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The Journal of Immunology, 1998, 160: 4979-4987.
Copyright © 1998 by The American Association of Immunologists

Phosphatidylinositol 3'-Kinase and SH2-Containing Inositol Phosphatase (SHIP) Are Recruited by Distinct Positive and Negative Growth-Regulatory Domains in the Granulocyte Colony-Stimulating Factor Receptor1

Melissa G. Hunter* and Belinda R. Avalos2,*,{dagger}

* The Molecular, Cellular and Developmental Biology Program, and {dagger} Division of Bone Marrow Transplantation, The Ohio State University, Arthur G. James Cancer Hospital and Research Institute, Columbus, OH 43210


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activation of both positive and "negative" or anti-proliferative signals has emerged as a common paradigm for regulation of cell growth through cell surface receptors that regulate immune responses. SHP-1 and -2 and the novel 5'-inositol phosphatase SHIP have recently been shown to function as growth inhibitory molecules in immune receptor signaling. In the current study, we have identified distinct regions in the granulocyte colony-stimulating factor receptor (G-CSFR) distal to the conserved box 2 motif necessary for mitogenesis, which exert positive and negative influences on growth signaling in Ba/F3 pro-B lymphoid cells. The region spanning amino acids 682 to 715 mediates activation of phosphatidylinositol 3'(PI3)-kinase. Activation of PI3-kinase leads to inhibition of apoptosis, promotion of cell survival, and enhanced proliferative responses to G-CSF. We show that the region of 98 amino acids in the distal tail of the class I G-CSFR down-modulates proliferative signaling, not only in myeloid cell lines, as previously reported, but also in Ba/F3 cells. This same region recruits SHIP to the signaling cascade through a mechanism involving Shc, with the formation of Shc/SHIP complexes. Our data suggest a model in which PI3-kinase and SHIP coordinately regulate growth signaling through the G-CSFR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neutrophils provide essential early defense against bacterial infections. The production of neutrophils is critically regulated by granulocyte colony-stimulating factor (G-CSF)3 through interactions with its receptor on responsive cells. The granulocyte colony-stimulating factor receptor (G-CSFR) belongs to the cytokine receptor superfamily whose members all lack tyrosine kinase activity. Despite the absence of intrinsic enzymatic activity, activation of Jak kinases and the Ras-MAP kinase cascade occur following ligand binding in association with the generation of mitogenic signals by the G-CSFR (1). Although these pathways contribute to growth regulation in a positive fashion, virtually nothing is known about the mechanisms of "negative" or anti-proliferative signaling by the G-CSFR. Growth inhibitory molecules recruited by the G-CSFR are postulated to play an important role in the maintenance of steady-state neutrophil levels.

Phosphatidylinositol 3'-kinase (PI3-kinase) is an enzyme that has been implicated in critically mediating mitogenic signaling by a variety of growth factors (2). Although its role in proliferative signaling by the G-CSFR has not been studied, PI3-kinase has been reported to prevent apoptosis in certain cell types (3). PI3-kinase phosphorylates phosphatidylinositols at the 3' position leading to increases in phosphatidylinositol (3, 4, 5)-triphosphate (PI(3, 4, 5)P3) and phosphatidylinositol (3, 4)-bisphosphate (PI(3, 4)P2). These phosphorylated intermediates may then act as second messengers and/or regulators of protein-protein interactions (4, 5). The mechanisms regulating PI3-kinase activity have become the subject of intense interest. A novel inositol polyphosphate 5'-phosphatase, referred to as SHIP (SH2-containing inositol phosphatase), was recently identified with unique substrate specificity for the PI3-kinase metabolite PI(3, 4, 5)P3 (6, 7, 8). This phosphatase was shown to hydrolyze PI(3, 4, 5)P3, resulting in inhibition of the function of PI3-kinase and down-regulation of its biologic effects on mitogenesis, cellular transformation, and immune cell function (6, 9, 10, 11).

In this article, we have examined the signaling pathways that are activated by the G-CSFR and a naturally occurring G-CSFR isoform that is highly expressed in placenta and some myeloid leukemia cell lines. We have identified distinct positive and negative growth-regulatory domains in the G-CSFR that recruit PI3-kinase and SHIP, respectively, to the G-CSFR signaling cascade. Activation of PI3-kinase promotes G-CSFR-mediated growth signaling through inhibition of apoptosis and requires the conserved region from residues 682 to 715 of the G-CSFR. A downstream carboxyl-terminal region absent in the class IV G-CSFR splice variant recruits Shc/SHIP complexes and down-regulates proliferative signaling. These studies are the first to demonstrate recruitment of a growth inhibitory molecule by the G-CSFR. Our data indicate functional differences in the class I and class IV G-CSFR isoforms that are likely to have important clinical relevance, and also provide a model for positive and negative growth regulation by the G-CSFR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Recombinant human G-CSF was a kind gift from Amgen (Thousand Oaks, CA). Recombinant mouse IL-3 was purchased from Becton Dickinson (Bedford, MA). For some experiments, WEHI-3 conditioned media (WEHI-3-CM) was used as a source of IL-3 (12). RPMI 1640, FBS, and other components for cell culture were obtained through Life Technologies (Grand Island, NY). Trizol, oligonucleotides, and reverse transcriptase (RT)-PCR reagents were purchased from Life Technologies, unless otherwise indicated. Radioactive isotopes were purchased from Amersham (Arlington Heights, IL). All other reagents were obtained through Sigma (St. Louis, MO) unless otherwise specified.

