From the
Northern blot analysis of polyadenylated RNA prepared from
RBL-2H3 cells revealed the presence of three distinct mRNAs encoding
p72
One of the primary responses observed following antigen-induced
cross-linking of the high affinity IgE receptor (Fc
Recent evidence suggests that engagement of the Fc
In
order to gain a better understanding of the role of p72
Sequencing of the original clones also indicated
that cDNAs encoding two different isoforms of the p72
Recent evidence
indicates the possibility that a family of p72
Fig. 5
shows the results of these
assays performed on immunoprecipitates from RBL-2H3 cells and
transfected COS-7 cells. The results of the immunocomplex kinase assays
(Fig. 5A) show that transfection of COS cells with
either p72
Fig. 6
shows the intron/exon boundaries identified
within the genomic DNA encoding this region of the protein. These data
indicate that the sequences encoding the 23-amino acid insert found
within the larger form of p72
Over the past several years, a great deal of data has
accumulated that strongly suggests a central role for non-receptor
protein-tyrosine kinase(s) in mediating the transmission of signals
originating from antigen-induced cross-linking of the high affinity IgE
receptor present on the surface of mast cells and basophils. One of the
protein-tyrosine kinases implicated to play a major role in signaling
through the Fc
The results of the
Northern blot analysis presented in Fig. 1prompted the cloning
of cDNAs encoding p72
While the 5.3-
and 3.8-kb messages appear to arise from the alternative use of two
poly(A) addition sites identified within the 3`-untranslated sequences
of the largest cDNA clone isolated, the molecular differences that give
rise to the 3.0-kb mRNA remain obscure. It is possible that, in
addition to that described below, an as yet unidentified alternative
RNA splicing event, perhaps within the 3`-untranslated region, results
in production of the 3.0-kb p72
Herein we
report the molecular cloning of two cDNAs encoding the rat homolog of
p72
The 23-amino acid insert appears to have little
effect upon p72
It should be noted that, during completion
of this work, potential alternative splicing of p72
It is interesting that
a comparison of the amino acid sequences of the rat, porcine, and human
p72
The second
spacer region, located between the second SH2 and the catalytic domain,
appears to be the least similar of the five domains compared. The fact
that all of the enzymes have been cloned from different species and
tissue sources (rat basophils, porcine and human B cells, and human T
cells) suggests at least two possible explanations for this
observation. The first would be that the only functional importance of
this region is to provide some minimal separation of the catalytic
domain from the SH2 domains, in order to maintain the ability of the
enzyme to function. The second, and more intriguing possibility, is
that the low level of similarity reflects divergent amino acid
subdomains involved in mediating the interaction of the different
p72
It is interesting that the rat
p72
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank C. Aversa for help with the Northern blot
analysis and A. L. Burkhardt and R. C. Penhallow for help with the
protein biochemistry.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, a protein-tyrosine kinase previously shown to be
associated with the high affinity IgE receptor present on the surface
of these cells (Hutchcroft, J. E., Geahlen, R. L., Deanin, G. G., and
Oliver, J. M.(1992) Proc. Natl. Acad. Sci. U. S.A. 89,
9107-9111). Here we report the full-length nucleotide sequence of
two of these messages, as well as the complete predicted amino acid
sequence of the rodent p72
protein-tyrosine kinase. In
addition, we report evidence indicating alternative splicing of
p72
mRNAs within RBL-2H3 cells. This splicing event
results in the expression of two distinct protein isoforms that differ
with respect to the presence of a 23-amino acid insert located within
the region of the protein that separates the two SH2 domains from the
catalytic domain. Both mRNAs arising from this splicing event appear to
encode functional protein-tyrosine kinases, as expression of the
corresponding cDNAs in COS cells results in the production of proteins
of the expected sizes that possess intrinsic tyrosine specific kinase
activity.
