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*
The Molecular, Cellular and Developmental Biology Program, and
Division of Bone Marrow Transplantation, The Ohio State University, Arthur G. James Cancer Hospital and Research Institute, Columbus, OH 43210
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
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-CSFR715 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 1
, 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-CSFR715 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-CSFR715 retains only the most proximal tyrosine at
residue 704. The G-CSFR682 truncation mutant terminates at residue
682 and lacks all four cytoplasmic tyrosine residues.
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. 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. 2A). As expected,
RT-PCR with G-CSFR715 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. 2A). Since the R22 primer corresponds to a region
that is deleted in G-CSFR682, no product was obtained. For PCR
amplification of the G-CSFR682 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. 2B). The integrity of the cDNA from each clone
was confirmed by amplification using primers corresponding to GADPH
(Fig. 2C). All samples lacking reverse transcriptase
were found to be negative for genomic DNA contamination (data not
shown).
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We next examined the mitogenic response of each G-CSFR form to
G-CSF. As shown in Figure 3A,
DNA synthesis was found to be maximal in class IV transfectants,
whereas class I and G-CSFR715 transfectants repeatedly exhibited a
50-fold lower response to G-CSF (Fig. 3A). An even
more marked reduction in DNA synthesis was consistently observed in
G-CSFR682 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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B, although class I, class IV, and G-CSFR715
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-CSFR715 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-CSFR682 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-CSFR682 and G-CSFR715
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-CSFR682 clones was due to accelerated
programmed cell death, LMWC DNA was isolated and examined for the
presence of an apoptotic ladder. Figure 4
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-CSFR715
transfectants, but not in G-CSFR682 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-CSFR682
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-CSFR715 transfectants in which an apoptotic ladder was not
evident was confirmed by agarose gel electrophoresis (data not
shown).
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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-CSFR682
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-CSFR682 (Fig. 5A). Although a very
faint tyrosine-phosphorylated p85 band is seen in the lane from
G-CSF-stimulated G-CSFR682 transfectants in Figure 5A,
this was not reproducible in multiple independent experiments. However,
IL-3 stimulation of G-CSFR682 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. 5B).
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C, activation of PI3-kinase could be detected in all
of the clones treated with G-CSF except G-CSFR682. Similar to the
results from Western blotting, IL-3 stimulation resulted in activation
of PI3-kinase activity in G-CSFR682 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-CSFR682
transfectants. As shown in Figure 5, C and D,
incubation of G-CSFR682 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 6, the anti-apoptotic effect of
G-CSF was nearly abolished in G-CSFR715 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-CSFR715
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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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 7A, 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. 7B). 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. 7A).
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A), 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. 8C). 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-CSFR715 transfectants but not in G-CSFR682 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
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. 9). 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 tyrosinephenylalanine 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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|
Acknowledgments |
|---|
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 |
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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:
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.
Received for publication September 4, 1997. Accepted for publication January 26, 1998.
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References |
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