Antibodies

The anti-SHIP rabbit polyclonal Ab used was generated from a GST-fusion protein corresponding to residues 886 to 955 of p130 SHIP as described earlier (13). This Ab also cross-reacts with the p110 and p145 SHIP isoforms. The 4G10 anti-phosphotyrosine Ab was a generous gift from Dr. Brian Druker (Oregon Health Sciences University, Portland, OR). In some experiments, anti-PI3-kinase (p85) from Transduction Laboratories (Lexington, KY) was used or a rabbit polyclonal anti-PI3-kinase (p85) from a GST-fusion protein encoding the N-terminal SH2 domain of p85 (generously provided by K. M. Coggeshall, Ohio State University, Columbus, OH). Rabbit polyclonal Ab to Shc was obtained from Upstate Biotechnology (Lake Placid, NY).

Cells

The class IV and G-CSFR{Delta}682 (also referred to as Cfr) cDNAs were stably transfected into the IL-3-dependent cell line Ba/F3, as previously described (12). For the construction of pCDM8-class I, the class I human G-CSFR cDNA (generously provided by A. Larsen (Immunex Corp., Seattle, WA)) was excised from pBluescript SK+, ligated to Bst XI linkers and inserted into the Bst XI site of pCDM8. The p309 plasmid containing the neomycin resistance gene, was co-transfected with the G-CSFR cDNA plasmid to establish stable clones. The transfectants were selected in G418 containing media as previously described (12). The pCDM8 vector was a gift from Dr. B. Seed (MIT, Cambridge, MA) and the p309 vector was generously provided by Jas Lang (The Ohio State University, Columbus, OH). The G-CSFR{Delta}715 clone was generated by introducing a stop codon with a C to T point mutation at nucleotide 2379 of the Class I cDNA (number corresponds to Larsen et al. published sequence (14)) by PCR. The oligonucleotides used to generate this mutant were: forward primer (F1) (5'-CCACCTAGCCCCAATCCCAGTCTGGC-3'), and reverse R4 (5'-GATCGCT GGTGCCAGACTGGGATTGGGGCTAGG-3'); the underlined nucleotides indicate the position of the point mutations. In the first PCR reaction, primer F2 containing a 5' restriction site for Bam HI and corresponding to nucleotides 2252 to 2268 of the Class I cDNA was used in conjunction with the R4 primer. In the second PCR reaction, primer F1 was used with primer R2 which was designed to contain an Xho I restriction site and corresponded to nucleotides 2581 to 2596 of the Class I cDNA. The extension products from both reactions were ligated and further amplified using primers F2 and R2. The PCR fragment was subcloned into pBluescript SK+, excised from the plasmid by Hind III and Bam HI digestion, and ligated into the Cfr101 and BstE II sites of pCDM8-Class I. DNA sequencing was done to confirm the introduced point mutation prior to the establishment of stable transfectants in Ba/F3 cells. Clones were selected by neomycin resistance and their ability to bind 125I-G-CSF. Receptor binding assays and Scatchard analyses were performed as described previously (12). Pools of three positive subclones for each G-CSFR clone were used for all experiments.

Reverse transcriptase-PCR

RNA was isolated from G-CSFR clones using Trizol, treated with 10 U DNase (Boehringer Mannheim Biochemical, Indianapolis, IN), and visualized by gel electrophoresis. To generate cDNA, 3 µg of RNA were used per reaction with 0.25 µg/µl random primer mix pdN6, 0.5 U RNase inhibitor (Boehringer Mannheim Biochemical) and 10 U reverse transcriptase (Boehringer Mannheim Biochemical), and incubated at 42°C for 2 to 4 h. Identical reactions were set up in the absence of reverse transcriptase to serve as negative controls for the PCR reactions. A total of 0.3 µg of cDNA was amplified by PCR using pairs of the following primers corresponding to different regions within the cytoplasmic domain of the G-CSFR: F21 (5'-CCCAACAGGAAGAATCCCCTCTGG-3'), R22 (5'-CAAGCACAAAAGGCCATTGGGTGG-3'), and R1 (5'-CCTCCTCCAGCACTGTGAG-3'). The R22 primer corresponds to shared sequences in the distal tail of the class IV cDNA and noncoding region of class I cDNA. Primers corresponding to the housekeeping gene, GADPH, were used to confirm the integrity of each cDNA. An annealing temperature of 62°C was used with primers F21/R22 and GADPH, whereas annealing was performed at 58°C with the F21/R1 primer pair. All PCRs were conducted for 28 cycles and the PCR products were visualized by gel electrophoresis.

Proliferation studies

Cells were serum and cytokine deprived for 2 to 4 h in RPMI 1640, 0.1% BSA, and 2 mM glutamine. The cells were then analyzed for DNA synthesis and long-term growth in response to G-CSF. For DNA synthesis experiments, the cells were washed once in PBS and resuspended at 1 x 105/ml in RPMI 1640 containing 10% FBS and 2 mM glutamine. A total of 5 x 103 cells/well were seeded in 96-well microtiter plates with varying concentrations of G-CSF (0.2–2000 pM). Duplicate plates were also set up in the presence of IL-3 without G-CSF. The plates were incubated for a total of 72 h at 37°C in 5% CO2, and pulsed with 0.5 µCi/well of [3H]TdR for the last 8 h of incubation. Samples were harvested onto glass fiber filters and counted in scintillation fluid. For long-term proliferation studies, the cells were serum and cytokine deprived as described above. The cells were then washed once in PBS and resuspended at 5 x 104/ml in 5 ml of RPMI 1640 media containing 10% FBS, 2 mM glutamine, and 1.9 ng/ml of G-CSF. Cell growth was assayed over a 4-day period. For each clone, viable cells and total cell number were determined daily by trypan blue exclusion. Media was added to the flasks to maintain the cell density of each clone at 5 x 104/ml when the cell density exceeded 5 x 105/ml.

