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(Received for publication, April 28, 1995; and in revised form, June 28, 1995) From the
The cDNA encoding the rat equivalent of the human hematopoietic
tyrosine phosphatase, also known as leukocyte phosphatase, was isolated
from a rat basophilic leukemia mast cell cDNA library. By
two-dimensional electrophoresis, the protein expressed in the mast
cells was of a size (40 kDa) and pI (6.9) predicted from the deduced
amino acid sequence. Thus, although previously shown to be
preferentially expressed in T cells and B cells, the phosphatase is
also found in mast cells. By immunofluorescence microscopy, rat
hematopoietic tyrosine phosphatase localized to discrete, globular
compartments within the cytoplasm and was not found either in the
nucleus or associated with the cell surface membrane. Aggregation of
high affinity IgE receptors in the mast cells induced tyrosine
phosphorylation of the phosphatase. The tyrosine phosphorylation was
mimicked by stimulation with calcium ionophore A23187 but not by direct
activation of protein kinase C. Since phosphorylation of the
phosphatase was dramatically reduced when the cells were activated in
Ca
Mast cells and basophils play a central role in allergic and
inflammatory reactions. They express high affinity IgE receptors
(Fc Regulation of the level of tyrosine phosphorylation of proteins
through dephosphorylation is an important aspect of the signal
transduction process. For example, the protein-tyrosine phosphatase
(PTP) CD45 is essential for signaling from receptors on T cells, B
cells, and mast
cells(22, 23, 24, 25, 26) .
However, we found that CD45 is absent from several RBL-2H3 cell
variants that have normal signaling through their Fc
The 5`-end was generated using the 5` rapid
amplification of cDNA ends system of Life Technologies, Inc.
Poly(A)
The whole cDNA sequence obtained contains a single open
reading frame encoding a putative protein 359 amino acids in length (Fig. 1). The presumptive initiation codon (nucleotides
107-109) is surrounded by a consensus Kozak sequence and is
preceded by stop codons in all three reading frames. The rat and human
sequences (27, 28) share 77 and 91% identity at the
nucleotide and amino acid levels, respectively. By Northern blotting,
rat HePTP was found to be restricted in its distribution to T cells and
RBL-2H3 cells (Fig. 2).
Figure 1:
Nucleotide and predicted amino acid
sequence of rat HePTP cDNA. The nucleotide sequence is numbered on the right. The predicted amino acid sequence of the coding region
is in single letter codes above the nucleotide
sequence.
Figure 2:
Northern blot analysis of HePTP mRNA
expression in cultured cell lines and rat tissues. Northern blotting of
30 µg/lane total RNA was with a 180-base pair fragment (nucleotides
227-406) from the 5`-noncatalytic domain of the coding sequence.
Samples were derived from: YAC-1 mouse T cell line (1),
RBL-2H3 cells (2), hindbrain (3), olfactory bulb (4), cerebellum (5), frontal lobe (6), liver (7), thymus (8), spleen (9), kidney (10), heart (11), testis (12), lung (13).
Figure 3:
One- and two-dimensional analysis of HePTP
in RBL-2H3 mast cells. Cell lysates were resolved either by one- (leftsinglelane) or by two-dimensional
analysis, electrotransferred to nitrocellulose membranes and blotted
with biotinylated anti-HePTP antibodies and horseradish
peroxidase-conjugated streptavidin (SA-HRP). The
proteins at the top of the blot were recognized by horseradish
peroxidase-conjugated streptavidin in the absence of anti-HePTP
antibody (data not shown). In cells solubilized for two-dimensional
analysis in urea lysis buffer, the HePTP was identified as a doublet,
whereas by one-dimensional SDS-PAGE, it migrated as a single
band.
Figure 4:
Immunofluorescence localization of HePTP
in resting and activated RBL-2H3 mast cells. RBL-2H3 cells were grown
on glass coverslips in 6-well tissue culture dishes. Following methanol
permeabilization, the cells were incubated with 10 µg/ml normal
rabbit IgG (A and C) or affinity-purified rabbit
anti-rat HePTP IgG (B, D, and E).
