From the
We obtained three types of cDNA clones homologous to
Ash/Grb2(Ash-l) cDNA from rats. One of these clones, Ash-
A novel gene isolated by Clark et al.(1992) was shown
to be a key molecule, which exists between downstream of an epidermal
growth factor receptor-like tyrosine kinase and upstream of a Ras
protein and is essential for signalings of vulval formation in the
nematode worm Caenorhabditis elegans. This gene,
sem-5, encodes the protein (Sem-5), which consists almost
exclusively of a single SH2
Balb/c 3T3 cells were cultured in Dulbecco's modified
Eagle's medium supplemented with 10% calf serum. Two days after
plating, the cells were deprived of serum in medium containing insulin
(5 µg/ml), transferrin (5 µg/ml), and 100 µg/ml bovine
serum albumin and then incubated for 24 h at 37 °C. Fusion proteins
(1 mg/ml) or GST (1 mg/ml) were microinjected into the cytoplasm of
cells using an Eppendorf microinjector and micromanipulator. One hour
after injection, the cells were exposed to 5 units/ml PDGF and cultured
in the presence of 50 µM 5-bromo-2-deoxyuridine (BrdUrd)
for 20 h. After labeling, the dishes were washed in phosphate-buffered
saline, and the cells were fixed in ethanol/acetic acid/H
Here we have demonstrated that there are at least two types
of splicing isoforms of Ash/Grb2(Ash-l) as well as one unusual
transcribed gene. These isoforms are generated by unusual splicings, as
demonstrated by the isolation and molecular characterization of the
Ash-l-encoding gene. The splicing site of Ash-m differs from that of
Ash-l in the sixth exon. The Ash-l-encoding gene has the sequence
5`-GACTTTGACCCCCAGG-3` in the sixth exon, which is similar to the
consensus sequence (5`-(T/C)
Ash-m is defective in the second SH3 domain. Although
Ash-l associates with Sos and dynamin by virtue of its SH3 domains, the
significance of the two SH3 domains is not necessarily clear. Our
results show that the binding ability of Ash-m to proteins including
Sos and dynamin is weaker than that of Ash-l. Specifically, Ash-m is
not able to bind with Sos effectively. In contrast, Ash-s binds to both
Sos and dynamin despite the fact that Ash-s is composed almost
exclusively of the first SH3 domain of Ash-l. Accordingly, the second
SH3 of Ash-l is not necessarily essential for binding to dynamin and
Sos, while the first SH3 domain seems to play an important role in
binding. Steric hindrance caused by the altered conformation of the
defective SH3 domain in Ash-m may account for the loss of association
with Sos. It has been proposed that the two SH3 domains in Ash-l are
not redundant and might have different functions since mutations in
either SH3 domain alter the sem-5 gene activity in C.
elegans (Clark et al., 1992). We are now considering the
following two possibilities for the role of Ash-m. Since Ash-m does not
associate with Sos effectively because of its defective SH3 domain, the
signals through Ash-m may be transduced mainly to dynamin and other
proteins without activating Ras. Then the signals possibly change cell
functions involving dynamin without inducing mitogenesis. If this is
the case, the direction of the signals may be regulated in the
cytoplasm by the ratio between the amounts of Ash-m and Ash-l. Another
possibility is that the second SH3 domain in Ash-m generates signals
differing from those of Ash-l. The amino acid residues of the deleted
region are similar to those ahead of the region (underlined regions in Fig. 1b). The newly created amino acid sequence
produced by the deletion is somewhat similar to the amino termini of
SH3 domains, although, as in Nck (Lehmann, 1990), it deviates from
typical SH3 domains (Koyama et al., 1993). If the second SH3
domain of Ash-m is functional, the signals may be transduced to other
pathways by unknown molecules. Indeed, newly formed SH3 domain by
splicing binds different proteins from those bound to the
carboxyl-terminal SH3 domain of Ash-l.
As for Ash-s, it associates
with both Sos and dynamin. The characteristics of its associations with
proteins in PC12 cell lysates differs from its associations with Balb/c
3T3 cell lysate proteins. Since a single SH3 domain constitutes Ash-s,
it neither binds to tyrosine kinase receptors nor has any other
functional domain to respond to extracellular signals. Accordingly,
Ash-s may exert inhibitory effects on Ash-l by competition.
Very
recently, a different type of Ash-l splicing product, Grb3-3 has been
described (Fath et al., 1994). Grb3-3 has two complete SH3
domains but has a portion deleted from its SH2 domain. It was reported
that microinjection of Grb3-3 into Swiss 3T3 cells causes apoptosis.