Analysis of apoptosis

The cells were washed twice in PBS, resuspended at 3 x 105/ml in RPMI 1640 media containing 10% FBS and 2 mM glutamine, and then incubated for 6 h in the presence of 10% WEHI-3-CM or 1.9 ng/ml G-CSF. Low m.w. chromosomal (LMWC) DNA was isolated as described by Kinoshita et al. (15). Briefly, the cells were lysed with 0.2% TX-100 (Boehringer Mannheim Biochemical), 10 mM EDTA, and 10 mM Tris (pH 7.5) for 10 min at 4°C, and spun at 10,000 x g for 10 min. The supernatants containing the LMWC DNA were purified by phenol:chloroform extraction and precipitated with ethanol. Intact high m.w. chromosomal (HMWC) DNA was isolated from the cell pellets by overnight incubation at 37°C in 10 mM Tris (pH 8.5), 1 mM EDTA, 1% SDS (Boehringer Mannheim Biochemical), and 15 µg/ml proteinase K (16). Both HMWC and LMWC DNA were treated with 0.3 mg/ml of RNase A for 30 min at 37°C, ethanol precipitated, and visualized on 1.5% agarose gels. The effect of the specific PI3-kinase inhibitor wortmannin on apoptosis was also assayed as described by Yao and Cooper (3) using a concentration of 200 nM wortmannin.

Immunoprecipitations and Western blot analyses

Cells were serum and cytokine deprived for 4 h in RPMI 1640 media containing 0.1% BSA and 2 mM glutamine, and during the last hour the cells were incubated with 10 µM sodium vanadate. The cells were then stimulated with 100 ng/ml of G-CSF or 20 ng/ml of IL-3 for 5 min, washed in PBS containing 1 mM sodium vanadate, and lysed in buffer containing 1% Nonidet P-40 (Boehringer Mannheim Biochemical), 1 mM EDTA (pH 8.0), 20 mM Tris (pH 8.0), 150 mM NaCl, 0.15 U/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 1 mM sodium vanadate, then cleared of insoluble material. Protein determinations of whole cell lysates were performed using the BCA assay (Pierce, Rockford, IL). For each sample, 1 mg of protein was precleared with protein A-Sepharose (Pharmacia, Piscataway, NJ) before immunoprecipitation with the indicated Abs. Western blot analyses were performed as previously described (17) with the exception of 5% nonfat dried milk for blocking, and analyzed by enhanced chemiluminescence per the manufacturer’s instructions (Amersham).

PI3-kinase assay

PI3-kinase assays were performed as previously described (18, 19). Before use, the phosphatidylinositol substrate was vacuum dried and suspended by sonication in 30 mM HEPES, pH 7.4. PI3-kinase immunoprecipitates were resuspended in assay buffer containing 30 mM HEPES (pH 7.4), 30 mM MgCl2, and 50 µM ATP. To each sample, a final concentration of 10 µg of phosphatidylinositol and 10 µCi of [32P]ATP was added. The reaction was incubated at room temperature for 20 min. The phospholipids were extracted from the reaction with a 1:1 solution of CH3OH:CHCl3 and resolved on CDTA-treated Silica Gel 60 TLC plates (E.M. Separations Technology, Gibbstown, NJ). The migration of PI(3)P corresponded to an Rf of 0.54.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of receptor expression in stable G-CSFR transfectants

To further investigate proliferative signaling through the G-CSFR, IL-3-dependent Ba/F3 cells were stably transfected with the class I and class IV isoforms as well as several G-CSFR truncation mutants. The class I G-CSFR (25-1 or wild type) has a cytoplasmic domain of 183 amino acids. The class IV G-CSFR (D7) has a 130-amino acid cytoplasmic tail in which the 87 carboxyl-terminal residues of the class I form are replaced by a distinct region of 34 residues (14). Both the class I and class IV isoforms have previously been shown to transduce mitogenic signals (20, 21).

As shown in Figure 1Go, all of the G-CSFR forms examined retain the conserved box 1 and 2 sequences that have been shown to be required for mitogenic signaling through the G-CSFR. The cytoplasmic tails in both the class I and class IV G-CSFR isoforms contain the identical membrane proximal 96 amino acids, and subsequently diverge. The class I G-CSFR also contains a conserved box 3 region that is shared with gp130 and four tyrosine residues at 704, 729, 744, and 764 (1). The distal carboxyl-terminal tail of the class I isoform that is absent in the other G-CSFR forms has been shown to mediate maturation signals (21). The class IV G-CSFR lacks box 3 as well as the tyrosine residues at amino acids 729, 744, and 764, and contains a unique tyrosine residue at amino acid 734 in its distinct carboxyl terminus. The G-CSFR{Delta}715 truncation mutant, which was initially described by Dong et al. (22) and isolated from a patient with severe congenital neutropenia, contains a C to T substitution at nucleotide 2379 (Larsen numbering) that introduces a premature stop codon resulting in deletion of the 98 carboxyl-terminal residues. G-CSFR{Delta}715 retains only the most proximal tyrosine at residue 704. The G-CSFR{Delta}682 truncation mutant terminates at residue 682 and lacks all four cytoplasmic tyrosine residues.