RI)
(
)
is an increase in the phosphorylation of specific cellular
proteins on tyrosine residues (1-4). Stimulation of
protein-tyrosine kinase activity appears to be necessary for induction
of the secondary events that culminate in the release of histamine from
intracellular stores
(5, 6) . Somewhat suprisingly,
initial studies involving molecular cloning and the structural analysis
of the receptor revealed a lack of intrinsic tyrosine kinase
activity
(7, 8, 9, 10, 11) .
Thus, identification of the mechanism by which the Fc
RI is able to
trigger an increase in protein-tyrosine kinase activity following cell
stimulation, and identification of the specific protein-tyrosine
kinases involved in this process has been the focus of intense study.
RI leads to
the activation of at least three protein-tyrosine kinases. The
activities of two members of the Src family of protein-tyrosine
kinases, pp60
and p56
(12) , as well
as that of p72
(13) , have been shown to be
increased following IgE/dinitrophenol-conjugated bovine serum
albumin-induced activation of rat basophilic leukemia cells (RBL-2H3).
Furthermore, both p56
and p72
can be
co-immunoprecipitated with the Fc
RI receptor from activated
cells
(12, 13) . While these results clearly suggest a
role for both of these kinases in mediating signal transduction through
the Fc
RI, the exact nature of their roles remains unclear.
in Fc
RI-stimulated mast cell activation, we have cloned and
analyzed p72
in RBL-2H3 cells. In this paper, we report
the complete amino acid sequence of rodent p72
and
present evidence for alternative mRNA splicing that results in the
generation of two distinct p72
protein isoforms.
Reagents
All tissue culture and molecular
biology reagents were purchased from Life Technologies, Inc. All
P-labeled nucleotides were purchased from DuPont NEN (3000
Ci/mmol). All other reagents were purchased from Sigma unless otherwise
indicated.
Cell Culture
RBL-2H3 cells were grown in minimum
essential medium supplemented with 20% fetal bovine serum, 1%
glutamine, and 1% penicillin/streptomycin. RBL-2H3 cells have been
previously described
(14) . COS-7 cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum, 1% glutamine, and 1% penicillin/streptomycin. All
cells were maintained at 37 °C in an atmosphere of 5%
CO.
Northern Blot Analysis
A probe corresponding to
amino acids 400-536 of the rat Syk open reading frame was used to
examine poly(A) RNA extracted from RBL-2H3 cells. The
probe was generated through nested PCR reactions utilizing
reverse-transcribed RBL-2H3 RNA as a template. The degenerate
oligonucleotide primers used were designed based upon the porcine
p72
cDNA sequence reported by Taniguchi et
al.(15) , corresponding to base pairs
1096-1110/1873-1859 for the first round and base pairs
1175-1190/1778-1763 for the second round of PCR reactions.
The 603-base pair PCR product from the second reaction was subcloned
into pBluescript and sequenced. Gel-purified cDNA insert was labeled
with [
-
P]dCTP by random priming (Life
Technologies, Inc.). Following transfer of RNA (10 µg) to Hybond
N
nitrocellulose membrane (Amersham), hybridization
was carried out under stringent conditions according to Maniatis et
al.(29) .
RNase Protection Assays
RNase protection assays
were performed using a kit purchased from Ambion Inc. (Austin, TX).
Restriction fragments isolated following digestion of the SykA and SykB
cDNAs with KpnI and NcoI were subcloned into
pBluescript (corresponding to nucleotides 1091-1260 of SykA and
nucleotides 1091-1329 of SykB). These plasmids were then used to
generate antisense RNA probes in order to examine poly(A) RNA prepared from RBL-2H3 cells.
Screening an RBL-2H3 cDNA Library
A custom
ZAP RBL-2H3 cDNA expression library was purchased from Stratagene.
Approximately 10
plaques were screened for Syk using the
P-labeled rat cDNA probe described above. Following three
rounds of screening, seven purified cDNA clones were isolated. Initial
sequencing data reduced the number of different clones to three. The
full nucleotide sequences of the clones isolated were determined
according to the method of Sanger et al.(16) , using a
Sequenase Version 2.0 sequencing kit (U. S. Biochemical Corp.).