Unstimulated RBL-2H3 cells (A and B) are
characteristically rounded with a bipolar shape. After activation (C-E), they spread out along the substratum and
take on a more fibroblast-like appearance. HePTP is distributed
throughout the cytoplasm, but it is not found either in the nucleus or
at the cell surface (B, D, and E). After
cell activation, HePTP localizes to globular-shaped subcellular
compartments. Original magnification of panelsA-D is 40
Figure 5:
Time course of Fc
Because rat HePTP became tyrosine-phosphorylated upon
Fc
Figure 6:
HePTP
is tyrosine-phosphorylated by calcium ionophore A23187 but not by
phorbol 12-myristate 13-acetate. RBL-2H3 cells were stimulated for 10
min with anti-receptor monoclonal antibody BC4 (BC4), phorbol
12-myristate 13-acetate (PMA), calcium ionophore A23187, or
buffer alone. Lysates were immunoprecipitated with normal rabbit IgG or
anti-rat HePTP antibodies and analyzed by immunoblotting with
anti-phosphotyrosine (upperpanel). The blots were
stripped and assayed for HePTP to confirm equivalent loading (lowerpanel). Each lane represents the precipitate
from 7.5
To more
fully explore the role of extracellular calcium in the Fc
Figure 7:
Importance of protein kinase C and
Ca
The cDNA we isolated is the rat equivalent of the human PTP
HePTP or LC-PTP(27, 28) . While the two human
sequences are nearly identical, they do differ in one major respect:
the location of the presumptive translation initiation codon. The cDNA
sequence of LC-PTP, which was confirmed by genomic cloning, suggests an
open reading frame that begins at nucleotide 105 (LC-PTP numbering). In
contrast, the cDNA sequence reported for human HePTP is lacking a
cytosine at nucleotide 115 (LC-PTP numbering), a position within the
putative coding sequence. The cDNA sequence that we present here
suggests that the translation initiation codon of rat HePTP is the same
as that of human LC-PTP. Thus, it may be that the cDNA library clone
from which the human HePTP sequence was obtained contained a deletion. By Northern blotting, human HePTP was detected in T cells and B
cells (27, 28) . In the present experiments, we also
found the rat equivalent of this PTP to be selectively expressed and
now extend the list of cell types to include RBL-2H3 mast cells. It is
interesting to note that T cells, B cells, and mast cells comprise a
limited set of cells that express on their surfaces multisubunit immune
response receptors(35) . Perhaps HePTP functions in a
receptor-dependent manner in each of these cell types. The
cytochemical distribution of rat HePTP is intriguing; it localizes to
globular or elongated cytoplasmic elements. This suggests that the
enzyme is compartmentalized to an organelle or to some subcellular
specializations. We speculate that the NH The signaling process initiated by Fc The rise in intracellular Ca The
requirement for calcium mobilization in the tyrosine phosphorylation of
HePTP indicates that it occurs late in the signaling
cascade(43) . That is, it is preceded by other events including
tyrosine phosphorylation and activation of phospholipase C- The SH2 domain-containing protein-tyrosine
phosphatase, Syp (also referred to as PTP1-D or SH-PTP2), was shown to
be tyrosine phosphorylated in response to epidermal growth factor and
platelet-derived growth factor receptor
activation(44, 45) . It was also constitutively
tryrosine-phosphorylated in cells transformed with v-Src(44) ,
suggesting that the Src family of kinases may be involved in
phosphorylating the PTP. Lyn is a Src family tyrosine kinase found in
abundance in RBL-2H3 cells; it coimmunoprecipitates with the Fc Because
protein-tyrosine phosphorylation is a prominent feature of signaling
through the IgE receptor in mast cells and basophils, protein-tyrosine
phosphatases must play an important regulatory role. We have identified
the rat equivalent of HePTP in RBL-2H3 cells, shown that it localizes
to a cytoplasmic compartment, that it becomes tyrosine phosphorylated
as a result of IgE receptor aggregation, and that this phosphorylation
is dependent on Ca
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
U28356[GenBank].
Volume 270,
Number 37,
Issue of September 15, pp. 21902-21906, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-free media, it is dependent on a rise in
intracellular Ca
. These data strongly suggest that
hematopoietic tyrosine phosphatase may be involved in the IgE
receptor-mediated signaling cascade.