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  		FIGURE 1. Schematic diagram of G-CSFR isoforms and truncation mutants. The extracellular domain (ED), transmembrane domain (TM), and intracellular domain (ID) are indicated for the class I and class IV G-CSFR isoforms as well as the truncation mutants G-CSFR{Delta}715 and G-CSFR{Delta}682. The length of the intracellular domain is indicated by the number in parentheses at the bottom right. The locations of the cytoplasmic tyrosine (Y) residues at positions 704, 729, 744, and 764 are indicated. The dotted line represents the alternative carboxyl tail in the class IV G-CSFR within which lies a unique tyrosine residue at position 734.

 
Receptor binding assays were performed on single clones and pools of three G-CSFR-positive subclones for each receptor form. Scatchard analyses indicated that all of the clones had similar binding affinities and receptor numbers as shown in Table IGo. To further confirm stable expression of the appropriate G-CSFR forms in each of the clones, RT-PCR was performed. The F21 and R22 primer set was used for amplification of the class I and IV G-CSFR forms, and produced fragments of 794 bp and 374 bp, respectively (Fig. 2GoA). As expected, RT-PCR with G-CSFR{Delta}715 that is identical to the class I G-CSFR except for a single point mutation at nucleotide 2379 resulted in amplification of a 794-bp fragment using primers F21 and R22 (Fig. 2GoA). Since the R22 primer corresponds to a region that is deleted in G-CSFR{Delta}682, no product was obtained. For PCR amplification of the G-CSFR{Delta}682 form, primer set F21/R1 was used and resulted in the amplification of a 151-bp product. The F21/R1 primer pair also amplified a 151-bp product from class IV G-CSFR transfectants (Fig. 2GoB). The integrity of the cDNA from each clone was confirmed by amplification using primers corresponding to GADPH (Fig. 2GoC). All samples lacking reverse transcriptase were found to be negative for genomic DNA contamination (data not shown).


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  		Table I. Binding properties of G-CSFR clones

 


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  		FIGURE 2. Confirmation of G-CSFR expression by RT-PCR. A, The primer set F21/R22 was used to amplify cDNA from class I, class IV, G-CSFR{Delta}715, and G-CSFR{Delta}682 transfectants. Note that an amplified product is only visualized for class I and IV and G-CSFR{Delta}715 transfectants as expected with the primers used. B, PCR amplification of cDNA from G-CSFR{Delta}682 and class IV clones was done using primers F21/R1. C, As a positive control, GADPH was amplified from each G-CSFR clone.

 
Proliferative signaling capacity of G-CSFR transfectants

We next examined the mitogenic response of each G-CSFR form to G-CSF. As shown in Figure 3GoA, DNA synthesis was found to be maximal in class IV transfectants, whereas class I and G-CSFR{Delta}715 transfectants repeatedly exhibited a 50-fold lower response to G-CSF (Fig. 3GoA). An even more marked reduction in DNA synthesis was consistently observed in G-CSFR{Delta}682 transfectants, even at concentrations of 200 pM G-CSF. As expected, Ba/F3 cells transfected with vector alone (CDM8) failed to proliferate in response to G-CSF.



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  		FIGURE 3. Proliferative responses of G-CSFR transfectants. Cells were serum and cytokine deprived for 4 h before cytokine stimulation. A, Analysis of DNA synthesis. The data are expressed as the percent [3H]thymidine uptake in cells grown in G-CSF vs IL-3. B, Long-term growth assays. G-CSFR transfectants were grown in 1.9 ng/ml of G-CSF for 4 days. At the indicated time points, the number of total viable cells was determined by trypan blue exclusion.

 
We further investigated the correlation between G-CSF-induced DNA synthesis and long-term growth of the transfectants. As shown in Figure 3GoB, although class I, class IV, and G-CSFR{Delta}715 transfectants could proliferate in 1.9 ng/ml of G-CSF, maximal growth responses were observed in class IV transfectants. Notably, class I, class IV, and G-CSFR{Delta}715 transfectants could proliferate in 1.9 ng/ml of G-CSF for periods exceeding 1 month, during which time the growth of class IV transfectants was consistently higher. G-CSFR{Delta}682 clones showed zero to minimal proliferation in G-CSF-containing media beyond 3 days. Thus, the observed differences in DNA synthesis and cell proliferation observed between G-CSFR{Delta}682 and G-CSFR{Delta}715 transfectants suggested the presence of a mitogenic-enhancing subdomain spanning residues 682 to 715 of the G-CSFR.