Antibody Production
A fragment of the rat SykA
cDNA, corresponding to amino acids 1-349, was subcloned into the
pGEX-2T plasmid (Pharmacia Biotech Inc.) for glutathione
S-transferase-fusion protein production. Using standard
protocols
(20) , antisera were produced against affinity-purified
glutathione S-transferase-Syk fusion protein in New Zealand
White rabbits.
Transfections
The open reading frames of both rat
p72 isoforms were subcloned into the SV40-based
expression plasmid pSVL (Pharmacia). COS-7 cells were transfected with
2.5 µg of plasmid DNA using lipofectamine (Life Technologies, Inc.)
according to the manufacturer's protocol and cultured for 48 h
prior to analysis.
Preparation of Cell Lysates
Cells were lysed in 50
mM Tris-HCl, pH 8.0, containing 150 mM NaCl, 1%
Nonidet P-40, 2 mM EDTA, 100 µM
NaVO
, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, and 100 µM phenylmethylsulfonyl fluoride.
Insoluble debris was removed by centrifugation at 10,000
g prior to analysis of cell extracts.
Immunoprecipitation, SDS-PAGE, Western Blotting, Peptide
Mapping, and Kinase Assays
Immunoprecipitations, Western
blotting, SDS-PAGE, and immunocomplex kinases were performed as
described previously
(17) . In order to resolve the different
isoforms of p72 immunoprecipitated from RBL-2H3 cell
extracts for Western blotting, the proteins were separated on a precast
10% Tricine gel (Novex, San Diego, CA). One-dimensional phosphopeptide
mapping was performed by excising
P-labeled protein bands
from polyacrylamide gels and subjecting the isolated protein to
re-electrophoresis in the presence of 20 ng of Staphylococcus
aureus V8 protease, according to the method of Cleveland et
al.(18) .
Northern Blot Analysis
The results of Northern
blot analysis of poly(A) RNA isolated from RBL-2H3
cells using a partial Syk cDNA probe is presented in Fig. 1. The
results show the presence of three distinct mRNA species at
approximately 3.0, 3.8, and 5.3 kb. This observation suggested the
possibility that multiple forms of p72
might exist within
RBL-2H3 cells. Thus, in order to gain a better understanding of the
molecular differences between these mRNAs, Syk-specific cDNAs were
cloned from an RBL-2H3 cDNA library.
Figure 1:
Northern blot analysis.
Ten µg of poly(A) RNA isolated from RBL-2H3 cells
was analyzed using a partial Syk cDNA probe. The sizes of the 5.3-,
3.8-, and 3.0-kb messages were determined by their migration within the
gel relative to commercial RNA size markers (Life Technologies, Inc.).
In addition, the positions of the 28 S and 18 S ribosomal RNAs are also
indicated.
Cloning and Sequencing
Approximately 10 recombinant clones from an RBL-2H3
ZAP cDNA library were
screened, in duplicate, by plaque hybridization. The seven clones
isolated following plaque purification were excised from the
phage as cDNA inserts in pBluescript plasmid. Initial sequencing data
indicated that several different, but overlapping, cDNAs containing the
p72
open reading frame had been identified. The combined
nucleotide sequences of these different cDNAs, and their predicted
amino acid sequences, are shown in Fig. 2.
Figure 2:
Nucleotide sequence and deduced amino acid
sequence. The nucleotide sequence of the 5.3-kb clone and the amino
acid sequence deduced from it are presented. The numbers on
the right refer to nucleotide positions, while those on the
left refer to amino acid positions. The 23-amino acid insert
unique to the p72 isoform is underlined. The
two polyadenylation signals identified within the 3`-untranslated
region are indicated in bold
letters.
