoprecipitates followed by anti-re1 immunoblotting (Fig. 4, A and B). Similar amounts of ~80""' could be immunoprecipitated from control and factor-treated cells (data not shown). Serial immunoprecipitations demonstrate that approximately 5 1 0 % of total cellular p80"-"' is inducibly tyrosine-phosphorylated and that the majority of the tyrosine-phosphorylated 80-kDa protein can be depleted with re1 antiserum (data not shown). Thus, p8OC-"' appears to account for most of the 80-kDa tyrosine-phosphorylated protein seen in neutrophils following G-CSF stimulation. G-CSF Deatment of Neutrophils Results in a n Increase in DNA Binding of p8@-re'-Nuclear lysates of untreated and factor treated neutrophils were examined in order to determine whether the G-CSF induced tyrosine phosphorylation of p80c-rn' resulted in any alteration in DNA binding of p8OC-"' . G-CSF treatment of neutrophils results in a n increase in the amount of p80""' which is capable of binding to a n HIV-1 kB site (Fig. 5, rel immunoblot). Tyrosine-phosphorylated p80""l is capable of binding to DNA and most of the increase in p80""' bound to DNA can be accounted for by the tyrosine-phosphorylated form of p80c"p'. A small amount of tyrosine-phosphorylated ~80""' was detected in unstimulated neutrophils; however, this is comparable to the background tyrosine phosphorylation observed in immunoblots (Fig. 2). DISCUSSION The results presented here demonstrate that stimulation of neutrophils with G-CSF results in the rapid tyrosine phosphorylation of p80""' . G-CSF treatment of neutrophils not only results in the tyrosine phosphorylation of p8OC-"' but results in an increase in the amount of ~80""' which is capable of binding to a kB site. Most of this increase can be accounted for by the tyrosine-phosphorylated ~8 0~-~' species. The tyrosine phosphorylation of p80""' is specific to stimulation of neutrophils with G-CSF. Although GM-CSF is similar to G-CSF in its ability to stimulate the function of neutrophils for tumor cell killing and an enhanced respiratory burst, the proteins which become tyrosine-phosphorylated in neutrophils following GM-CSF treatment are different than those seen inducibly phosphorylated after G-CSF treatment. These data suggest that different kinases become activated following binding of ligand to their cognate receptors. Alternatively, the same kinase could have differing substrate specificity depending on the manner in which it is activated. These data also suggest that the tyrosine kinase pathway of signal transduction utilized by G-and GM-CSF may be responsible for the unique G-CSF Stimulates Tjrosine Phosphorylation of re1 effects of each of these factors rather than for their overlapping functions. However, it remains possible that these two pathways could converge on common downstream effectors. The major protein which becomes tyrosine-phosphorylated following stimulation of neutrophils with G-CSF is p80c"'e'. The p80""' protein is encoded for by a proto-oncogene which belongs to a family of transcriptional regulatory proteins which include v-rel, the oncogene product of the avian reticuloendotheliosis virus strain T (rev-T) (9). Other members of this family include the maternal effects gene dorsal of Drosophila which has a role in the establishment of dorsaVventra1 polarity of the Drosophila embryo (10)(11)(12). Another well characterized member of this family is the transcription factor NF-kB which is composed or two rel-related proteins (13)(14)(15). NF-kB activity is induced by a variety of agents including cytokines, mitogenic lectins, and phorbol esters (24,25). The re1 proteins are regulated in part by subcellular localization. NF-kB appears to be sequestered in an inactive form in the cytoplasm through its association with IkB which in turn is associated with the cytoskeleton by virtue of ankryin repeats (14, 26,27). Cellular activation results in the phosphorylation of IkB, releasing NF-kB which translocates to the nucleus where it can bind to and modulate the transcriptional activity at kB sites (24,28). Although similar interactions have been demonstrated for p8OC-"', it has also been shown that p8OC-"' function can be modified directly by phosphorylation of ~80""' (26, 27,29).
Tyrosine phosphorylation of p80""' has been demonstrated in T cells following stimulation with PHA and PMA and was associated with translocation of p8OC-"' to the nucleus (18). The present study extends these findings by showing that G-CSF stimulation of neutrophils results in an increase in the binding of tyrosine phosphorylated ~8 0~"~' isoforms to a kB site. The functional consequences of p8OC-"' tyrosine phosphorylation have not been determined. As the increase in DNA binding of p80""' could be accounted for largely by the tyrosine-phosphorylated form, it is possible that tyrosine phosphorylation results directly in an increase in the DNA binding ability of p8OC-"' . Alternatively, tyrosine phosphorylation might alter the ability of p8OC-"' to modulate transcriptional activity of specific promoters or may regulate subcellular localization of p80""'.
The results presented here implicate p8OC-"' as a n important signal-transducing protein in G-CSF-stimulated pathways in neutrophils. The specific transcriptional program modified by p80""' following G-CSF treatment of neutrophils remains to be determined as does the kinase responsible for the tyrosine phosphorylation of p8OC-"' . However, this study provides some initial clues which should be helpful in the dissection of the signal transduction pathways utilized by the G-CSF receptor.