But microinjection of Ash-m or Ash-s did not cause significant cell
death within 24 h. Therefore, Ash-m and Ash-s may act as differently
from Grb3-3 as regulators. We believe our findings provide a clue to be
used in elucidating the regulatory mechanisms of signals from tyrosine
kinase receptors to molecules downstream of Ash-l.
The beginning and the end of each exon are indicated by
opening and closing brackets, respectively. The stop codon at the
splicing donor site of the third exon is underlined. The two splicing
sites at the sixth exon generating Ash-l and Ash-m are indicated as the
numbers 5 and 5`, respectively.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We are greatly indebted to Yoko Kagawa and Kyoko
Takahashi for their excellent technical assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, was an
unusual transcribed gene having 93% identity in the nucleotide sequence
to Ash-l. The other two clones, Ash-m and -s, had nucleotide sequences
identical with Ash-l cDNA in the amino-terminal region. The coding
sequence of Ash-m cDNA is 42 nucleotides shorter than that of Ash-l
cDNA. The defective region of Ash-m cDNA encodes 14 amino acid residues
(157 to 170 of Ash-l), which comprise the most conserved region of the
second SH3 domain. On the other hand, the coding sequence of Ash-s
terminated at the end of the first SH3 domain due to a stop codon at
the boundary of the sequence, thereby differing from Ash-l cDNA.
Cloning of the genomic DNA of the Ash-l-encoding gene, determination of
the gene organization, and nucleotide sequencing revealed that the two
isoforms, as well as Ash-l, are generated from a single gene by unusual
alternative splicings. The gene spans more than 16 kilobases and
contains 6 exons and 5 introns. Ash-m and Ash-s mRNAs were detected in
various tissues by reverse-transcribed polymerase chain reaction. Ash-m
physically associated with dynamin, but the association with Sos was
less effective than that of Ash-l in rat pheochromocytoma PC12 cell
lysates, irrespective of treatment with nerve growth factor. In
contrast, Ash-s formed a complex with dynamin and Sos in cell lysates.
Moreover, the newly formed carboxyl-terminal SH3 of Ash-m by splicing
bound different proteins from those bound to the carboxyl-terminal SH3
domain of Ash-l, suggesting that Ash-m generates different signals.
Microinjection of Ash-m or Ash-s into Balb/c 3T3 cells inhibited DNA
synthesis induced by platelet-derived growth factor. These results show
that these isoforms act as dominant negative regulators of mitogenic
signals by Ash-l.
(
)
domain and two SH3
domains. Since SH2 domains recognize phosphotyrosine residues in
tyrosine kinase receptors, Sem-5 became the most likely candidate for
the molecule that couples the tyrosine kinase receptor encoded with Ras
signaling. A mammalian counterpart of Sem-5, Ash-l/Grb-2, was
identified independently by two groups using different strategies.
Matuoka et al.(1992) cloned cDNAs from rats and humans by
hybridization with mixed oligonucleotides encoding a consensus sequence
to the SH2 domain. On the other hand, Lowenstein et al. (1992)
utilized the CORT (Cloning Of Receptor Targets) method for the human
cDNA in which the phosphorylated tyrosine residues in epidermal growth
factor receptors were used as a probe and
gt11 phages were
screened for the proteins binding to the probe. The evidence that
Ash-l/Grb-2 links tyrosine kinase receptors with Ras signaling has been
obtained from the direct association of Ash-l with Sos (guanine
nucleotide exchange factor for Ras) (Egan et al., 1993;
Rozakis-Adcock et al., 1993; Li et al., 1993; Simon
et al., 1993; Olivier et al., 1993; Chardin et
al., 1993). Thus, it is demonstrated that Ash-l is the adaptor
molecule that physically associates with mSos, allowing
ligand-activated tyrosine kinase receptors to modulate Ras activity. A
recent study has shown that injection of the antibody against Ash-l
into cells abolishes the reorganization of actin stress fibers (Matuoka
et al., 1993), controlled by Rac proteins (Ridley and Hall,
1992; Ridley et al., 1992). In addition, dynamin, which
stimulates GTPase activity by binding to microtubules, also binds to
Ash-l through the SH3 domains (Gout et al., 1993; Herskovits
et al., 1993; Miki et al. 1994). These findings raise
the possibility that Ash-l functions not only in Ras signaling but also
in other pathways. Since the SH3 domains function to mediate signals
downstream of Ash-l, differences in the binding specificities of the
two SH3 domains to various signaling molecules will regulate the signal
direction. Therefore, we thought that it would be worthwhile to search
for Ash-l isoforms, especially those having SH3 domains different from
the original. In the present paper, we describe the isolation of
splicing isoforms of Ash-l, the generation mechanism of the isoforms as
revealed by genomic cloning of the Ash-l-encoding gene, and the roles
of these isoforms in mitogenic signaling.