Identification of an anti-apoptotic domain in the G-CSFR

To determine whether the decreased level of G-CSF-induced proliferation observed in G-CSFR{Delta}682 clones was due to accelerated programmed cell death, LMWC DNA was isolated and examined for the presence of an apoptotic ladder. Figure 4Go shows that growth factor deprivation for 6 h resulted in apoptosis of all the transfectants. The addition of G-CSF, however, inhibited apoptosis in class I, class IV, and G-CSFR{Delta}715 transfectants, but not in G-CSFR{Delta}682 transfectants. Since Ba/F3 cells are IL-3 dependent, incubation with media containing 10% WEHI-3-CM inhibited apoptosis in all of the clones including G-CSFR{Delta}682 transfectants. In cells transfected with vector alone (CDM8), an expected ladder was seen in the presence of G-CSF and in the absence of added growth factor, but not in cells grown in 10% WEHI-3-CM (data not shown). The presence of intact HMWC DNA in G-CSF-treated class I, class IV, and G-CSFR{Delta}715 transfectants in which an apoptotic ladder was not evident was confirmed by agarose gel electrophoresis (data not shown).



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  		FIGURE 4. DNA fragmentation of extracts from G-CSFR transfectants. LWMC DNA was isolated from class I, class IV, G-CSFR{Delta}715, G-CSFR{Delta}682, and vector alone (CDM8) transfectants. Samples were cultured for 6 h in media containing 10% FBS and no added cytokine (0), 1.9 ng/ml of G-CSF (G), or 10% WEHI-3-CM (W). LMWC DNA was visualized on a 1.5% agarose gel stained with ethidium bromide. DNA markers are shown to the left of each gel.

 
The anti-apoptotic domain in the G-CSFR mediates PI3-kinase activation

Since activation of PI3-kinase has been implicated in preventing apoptosis in several growth factor receptor systems (3), we were interested in investigating whether the anti-apoptotic domain we identified in the G-CSFR might also mediate activation of PI3-kinase to account for the inability of G-CSF to inhibit apoptosis in G-CSFR{Delta}682 transfectants. PI3-kinase was immunoprecipitated from samples stimulated for 5 min with 100 ng/ml of G-CSF. Western blot analysis of the immunoprecipitates with the 4G10 anti-phosphotyrosine Ab showed that G-CSF induces tyrosine phosphorylation of PI3-kinase in all of the transfectants except G-CSFR{Delta}682 (Fig. 5GoA). Although a very faint tyrosine-phosphorylated p85 band is seen in the lane from G-CSF-stimulated G-CSFR{Delta}682 transfectants in Figure 5GoA, this was not reproducible in multiple independent experiments. However, IL-3 stimulation of G-CSFR{Delta}682 transfectants consistently induced rapid tyrosine phosphorylation of PI3-kinase. Confirmation of equal protein loading was done by stripping the blot and reblotting with anti-PI3-kinase (Fig. 5GoB).



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  		FIGURE 5. Tyrosine phosphorylation and activation of PI3-kinase in G-CSFR transfectants. A, Tyrosine phosphorylation of PI3-kinase. Lysates from unstimulated (0) and G-CSF stimulated (+) cells were immunoprecipitated with anti-PI3-kinase overnight and resolved on a 10% acrylamide gel (Novex, San Diego, CA). Immunoblotting with the anti-phosphotyrosine Ab 4G10 was performed as described. The arrow on the right indicates the tyrosine-phosphorylated PI3-kinase band. B, Equal loading of protein was confirmed by reblotting with anti-PI3-kinase. C, Measurement of PI3-kinase activity. PI3-kinase activity in unstimulated (0) and G-CSF-stimulated (+) G-CSFR transfectants was determined by the formation of PI(3)P. Wortmannin (Wort) and normal rabbit serum (NRS) were used as controls. Arrow indicates migration of PI(3)P. D, Calculation of PI3-kinase activation by densitometry. The data are expressed as the percent increase in PI3-kinase activity in cytokine-stimulated samples vs unstimulated samples from the average of two independent experiments.

 
Since several investigators have suggested that tyrosine and/or serine/threonine phosphorylation are necessary for PI3-kinase activation (19, 23, 24, 25), we examined activation of the enzymatic activity of PI3-kinase in our G-CSFR clones by in vitro kinase assays. Aliquots from the same PI3-kinase immunoprecipitates used for Western blot analysis were analyzed in in vitro kinase assays. As shown in Figure 5GoC, activation of PI3-kinase could be detected in all of the clones treated with G-CSF except G-CSFR{Delta}682. Similar to the results from Western blotting, IL-3 stimulation resulted in activation of PI3-kinase activity in G-CSFR{Delta}682 clones. The effect of G-CSF on activation of PI3-kinase appeared to be specific, since the addition of the specific PI3-kinase inhibitor, wortmannin, abolished the effect of G-CSF on PI3-kinase activation (data not shown). Wortmannin also inhibited IL-3-stimulated PI3-kinase activation in G-CSFR{Delta}682 transfectants. As shown in Figure 5Go, C and D, incubation of G-CSFR{Delta}682 transfectants in IL-3 and 200 nM wortmannin leads to near complete (99% by densitometry) inhibition of PI3-kinase activity.

We further investigated the role of PI3-kinase activation in G-CSFR-mediated inhibition of apoptosis. As shown in Figure 6Go, the anti-apoptotic effect of G-CSF was nearly abolished in G-CSFR{Delta}715 transfectants when the cells were also incubated with 200 nM wortmannin for 6 h. Wortmannin also inhibited the anti-apoptotic effect of WEHI-3-CM in G-CSFR{Delta}715 clones. Similar results were also obtained with class I and class IV clones (data not shown). These results suggest that activation of PI3-kinase by the G-CSFR is required for inhibition of apoptosis.