Upon examination of
the complete rat p72 nucleic acid sequence, it became
apparent that the 5.3-kb and 3.8-kb messages identified by Northern
blot analysis most likely arise from alternative use of the two
polyadenylation sites contained within the 3`-untranslated sequence. As
described above, an additional message, which migrates at approximately
3.0 kb, was also observed. While sufficient in size to encode
full-length p72
protein, the nature of this message
remains unclear.
protein, designated p72
and p72
,
had also been identified. When the amino acid sequences of these
different forms were compared with the porcine p72
amino
acid sequence reported by Taniguchi et al.(15) , it was
apparent that while the p72
form showed high identity to
the porcine enzyme, the p72
form contains a deletion of
23 amino acids. These 23 amino acids are located in the second
so-called ``spacer'' region (spacer B) between the second Src
homology 2 (SH2) domain and the catalytic domain.
-like
protein-tyrosine kinases might exist. This has been suggested by the
finding that human p70
, a tyrosine-specific kinase
associated with the T cell antigen receptor, is structurally related to
p72
(19) . Alignment of the predicted amino acid
sequences of the rat, porcine, and human
(21) p72
proteins, as well as human p70
, illustrates the
similarities between the four proteins (Fig. 3). Each protein
contains two SH2 domains and a single catalytic domain separated by
spacer domains of different lengths. The domain over which the rat and
porcine proteins display their greatest differences is the spacer
region located between the second SH2 domain and the catalytic domain.
Porcine p72
and rat p72
contain an insert
of 23 amino acids that is not found in either the rat p72
homolog or human p70
. At present, the significance
of this additional sequence is unknown. Its conservation between
species does however suggest the possibility that this spacer region
may play a role in the biology of this enzyme.
Figure 3:
Homology comparison of rat, porcine, and
human p72, with human p70
. Alignment of the
amino acid sequences of rat, porcine, and human p72
, as
well as human p70
. The numbers at the left refer to amino acid position. Dashes indicate amino acid
identity. Spaces represent gaps that have been introduced in
order to maintain sequence alignment. The 23-amino acid sequence
deleted in the smaller p72
isoform is
underlined. The SH2 and catalytic domains are presented within
the shaded and boxed
areas.
Expression of Rat Syk Protein Isoforms
In order to
verify the presence of two mRNAs encoding p72 within the
cell, RNA protection assays were performed. Partial cDNAs encoding the
spacer B region of the rat protein, with and without the insert (see
Fig. 4
), were generated and used to prepare antisense RNA
templates. The results presented in Fig. 4show that two messages
encoding p72
could be detected in poly(A)
RNA samples isolated from RBL-2H3 cells. The larger protected
fragment of 237 nucleotides represents the band resulting from
protection by mRNA encoding p72
containing the larger
spacer B region (p72
), while the band of approximately
168 nucleotides results from protection of message-encoding protein
containing the smaller spacer B region (p72
). The band
migrating at approximately 110 nucleotides, which was protected from
digestion by both probes, represents a fragment containing sequences
common to both probes. (The slight variation in size observed results
from differences in the methods used in subcloning of the cDNA
fragments used to generated the riboprobes.) A second common fragment
of approximately 70 nucleotides could also be detected, but was
difficult to see over background (data not shown). These results
suggest that RNAs encoding both forms of p72
are
expressed within RBL-2H3 cells.
Figure 4:
RNase protection assays. Ten µg of
poly(A) RNA isolated from RBL-2H3 cells (lanes 4 and 5) or 25 µg of yeast total RNA (lanes 3 and 6) were analyzed by RNase protection assay using the
previously described SykA (lanes 2, 3, and
4) and SykB (lanes 5, 6, and 7)
specific antisense RNA probes. Undigested
P-labeled SykA
and SykB probes are shown in lanes 2 and 7,
respectively. The fragment sizes expected to result from complete
protection by the SykA and SykB probes are approximately 168 and 237
nucleotides, respectively. Molecular weight size markers are shown in
lanes 1 and 8.