Isolation of Ash/Grb2 Isoform cDNAs
A rat fetal
brain cDNA library (E15) in Zap II vector was screened with
P-labeled Ash-l cDNA. Conditions for the transfer of phage
plaques to nitrocellulose filters and fixation of DNA were essentially
the same as the standard protocols (Maniatis et al., 1982).
Hybridization was performed in 5
SSC, 30% formamide, 5
Denhardt's solution, 0.1% SDS, 100 µg/ml salmon sperm DNA,
10% dextran sulfate at 42 °C overnight. The filters were washed
three times in 2
SSC, 0.1% SDS at room temperature and then at
55 °C twice. Autoradiography was conducted at -70 °C
overnight using Kodak XAR film with an intensifying screen. Conversion
of phage to plasmid was performed according to the manufacturer's
instructions (Stratagene). The plasmids were amplified in
Escherichia coli XL1-Blue. Preparation of the plasmid DNA was
carried out as described (Watanabe et al., 1992).
Isolation of Ash-l Genomic Clones
2
10
recombinant phages of a Wistar rat genomic library,
previously reported (Watanabe et al., 1992), were screened
with
P-labeled Ash-l and Ash-m cDNAs. The conditions of
phage transfer to filters, hybridization, and washing were the same as
those used in the cDNA screening described above.
Nucleotide Sequencing
Recombinant pBluescript
plasmids containing the cDNA inserts were digested with appropriate
pairs of restriction enzymes to generate 5`- and 3`-protruding ends at
unique sites in the polylinker region. The resulting linearized DNA was
treated with exonuclease III and S1 nuclease to effect various extents
of deletion (Guo and Wu, 1983). Sequence analyses were performed using
double-stranded DNA by the dideoxy chain termination method (Sanger
et al., 1977; Messing et al., 1981).
PCR Experiment
Tissues used to extract total RNAs
were cerebrum, cerebellum, thymus, spleen, heart, lung, liver, kidney,
stomach, small intestine, large intestine, skeletal muscle, and testis.
The total RNAs were isolated by the method of Chirgwin et
al.(1979). RNA PCR was performed using GeneAmp RNA
PCR kit (Perkin-Elmer Cetus). The oligonucleotide primers used to
discriminate Ash-l and Ash-m mRNAs in the total RNAs were
5`-ATGAAACCACATCCGTGGTTTTTTGGCAAAATCC-3` (oligo-2 in
Fig. 1a) and 5`-ATCTCTTTTGCTTCTTAGACGTTCC-3` (oligo-5).
The regions chosen to synthesize the primers are indicated by
convergent arrows in Fig. 1a. The PCR cycle condition
was as follows: one cycle of 94 °C for 4 min, 50 °C for 5 min,
and 72 °C for 5 min, followed by 35 cycles of 94 °C for 1 min,
50 °C for 2 min, and 72 °C for 3 min, and finally followed by
72 °C for 7 min. The reaction products were analyzed by agarose gel
electrophoresis. The primers used to detect Ash-s mRNAs were
5`-GCTCAGAATGGAAGCCAT-3` and 5`-CTATATAGGCCTTGTTCCCCTAAGC-3`. The
products were analyzed by Southern blot hybridization with the labeled
probe, 5`-CATAGAAATGAAACCACATCC-3`.
Figure 1:
a, comparison of the nucleotide
sequences among Ash-l, Ash-m, Ash-s, and Ash-. The coding region
of Ash-l was aligned with the corresponding regions of the other cDNAs.
Nucleotide residues identical with Ash-l are indicated by
dots. The open reading frame of Ash-l starts from the ATG
boxed in the nucleotide sequence. The stop codons terminating
coding sequences of Ash-l, Ash-m, and Ash-s are shown in shaded
boxes. Arrows above the sequence indicate the oligonucleotides
used for PCR primers or hybridization. b, amino acid sequences
of Ash-l, Ash-m, and Ash-s. The amino acid sequences in open and shaded boxes are the SH2 and SH3 domains of Ash-l,
respectively. Ash-m lacks in the amino acid sequence of
QPTYVQALFDFDPQ.