RI) (
)on their cell surfaces that, when aggregated,
initiate biochemical events that lead to the release of inflammatory
mediators. In the rat basophilic leukemia (RBL-2H3) mast cell line,
aggregation of FcRI induces activation of phospholipases
A
, C, and D, an increase in intracellular Ca concentration, and activation and translocation of protein kinase
C from the cytosol to the plasma
membrane(1, 2, 3, 4, 5) .
In addition, numerous proteins become tyrosine phosphorylated following
receptor aggregation. These include the
and subunits of the
receptor, phospholipase C-
1, Vav, Nck, and paxillin;
protein-tyrosine kinases such as Lyn, Syk, Fak, and Btk; and other
unidentified
proteins(6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) .
RI. (
)Therefore, we used a molecular cloning approach to
identify other PTPs that may be involved. The rat equivalent of human
hematopoietic tyrosine phosphatase (HePTP), (
)also known as
leukocyte protein-tyrosine phosphatase (LC-PTP; (27) and (28) ) was isolated from an RBL-2H3 cell cDNA library. HePTP
mRNA was present only in RBL-2H3 mast cells, the YAC-1 T cell line, and
the thymus in Northern blots. Moreover, the protein became tyrosine
phosphorylated upon aggregation of the FcRI in RBL-2H3 cells. This
phosphorylation was Ca
-dependent and accordingly
would be considered a ``late'' event in the activation
process. Thus, HePTP may be involved in the signaling cascade initiated
by IgE receptor aggregation.
Molecular Cloning
Two degenerate primers were
designed based on common sequences in the catalytic domains of several
PTP
((His/Asp)-Phe-Trp-(Arg/Lys/Leu/Glu)-Met-(Val/Cys/Ile)-Trp-(Glu/Gly) to
Val-His-Cys-Ser-Ala-Gly-(Val/Ile)-Gly)(29) . Forward 5`-GC GAA
TTC (C/G)A(T/C) TT(C/T) TGG I(G/A/T)I ATG (G/A)TI TGG (G/C)A-3` and
reverse 5`-GC GAA TTC CCI A(T/C)I CCI GC(G/A) CT(G/A) TGI AC-3`
oligonucleotide primers were synthesized on an Applied Biosystems Inc.
392 DNA/RNA synthesizer. RBL-2H3 cell cDNA, prepared as described
previously (30) , served as template in PCR reactions. The PCR
products were subcloned into the EcoRI site of pBluescript SK
(Stratagene, La Jolla, CA), and the sequences of both strands were
determined by automated sequence analysis (Applied Biosystems Inc.,
Foster City, CA). The HePTP PCR product was used to screen a previously
characterized RBL-2H3 cell cDNA gt11 library(30) .
Positive plaques were purified, their inserts were subcloned in
pBluescript SK, and the sequence of both strands was determined by
automated sequencing.
RNA from RBL-2H3 cells and the PTP
gene-specific primer 5`-TAGAGTCCAGCGTGTA-3` corresponding to
nucleotides 367-352 in the rat PTP were used. The rapid
amplification of cDNA ends products were subcloned into pBluescript SK
and sequenced on both strands by automated sequence analysis.
Antibody Preparation
A rat PTP-specific peptide
comprising amino acids 121-134 (SKDRYKTILPNPQS) with a cysteine
residue at the carboxyl end was synthesized by Chiron Mimotopes
(Emeryville, CA). Antibodies were generated in rabbits as described
previously(15) . Some affinity-purified anti-PTP antibodies
were biotinylated using NHS-LC-biotin (Pierce) according to the
manufacturer's recommendations.Two-dimensional Gel Electrophoresis
Whole cell
lysates from both nonstimulated and activated RBL-2H3 cells were used
as described previously(20) . Blots were probed with 100 ng/ml
biotinylated anti-rat HePTP and 10 pg/ml horseradish
peroxidase-conjugated streptavidin (Pierce).Immunofluorescence Microscopy
Immunofluorescence
microscopy was as described previously (20) using 10 µg/ml
nonimmune rabbit IgG or rabbit anti-PTP (described above) and
fluorescein isothiocyanate-conjugated F(ab`) donkey
anti-rabbit IgG (Jackson ImmunoResearch).Cells and Cell Activation
RBL-2H3 cells were
maintained as monolayer cultures as described previously(31) .