In order to determine if both forms
of Syk cDNA encoded functional protein-tyrosine kinases, the coding
regions of these messages were subcloned into the SV40-based expression
vector pSVL and transfected into COS-7 cells. Expression of p72 protein was determined through the use of immune-complex kinase
assays, as well as by Western blotting. The rabbit antibodies used had
been generated against a bacterially produced glutathione
S-transferase-fusion protein containing the entire
noncatalytic amino-terminal portion of rat p72
protein
(amino acids 1-348).
cDNA resulted in the appearance of
phosphoproteins that were not present in immunoprecipitates prepared
from COS cells transfected with vector alone. These phosphoproteins
were found to comigrate with a diffuse set of phosphoprotein bands
detected in immunoprecipitates prepared from RBL-2H3 cells (data not
shown). One-dimensional phosphopeptide mapping experiments were
performed in order to confirm that the phosphoproteins observed in COS
cell immunocomplex kinase assays represented p72
. The
P-labeled bands were excised from SDS-polyacrylamide gels
of immunoprecipitates prepared with transfected COS cells and RBL-2H3
cells, digested with S. aureus V8 protease, and reanalyzed by
SDS-PAGE. The phosphopeptide patterns generated appeared to be
identical (data not shown).
Figure 5:
Expression of Syk cDNAs. A,
lysates prepared from COS cells transfected with either the
p72 cDNA (lane 2), the p72
cDNA
(lane 3), or vector alone (lane 1) were
immunoprecipitated with a polyclonal antisera directed against the
amino-terminal 349 amino acids of p72
. These
immunoprecipitates were then used to perform immunocomplex kinase
assays. A control immunoprecipitation, in which antisera were omitted,
is shown in lane 4. B, lysates prepared from RBL-2H3 cells
(lanes 4 and 5) or COS cells transfected with either
the p72
cDNA (lane 2), the p72
cDNA (lane 3), or vector alone (lane 1) were
immunoprecipitated with a polyclonal antiserum directed against the
amino-terminal 349 amino acids of p72
. Antisera were
omitted from the RBL-2H3 control immunoprecipitations (lane
4). These immunoprecipitates were then subjected to Western blot
analysis using the anti-p72
polyclonal
antisera.
Fig. 5B shows that when
immunoprecipitates prepared from COS cells transfected with either the
SykA or SykB cDNA were examined by Western blotting, only a single
protein band corresponding to each isoform could be detected. However,
when similar immunoprecipitates prepared from RBL-2H3 cells were
examined, protein bands corresponding to both p72 isoforms could be detected. The two protein bands observed in
RBL-2H3 immunoprecipitates comigrated with the single bands observed in
immunoprecipitates prepared from the SykA and SykB transfected COS
cells. The results of these expression studies suggest that different
isoforms of p72
protein appear to be expressed in RBL-2H3
cells as the result of alternative mRNA splicing. The results obtained
by both RNase protection and Western blot analysis suggest that the
larger form of p72
(SykB), that contains the 23-amino
acid insert, represents the isoform most highly expressed in RBL-2H3
cells.
Syk Genomic DNA Analysis
In order to verify that
the insert present in p72 represents an exon resulting
from an alternative splicing event, the partial cDNA clone used to
generate antisense RNA for RNase protection assays was used as a probe
to screen a mouse genomic DNA library. Genomic DNA encoding the
``spacer B'' region of p72
was isolated and
sequenced.
protein are contained
within an independent exon. This observation strongly supports the
hypothesis that the two distinct forms of p72
identified
by cDNA cloning are the result of alternative RNA splicing.
Furthermore, this finding suggested the possibility that the
alternative splicing of mRNA encoding p72
might not be
cell type- or species-restricted. This was verified by performing
reverse-transcribed PCR reactions using total RNA prepared from PT18
cells, a mouse Il-3-dependent mast cell line. Products corresponding to
partial messages encoding both isoforms of p72
were
detected using this technique (data not shown).
Figure 6:
Analysis of mouse Syk genomic DNA. This
sequence of amino acids represents part of the spacer B region located
between the second SH2 domain and the catalytic domain of
p72. The triangles denote the position at which
sequence analysis has indicated the presence of introns within the
genomic DNA encoding this region of the protein. The approximate sizes
of the introns and the RNA splice donor and acceptor sites are also
presented.
RI is p72
.