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  		FIGURE 6. Specificity of PI3-kinase activation in prevention of apoptosis by G-CSF. LMWC DNA was isolated from G-CSFR{Delta}715 transfectant cells that were incubated for 6 h in media containing no added growth factors (0), 1.9 ng/ml of G-CSF (G), or 10% WEHI-3-CM (W) in the absence or presence of 200 nM wortmannin.

 
G-CSF induces tyrosine phosphorylation of SHIP in class I transfectants

The novel 145-kDa 5' inositol phosphatase, SHIP, has recently been reported to be tyrosine phosphorylated in hematopoietic cells following treatment with IL-3, granulocyte-macrophage CSF, and macrophage CSF (6, 7, 8). It has been suggested that SHIP functions as a negative regulator of cell growth through degradation of PI3-kinase metabolites (6, 9). We were therefore interested in examining whether SHIP was differentially recruited by class I and class IV transfectants to explain the observed differences in their proliferative responses to G-CSF.

SHIP was immunoprecipitated from class I and class IV G-CSFR clones before and after stimulation with 100 ng/ml of G-CSF for 5 min and the immunoprecipitates were immunoblotted with the 4G10 anti-phosphotyrosine Ab. As shown in Figure 7GoA, G-CSF rapidly induced tyrosine phosphorylation of SHIP in class I transfectants. A very faint band was seen in G-CSF-treated class IV transfectants. Reblotting the same blot with anti-SHIP Ab confirmed equal loading of SHIP in all lanes (Fig. 7GoB). Notably, a tyrosine-phosphorylated 52-kDa band was consistently observed in SHIP immunoprecipitates from G-CSF-stimulated class I clones but not in G-CSF-stimulated class IV clones (Fig. 7GoA).



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  		FIGURE 7. Tyrosine phosphorylation of SHIP in G-CSFR transfectants. A, Lysates from unstimulated (0) and G-CSF stimulated (+) class I and class IV clones were immunoprecipitated with anti-SHIP Ab, and then immunoblotted with the 4G10 Ab. Arrows indicate the migration of tyrosine-phosphorylated SHIP (top) and a tyrosine-phosphorylated 52-kDa species that coprecipitates with SHIP (bottom). The broad lower band at the bottom is the Ig heavy chain. B, Equal loading of SHIP in A was confirmed by stripping and reblotting with anti-SHIP Ab.

 
Since SHIP has previously been reported to associate with Shc (6, 7, 9, 11, 26), we further investigated whether the 52-kDa band that associated with tyrosine-phosphorylated SHIP from G-CSF-treated class I immunoprecipitates was Shc. Reblotting SHIP immunoprecipitates with Ab to Shc resulted in the appearance of a broad IgG band in the 52-kDa region due to cross-reactivity of both rabbit Abs (data not shown). However, when the converse experiment was performed, tyrosine phosphorylation of Shc was only observed in G-CSF-stimulated class I transfectants (Fig. 8GoA), identifying the 52-kDa species we previously observed as Shc. An additional 145-kDa tyrosine-phosphorylated band corresponding to the migration of SHIP was also observed only in the G-CSF-stimulated class I transfectants. Reblotting Shc immunoprecipitates with Ab to SHIP confirmed the identity of the 145-kDa band in G-CSF-stimulated class I clones as SHIP (Fig. 8GoC). Thus, tyrosine-phosphorylated SHIP coimmunoprecipitates with tyrosine-phosphorylated Shc in class I transfectants but not in class IV transfectants following G-CSF stimulation.



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  		FIGURE 8. Association of SHIP with tyrosine-phosphorylated Shc in G-CSFR transfectants. A, Shc immunoprecipitates from unstimulated (0) or G-CSF-stimulated (+) class I and class IV transfectants were blotted with the 4G10 Ab. Arrows indicate the migration of tyrosine-phosphorylated Shc (bottom) and a 145-kDa species. B, Equal loading of Shc was confirmed by stripping the blot in A and reblotting with anti-Shc. C, The blot in A was stripped and reblotted with anti-SHIP Ab.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Significant advances have been made in understanding the molecular mechanisms used by various cell surface receptors to transduce mitogenic signals. Enzymes such as Ras, protein kinase C, and PI3-kinase have been shown to be positive effectors of mitogenic signaling by several tyrosine kinase receptors. More recently, studies have focused on the identification of negative or anti-proliferative signaling molecules. The tyrosine phosphatases SHP-1 and -2 and the inositol 5'-phosphatase SHIP have been reported to function as growth inhibitors for receptors that regulate immune responses (10). To date, little is known about the mechanisms regulating mitogenic signaling by cytokine receptors, particularly the G-CSFR. Although activation of the Jak-Stat and Ras-MAPK cascades have been shown to correlate with G-CSFR-mediated proliferative responses, the contribution by other signaling molecules to positively and negatively modulate this response has not been elucidated (1).

In the current study, we have identified a domain in the G-CSFR spanning residues 682 to 715 that mediates activation of PI3-kinase. This domain maps to the same region in the G-CSFR previously identified by us and others as a mitogenic-enhancing subdomain (12, 21). Activation of PI3-kinase through the G-CSFR in Ba/F3 transfectants correlated with inhibition of apoptosis and enhanced proliferation.