For activation, 150-mm diameter Petri dishes were seeded with
1.2-2 
10
cells. After overnight culture, the
cell monolayers were washed twice with 30 ml of phosphate-buffered
saline at room temperature. The cells were then incubated at 37 °C
for 10 min unless otherwise indicated in 10 ml of Eagle's minimum
essential medium with Earle's salts containing 0.1% bovine serum
albumin, 10 mM Tris (pH 7.4), and 0.03 µg/ml
anti-FcRI monoclonal antibody BC4; 0.5 µM calcium
ionophore A23187; or 40 nM phorbol 12-myristate 13-acetate.
The supernatants were removed and assayed for histamine by automated
analysis(32) .
Immunoprecipitations
After activation, the
monolayers were washed once with 10 ml of ice-cold phosphate-buffered
saline containing protease inhibitors (concentrations as in lysis
buffer), solubilized in 1 ml of lysis buffer (3% Brij 96, 20 mM Tris, pH 7.4, 100 mM NaCl, 1 mM
NaVO
, 2 mM phenylmethylsulfonyl
fluoride, 90 milliunits/ml aprotinin). Postnuclear lysates were
precleared 1 h at 4 °C with protein A-agarose, and proteins were
immunoprecipitated at 4 °C for 1 h with 5 µg of affinity
purified anti-rat HePTP antibodies bound to protein A-agarose. The
beads were washed 7 times with ice-cold lysis buffer, and proteins were
eluted by boiling 5 min in SDS-PAGE sample buffer. Brij 96, protease
inhibitors, and protein A-agarose beads were from Sigma.Immunoblotting
Whole cell lysates and
immunoprecipitated proteins were separated by SDS-PAGE using
4-20% linear gradient gels (Novex) under reducing conditions.
Separated proteins were electrotransferred to polyvinylidene difluoride
membranes (Immobilon) and blocked by overnight incubation in 4%
protease-free bovine serum albumin (Intergen). For detection of
tyrosine-phosphorylated proteins, membranes were probed with 40 ng/ml
anti-phosphotyrosine mAb PY-20 coupled to horseradish peroxidase (ICN).
Signals were detected by chemiluminescence using a Renaissance kit
(DuPont NEN) and Kodak X-Omat radiographic film (Eastman Kodak Co.).
Membranes were then stripped of antibodies according to the protocol of
the Amersham ECL kit and reprobed for PTP with 100 ng/ml biotinylated
anti-rat HePTP IgG followed by 10 pg/ml horseradish
peroxidase-conjugated streptavidin (Pierce).
Isolation and Analysis of Rat HePTP cDNA
Part of
the catalytic domain of rat HePTP was amplified from RBL-2H3 cell cDNA
using reverse transcriptase-based PCR and degenerate oligonucleotide
primers. The amplified product was used to screen an RBL-2H3 cDNA
gt11 library. Out of 5
10
plaques surveyed, 5
hybridized to the probe. The 5 clones yielded similar sized cDNA, one
of which was completely sequenced on both strands. Because the 5`-end
(nucleotides 1-226) of the rat HePTP cDNA was missing, the
remaining four clones, the RBL-2H3 cell gt11 library, and a rat
spleen library were all screened by PCR using phage-specific and
HePTP-specific primers. However, no new products were found. Therefore,
the 5`-end was generated using the 5` rapid amplification of cDNA ends
technique.
Rat HePTP Protein Expression and Distribution in RBL-2H3
Mast Cells
To determine if RBL-2H3 cells expressed the HePTP
protein, cell lysates were separated by one- and two-dimensional
electrophoresis and analyzed by immunoblotting. The rat HePTP had a
molecular mass of 40 kDa and an isoelectric point of
6.9 (Fig. 3), in agreement with values predicted from the deduced
amino acid sequence. The deduced amino acid sequence also suggested
that the phosphatase was a cytosolic protein. Therefore, we examined
its subcellular localization by immunofluorescence microscopy (Fig. 4). In unstimulated RBL-2H3 cells, the protein was evenly
distributed throughout the cytoplasm, with little if any appearing at
the plasma membrane or in the nucleus (Fig. 4B).