, with the intention of identifying
the molecular differences underlying the presence of three apparent
messages encoding p72
in RBL-2H3 cells.
message.
from RBL-2H3 cells. The cDNAs differ by 69 base
pairs, resulting in the insertion of 23 amino acids within the
``spacer'' region located between the second SH2 and the
catalytic domain of p72
. Examination of mouse genomic DNA
encoding this region of the protein suggests that the sequences
corresponding to the 23-amino acid insert comprise a distinct exon that
is alternatively spliced in the SykB mRNA. While the results of the
RNase protection assays indicate that messages encoding both forms of
p72
protein are expressed in RBL-2H3 cells, it is quite
clear that this splicing event cannot account for the generation of the
3.0-kb message.
enzymatic activity in vitro, as
expression of cDNAs encoding either p72
or p72
in COS cells resulted in the production of a functional
protein-tyrosine kinase. While both forms of the protein appear to be
expressed in RBL-2H3 cells, whether or not the presence of these
additional amino acids has any significant effect upon the role of
p72
as a signal transducing protein in vivo remains to be determined. Alternative splicing of RNAs encoding
other protein-tyrosine kinases, in particular members of the Src family
of protein-tyrosine kinases, has been demonstrated by several
laboratories. Two distinct forms of the c-Lyn protein-tyrosine kinase
are expressed in various hematopoietic cells
(22, 23) ,
while the cDNAs encoding c-Fyn and c-Src appear to be alternatively
spliced in hematopoietic cells and neuronal cells, respectively
(24-27). As is the case with p72
, the functional
significance of these splicing events remains unclear. While the
alternative splicing of Src family protein-tyrosine kinase RNAs
exhibits a pattern consistent with restriction of such events to
specific tissue types, this does not appear to be the case with Syk RNA
splicing. Using antisense RNA probes prepared from partial mouse Syk
cDNAs, we have observed RNase protection patterns consistent with the
presence of both forms of Syk mRNA in all tissues expressing this
protein (data not shown).
RNA
in human basophils was reported by Yagi et al.(28) .
Using reverse-transcribed PCR, this group detected a partial cDNA
encoding a form of p72
containing a deletion of 23 amino
acids within the second spacer region. The amino acids deleted
correspond exactly with those we have identified as defining the
alternatively spliced exon that encodes the 23-amino acid insert
present in the larger form of p72
(SykB) expressed in rat
basophils. The data presented here both confirm and extend the findings
reported by Yagi et al.(28) .
proteins with that of human p70
shows
that the domains exhibiting the highest and lowest levels of identity
are the two spacer domains. The first spacer region, separating the two
SH2 domains, exhibits the highest level of amino acid identity between
the three protein sequences. This observation suggests the possibility
of a significant functional role for this domain, other than simply
separation of the SH2 domains. This conservation of amino acid
sequences may be required in order to maintain necessary secondary
structural elements, or, alternatively, to provide sites for
p72
or p70
to interact with other cellular
proteins. It is interesting that a tyrosine residue located toward the
center of this domain is conserved in all four proteins and that the
sequence surrounding this residue conforms well to consensus tyrosine
phosphorylation sites. This observation is suggestive of a role for the
phosphorylation of this tyrosine residue in the regulation of either
p72
's protein-tyrosine kinase activity or its
ability to associate with other proteins. Experiments designed to
investigate this hypothesis are currently in progress.
family member protein-tyrosine kinases with other
proteins in a tissue-specific manner.
protein, as well as both the human and the porcine
p72
proteins, contains an insertion of 23 amino acids
within this second spacer region. The fact that the amino acids
contained within this insert, including a potential tyrosine
phosphorylation site, have been very highly conserved in all forms of
p72
identified to date, suggests that this sequence may
define a significant structural and/or functional domain within
p72
. Experiments designed to investigate this possibility
are presently underway.
/EMBL Data Bank with accession number(s) U21683 (SykA)
and U21684 (SykB).
RI, high affinity IgE receptor; SH, src homology; PAGE,
polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; kb,
kilobase(s).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.