The requirement for activation of PI3-kinase for prevention of apoptosis was shown to be specific since treatment with wortmannin at a concentration that inhibited PI3-kinase activation nearly completely abolished the anti-apoptotic effect of G-CSF in cells transfected with G-CSFR forms containing an intact PI3-kinase activation domain. A role for PI3-kinase in the prevention of apoptosis has previously been reported. Yao and Cooper showed that nerve growth factor and platelet-derived growth factor both activated PI3-kinase to prevent apoptosis in PC-12 pheochromocytoma cells (3).

We observed activation of PI3-kinase in class I, class IV, and G-CSFR{Delta}715 transfectants but not in G-CSFR{Delta}682 transfectants that lack the tyrosine residue at position 704 that is present in the other G-CSFR forms. Once phosphorylated, Tyr704 in the G-CSFR could provide a potential binding site for SH2-containing proteins. We have not addressed the precise mechanism by which PI3-kinase is recruited to the G-CSFR signaling cascade. It is possible that PI3-kinase either binds directly to the G-CSFR via Tyr704 or that PI3-kinase is recruited to the G-CSFR signaling cascade via an intermediate molecule that may interact with Tyr704 in the G-CSFR. We have been unable to directly examine whether PI3-kinase directly binds to the G-CSFR in G-CSFR mutants containing single tyrosine->phenylalanine substitutions at each of the four tyrosine residues in the class I G-CSFR due to the lack of available high affinity Abs to the G-CSFR that work well for immunoprecipitation and immunoblotting. Future studies with biotinylated phosphopeptides corresponding to the regions flanking the cytoplasmic tyrosine residues in the G-CSFR should help to further address this.

PI3-kinase is a heterodimeric protein that consists of a p85 adapter subunit containing two SH2 domains and a p110 catalytic subunit. The p85 subunit can interact with phosphotyrosine residues on growth factor receptors through its SH2 domains to recruit the p110 subunit to the receptor complex. Recruitment of p110 leads to phosphorylation of phosphatidylinositols PI(4)P and PI(4, 5)P2 at the 3' position within the inositol ring (23, 27, 28, 29). A consensus binding motif for the SH2 domains of the p85 subunit of PI3-kinase corresponding to the sequence Tyr-Met/Val-X-Met has previously been reported (27, 28).

Examination of the residues at the +1, +2, and +3 positions from Tyr704 of the G-CSFR indicate the sequence Tyr-Val-Leu-Gln. Although the valine at position +1 and leucine at position +2 fit the optimal sequences predicted for binding of PI3-kinase via either its N-terminal or C-terminal SH2 domains, the presence of glutamine at position +3 instead of the highly preferred methionine at this position (27, 28) argues against recruitment of PI3-kinase via direct binding to the G-CSFR. More recently, a new recognition motif for binding of PI3-kinase was reported for the erythropoietin receptor (EPO-R) in which the residue at position +3 was found to be cysteine instead of methionine (29). Thus, the mechanism for recruitment of p85 to the G-CSFR appears to be indirect via an adapter protein that encodes a high affinity p85 binding site. Potential candidate adaptor proteins include Vav, Stat3, and Tec, which have recently been shown to link some of the cytokine receptors to PI3-kinase (30, 31, 32), and have also been reported to undergo tyrosine phosphorylation in response to G-CSF (33, 34, 35, 36). Irrespective of the mechanism for recruitment of PI3-kinase, our results indicate that PI3-kinase positively regulates growth signaling through the G-CSFR.

We have also identified a region in the distal tail of the class I G-CSFR isoform that negatively regulates growth signaling. In repeated experiments, we consistently observed greater G-CSF-induced proliferative signaling in class IV transfectants compared with class I transfectants. Given our observation that PI3-kinase positively regulates G-CSF-mediated mitogenic signaling, we were interested in determining whether potential phosphatases might be recruited to the carboxyl-terminal region of the class I G-CSFR to down-modulate proliferative signaling. The tyrosine phosphatase Syp (SHP-2) was previously reported to associate with Grb2 via the distal tail of the G-CSFR (37). Another candidate phosphatase was the novel 145-kDa 5' inositol phosphatase, SHIP (SH2 inositol phosphatase) that can dephosphorylate the active PI3-kinase metabolite phosphatidylinositol (3, 4, 5)-P3 at the 5' position (7, 8). SHIP has been shown to function as a negative regulator of mitogenic signaling through the M-CSFR (Fms receptor) and Fc{gamma}RIIB (6, 9, 10, 11). More recently, overexpression of SHIP in an IL-3-dependent cell line was reported to decrease cell viability by induction of apoptosis (38). Thus, since G-CSF was shown to promote cell growth by activation of PI3-kinase and inhibition of apoptosis, we postulated that SHIP might also down-regulate mitogenic signaling through its ability to hydrolyze the primary in vivo product of PI3-kinase, PI(3, 4, 5)P3.

Strong induction of tyrosine phosphorylation of SHIP was consistently observed only in class I transfectants stimulated with G-CSF. Tyrosine-phosphorylated Shc was found to coprecipitate with SHIP following G-CSF stimulation in class I but not class IV transfectants, consistent with previous reports that SHIP forms signaling complexes with Shc (6, 7, 9, 11, 26). The appearance of tyrosine-phosphorylated Shc only in class I transfectants is consistent with a previous report that Shc is uniquely recruited to the G-CSFR signaling cascade by Tyr764, which is only present in the class I G-CSFR. Shc was also shown to form complexes with p145 in class I transfectants following G-CSF stimulation (37). We have now identified the p145 species in the Shc complexes as SHIP.