However, after stimulation through the high affinity IgE receptors, the
cells spread (Fig. 4D) and it became apparent that the
phosphatase was localized to globular-shaped subcellular compartments (Fig. 4E). As the immunofluorescent globules were more
abundant than the secretory granules, it is unlikely that HePTP is
present in secretory granules.
, of panelE is 60.
Fc
A prominent feature of signaling in RBL-2H3 cells is the
phosphorylation of numerous proteins on tyrosine residues. To determine
if rat HePTP also became tyrosine-phosphorylated in IgE
receptor-activated cells, HePTP was immunoprecipitated from resting and
from stimulated cells, separated by SDS-PAGE, and immunoblotted with
anti-phosphotyrosine antibodies. As shown in Fig. 5, HePTP was
at best only weakly tyrosine phosphorylated in nonactivated cells. By
contrast, after FcRI Aggregation Induces Tyrosine Phosphorylation of
HePTP
RI aggregation, HePTP underwent time-dependent
tyrosine phosphorylation (Fig. 5). Phosphorylation was
detectable by 1 min, peaked at 5-10 min, and declined thereafter.
It did, however, remain detectable up to 60 min, the longest time point
tested.
RI-induced HePTP
tyrosine phosphorylation. Monolayer cultures of RBL-2H3 cells were
stimulated with 30 ng/ml anti-receptor monoclonal antibody BC4 at 37
°C for the indicated times. Lysates were immunoprecipitated with
anti-rat HePTP antibodies, and the precipitated proteins analyzed by
immunoblotting with anti-phosphotyrosine antibodies (upperpanel). The blots were stripped and assayed for HePTP to
confirm equivalent loading (lowerpanel). Each lane
represents the precipitate from 7.5
10
cells.
Percent histamine release (HR%) results are at the bottom of each lane. Arrow indicates position of
HePTP.
RI aggregation, it was possible that the phosphatase might
interact with the receptor or one of the receptor-associated proteins.
However, in immunoblotting experiments, we could not detect any HePTP
in immunoprecipitates of Lyn, Syk, or of the IgE receptors, and
conversely, we found no evidence of Lyn, Syk or of the IgE receptor
subunits in HePTP immunoprecipitates. Thus, although HePTP is
tyrosine-phosphorylated by receptor aggregation, it does not appear to
physically associate with either Lyn, Syk, or the IgE receptors.
Characteristics of the Tyrosine Phosphorylation of
HePTP
Some proteins are phosphorylated on tyrosine very early
after IgE receptor aggregation, and others are phosphorylated only
after a rise in intracellular Ca and/or after
activation of protein kinase C(9, 33, 34) .
Experiments therefore examined the relationship between tyrosine
phosphorylation of the HePTP, Ca
influx, and
activation of protein kinase C. Stimulation with the Ca
ionophore A23187 was as effective as Fc
RI cross-linking at
inducing tyrosine phosphorylation of HePTP (Fig. 6). In
contrast, direct activation of protein kinase C by the addition of
phorbol 12-myristate 13-acetate failed to do so (Fig. 6).
Therefore, an increase in intracellular calcium but not PKC activation
can directly stimulate tyrosine phosphorylation of HePTP.
10 cells. Percent histamine release (HR%) results are at the bottom of each lane. Arrow indicates position of
HePTP.
RI- and
ionophore-induced tyrosine phosphorylation of HePTP, RBL-2H3 cells were
first rinsed and then activated in Ca
-free media
containing EDTA (Fig. 7, lanes4-6). In
the absence of extracellular Ca
, tyrosine
phosphorylation of HePTP was substantially diminished when compared
with controls (lanes6 and 3). However,
because some tyrosine-phosphorylated HePTP was still detected after
receptor aggregation or after ionophore stimulation in
Ca
-free media (lanes5 and 6), the small rise in intracellular Ca
due
to release from intracellular compartments probably contributed the
requisite divalent ions. Identical results were obtained when the
concentration of EDTA in the wash and incubation media was 40
µM or 4 mM. Thus, Fc
RI-mediated tyrosine
phosphorylation of HePTP is a Ca
-dependent process
and is one of the late signaling events.
in the tyrosine phosphorylation of HePTP. RBL-2H3
cells were incubated for 10 min with buffer alone (lanes1 and 4), with calcium ionophore A23187 (lanes2 and 5), or with anti-Fc
RI monoclonal
antibody BC4 (lanes3 and 6). In some cases (lanes4-6), monolayer cultures were washed
with Ca
-free medium containing 40 µM
EDTA and then activated in the same medium. Lysates were
immunoprecipitated with anti-rat HePTP antibodies and analyzed by
immunoblotting with anti-phosphotyrosine (upperpanel). The blots were stripped and assayed for HePTP to
confirm equivalent loading (lowerpanel). Each lane
represents the precipitate from 7.5
10 cells.
