(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-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.
Mast cells and basophils play a central role in allergic and
inflammatory reactions. They express high affinity IgE receptors
(FcRI) (
)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 Fc
RI 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) .
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 FcRI. (
)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 Fc
RI 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.
The 5`-end was generated using the 5` rapid
amplification of cDNA ends system of Life Technologies, Inc.
Poly(A) 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.
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, of panelE is 60
.
Figure 5:
Time course of FcRI-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.
Because rat HePTP became tyrosine-phosphorylated upon
FcRI 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.
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 10
cells. Percent histamine release (HR%) results are at the bottom of each lane. Arrow indicates position of
HePTP.
To more
fully explore the role of extracellular calcium in the FcRI- 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.
Figure 7:
Importance of protein kinase C and
Ca 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.
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-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.
The signaling process initiated by FcRI
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.
The rise in intracellular Ca 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.
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-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.
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 FcRI,
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.
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. These results strongly suggest
that HePTP may be involved in the IgE receptor-mediated signaling
cascade in these cells.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U28356[GenBank].