The enhanced proliferative signaling capacity we observed in Ba/F3 cells transfected with the class IV G-CSFR form compared with the class I isoform is in direct contrast to previously published data. Although Dong et al. reported down-modulation of mitogenic signaling by the distal tail of the class I G-CSFR in the two myeloid cell lines, L-GM and 32D, the same effect was not observed in Ba/F3 cells, which are a pro-B lymphoid cell line (21). Furthermore, Dong reported that growth signaling by the class IV receptor was significantly weaker in Ba/F3 cells than that transduced by the class I isoform, and suggested that the hydrophobic residues present in the class IV G-CSFR might function to fold the alternative carboxyl tail toward the cell membrane and thereby hinder the association of growth-signaling molecules.

The reasons for the discrepancies in our results and those previously reported by Dong are not readily apparent. However, negative regulation of proliferative signaling by carboxyl-terminal sequences has been reported with the EPO-R transfected into Ba/F3 cells (39, 40). Notably, both the EPO-R and G-CSFR exist as single chain molecules that homodimerize to transduce signals. In the case of the EPO-R, recruitment of SHP-1 (also called HCP) was shown to play a role in termination of proliferative signals by inactivation of Jak 2 (41). Additionally, erythropoietin has been reported to induce association of SHIP with Shc. Signaling events mediated by the carboxyl-terminal portion of the EPO-R have been shown to contribute to maximal tyrosine phosphorylation of Shc (7, 42, 43). Our results suggest that similar growth inhibitory molecules may be recruited by the carboxyl-terminal sequences of the G-CSFR to down-regulate mitogenic signaling. Since aberrant G-CSFR isoform expression has been postulated to play a role in disorders of myelopoiesis (1, 44), the enhanced proliferative signaling capacity of the maturation-defective class IV isoform may have important clinical relevance.

Previous studies by Wang, Paul, and Keegan have also demonstrated the presence of distinct regions in the cytoplasmic tail of the IL-4 receptor that appear to positively and negatively regulate cell growth (45). A role for cytoplasmic tyrosine residues present in the IL-4 receptor in the promotion of negative signaling was suggested, although the mechanism of negative regulation was not elucidated.

Taken together, our data support a model in which distinct regions of the G-CSFR positively and negatively regulate mitogenic signaling (Fig. 9Go). The membrane proximal 53 amino acids containing the conserved box 1 and 2 regions are absolutely required for proliferative signaling and activation of Jak kinases, as previously reported by us and other investigators (35, 46, 47, 48). The immediate downstream sequence of 33 amino acids enhances mitogenic signaling by prevention of apoptosis and activation of PI3-kinase. Recruitment of PI3-kinase to the G-CSFR signaling cascade likely occurs via Tyr704 through an indirect mechanism involving another signaling intermediate. Shc binds to Tyr764 in the distal tail of the class I G-CSFR, as previously reported (37), and recruits SHIP as indicated by our data. Activation of SHIP could lead to degradation of the substrates generated from PI3-kinase activation and a resultant down-regulation in proliferative signaling. The absence of the critical tyrosine residue at position 764 that appears to be required for Shc/SHIP recruitment would explain the stronger proliferative signaling we observed in class IV transfectants. In preliminary experiments with G-CSFR mutants containing tyrosine->phenylalanine substitutions at each cytoplasmic tyrosine residue, we have observed increased growth in mutants harboring a substitution at Tyr764, further supporting a negative regulatory role for SHIP. Final confirmation of the role of SHIP in G-CSFR-mediated growth signaling will require additional studies that are currently underway to examine the 5'-phosphatase enzymatic activity of SHIP in unstimulated and G-CSF-stimulated cells.



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  		FIGURE 9. Proposed model for regulation of proliferative signaling through the G-CSFR. The membrane proximal region encompassing boxes 1 and 2 is absolutely required for mitogenic signaling and activation of Jak kinases. PI3-kinase is recruited via Tyr704 (Y 704), which lies in the immediate downstream region and its activation enhances mitogenic signaling. Shc binds to Y (764) in the distal tail and recruits SHIP, a negative regulator of cell growth.

 


   Acknowledgments
 
We are grateful to Dr. Sunita Chaudhary and Jennifer Parker for their assistance in generation of the G-CSFR{Delta}715 mutant and SHIP/Shc immunoblotting, respectively. We thank Kerry Sibert and Roy Pitman for assistance in cell culture and proliferation analyses. We also thank Norma Haas for preparation of the manuscript.


   Footnotes
 
1 This work was supported by Grant CA75226 from the National Cancer Institute. Back

2 Address correspondence and reprint requests to Dr. Belinda R. Avalos, The Ohio State University, Division of Bone Marrow Transplantation, 303 East Doan Hall, 410 West Tenth Avenue, Columbus, OH 43210. E-mail address: Back

3 Abbreviations used in this paper: G-CSF, granulocyte colony-stimulating factor; SHIP, SH2-containing inositol phosphatase; PI3, phosphatidylinositol 3'; PI(3,4,5)P3, phosphatidylinositol (3,4,5) triphosphate; PI(3,4)P2, phosphatidylinositol (3,4) bisphosphate; WEHI-3-CM, WEHI-3 conditioned media; RT, reverse transcriptase; LMWC, low m.w. chromosomal; HMWC, high m.w. chromosomal; EPO-R, erythropoietin receptor. Back

Received for publication September 4, 1997. Accepted for publication January 26, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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