Percent histamine release (HR%) results are at the bottom of each lane. Arrow indicates position of
HePTP.
-terminal
noncatalytic domain of HePTP may play a role in targeting the enzyme to
its intracellular locales. PTP1B (36) and DPTP61F(37) are nontransmembrane phosphatases that contain
carboxyl-terminal sequences involved in directing these proteins to the
endoplasmic reticulum. PTPMEG1(38) , PTPH1(39) , and
PTPD1 (40) are other cytosolic PTP that contain amino-terminal
sequences with homology to proteins that associate with the
cytoskeleton. This has lead some to conjecture that these or other such
PTP may be involved in focal adhesions(41) . Although the amino
terminus of HePTP does not share homology with cytoskeleton-associated
proteins, we nonetheless examined adherent RBL-2H3 cells for
colocalization of the enzyme to sites of cellular attachment to the
substratum. By laser confocal microscopy, HePTP did not accumulate
along the basal (adherent) surface of RBL-2H3 cells. ()Thus,
it is unlikely that HePTP is associated with focal adhesion sites in
these cells.RI
aggregation involves tyrosine phosphorylation of several proteins. Here
we report that Fc
RI aggregation induces tyrosine phosphorylation
of the cytosolic protein-tyrosine phosphatase, HePTP. The results
suggest that the Fc
RI-induced tyrosine phosphorylation of HePTP is
dependent on an elevation in the intracellular Ca
concentration. First, cells activated through the IgE receptors
in media lacking Ca
showed a dramatic diminution in
the level of HePTP tyrosine phosphorylation. The residual low level of
HePTP phosphorylation seen under these conditions may be attributed to
the relatively small amount of calcium stored within intracellular
compartments and released upon cell stimulation(42) . Second,
triggering of the cells with Ca
ionophore in either
calcium containing or calcium-free media mimicked the results obtained
by aggregating the Fc
RI. Thus, elevated intracellular
Ca
concentrations are needed for the tyrosine
phosphorylation of HePTP.
concentration that results from receptor engagement in many cell
types is accompanied by activation of protein kinase C(42) .
Optimal tyrosine phosphorylation of some mast cell proteins, such as
the focal adhesion kinase (p125
) and the
cytoskeletal protein paxillin, require both protein kinase C activation
and influx of extracellular calcium (16, 18) .
However, in the present experiments, direct activation of protein
kinase C with 40 nM phorbol 12-myristate 13-acetate failed to
elicit HePTP phosphorylation. Thus, tyrosine phosphorylation of HePTP
can occur independent of protein kinase C activation.
1, Lyn,
and Syk. The time course experiments showing that HePTP became tyrosine
phosphorylated between 1 and 5 min after Fc
RI aggregation also
bear this out.
RI,
and is believed to be critically important in the IgE receptor-mediated
signal transduction
process(10, 21, 46, 47) . However,
we found no evidence that Lyn and HePTP interacted. Likewise, we found
no evidence for an association between HePTP and the other
Fc
RI-associated protein-tyrosine kinase, Syk.
. These results strongly suggest
that HePTP may be involved in the IgE receptor-mediated signaling
cascade in these cells.
)RI, high affinity IgE receptor; PCR, polymerase chain reaction;
PTP, protein-tyrosine phosphatase(s); LC-PTP, leukocyte
protein-tyrosine phosphatase; HePTP, hematopoietic tyrosine
phosphatase; PAGE, polyacrylamide gel electrophoresis; RBL-2H3, rat
basophilic leukemia 2H3 cells.
)))
We thank Drs. Majed Hamawy and Nick Ryba for helpful
discussions and for reviewing this manuscript. We also thank Greta
Bader for histamine analysis and Nikki Hayes for DNA sequence analysis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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