Production and Purification of Glutathione S-Transferase
Fusion Proteins of Ash-m, Ash-s, and Carboxyl-terminal SH3s of Ash-l
and Ash-m
The coding regions of Ash-s and -m were amplified by
PCR. The oligo-1 primer was synthesized with a BamHI site at
the 5`-end. Ash-m coding fragments were amplified in the same manner as
Ash-s except that the oligonucleotide from the carboxyl-terminal region
of Ash-l (oligo-5) was utilized as a 3`-primer. Both PCR fragments for
Ash-s and -m were inserted between the BamHI and SmaI
sites of pBluescript KS, amplified in XL1-Blue, and
excised between the BamHI site in the PCR primer and an
EcoRI site in the polylinker region of the vector. The
fragments were inserted between the BamHI and EcoRI
sites of a pGEX-3X expression vector. Similarly, DNA fragments coding
the carboxyl-terminal SH3 domains of Ash-l and Ash-m were amplified
using synthetic oligonucleotides 5`-GTTGGATCCTGTCCAGGAACCAGCAG-3`
(nucleotide 419-435 of Ash-l with the BamHI restriction
site) and 5`-AAAGAATCCTTAGACGTTCCGGTTC-3` (nucleotide 639-654 of
Ash-l with the EcoRI site) as primers. The resulting 254-bp
(Ash-l) and 212-bp (Ash-m) products were digested with BamHI
and EcoRI, then ligated into the BamHI and
EcoRI sites of PGEX-3X expression vector. Bacteria-harboring
plasmids were precultured in M9-ZB medium at 37 °C overnight. On
the next day, the bacteria were diluted to A
= 0.1 with medium, and culture was continued until the
A
value was between 0.5 and 0.6.
Protein production was then induced by adding 1 M
isopropyl-1-thio-
-D-galactopyranoside (0.5 ml/500 ml
culture medium) to the culture medium. After 5 h, the bacteria were
harvested and suspended in a lysis buffer containing 1 mM
Tris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl, 0.1
mM phenylmethylsulfonyl fluoride, diisopropylfluorophosphate,
and 1% Triton X-100. The extracts were sonicated vigorously and then
centrifuged at 100,000
g for 40 min. The resultant
supernatants were applied to a glutathione-agarose (Pharmacia Biotech
Inc.) column. Glutathione S-transferase (GST)-Ash fusion
proteins were eluted with buffer containing 20 mM Tris-HCl (pH
8.0) and 25 mM glutathione.
Production of GST-Phospholipase C
The cDNA
encoding phospholipase C 1(SH3) and
GST-Phosphatidylinositol 3-kinase 85-kDa Subunit (SH3)
1 was digested with HincII at
nucleotide 2204 and HpaII at nucleotide 2560, respectively.
The resulting cDNA fragment was inserted into the pGEX-3X vector. The
recombinant plasmid was transformed into the XL-I Blue E. coli strain. Methods for recombinant protein expression were the same
as described above. GST-phosphatidylinositol (PI) 3-kinase was prepared
as an HindIII restriction fragment containing the amino
terminus to residue 388 (Shibasaki et al., 1994).
In Vitro Binding Experiments on Ash Isoforms with
Proteins in Cell Lysates
Rat pheochromocytoma PC12 cells were
metabolically labeled with [S]methionine (100
µCi/15-cm dish/10 ml) by incubating in methionine-free medium for 3
h. Subsequently, the cells were, if necessary, exposed to 10 ng/ml
nerve growth factor for 3 min. The cells were washed with
phosphate-buffered saline, harvested in LIPA buffer (20 mM
Tris-HCl, pH 7.4, 1.0% Triton X-100, 150 mM NaCl, 100
µM leupeptin, 100 µM aprotinin, 1 mM
EDTA, and 1 mM EGTA), lysed by sonication, and finally
centrifuged at 100,000
g for 30 min at 4 °C. The
cell lysates thus obtained were mixed with 50 µm of glutathione
beads binding GST-fusion proteins (50 µg of protein) and incubated
at overnight 4 °C. The beads were washed in 20 mM
Tris-HCl, 0.5 M NaCl, and 1.0% Triton X-100, and suspended in
SDS sample buffer. After SDS-PAGE was carried out, autoradiograms were
obtained using Kodak X-Omat film exposed for 2 days. The protein bands
of dynamin and Sos were determined by Western blot using anti-dynamin
antibody (Miki et al., 1994) and anti-mouse Sos 1 antibody
(UBI, New York), respectively.
GST-Ash Fusion Protein Injection into Balb/c 3T3
Cells
GST-Ash-l, GST-Ash-m, and GST-Ash-s eluted from the
glutathione-agarose column were dialyzed against 140 mM
potassium phosphate (pH 7.25). Protein concentrations were adjusted to
1 mg/ml.
O
(90:5:5) for 20 min at room temperature. The proportion of cells
synthesizing DNA was determined using a cell proliferation kit
(Amersham Life Science).
Isolation and Characterization of cDNA Clones from
Ash/Grb2-splicing Isoforms and Transcribed Pseudogene
Initial
screening of the 2 10
recombinant phages with
P-labeled Ash/Grb2(Ash-l) cDNA as a probe yielded 57
positive clones. Ten recombinant phage DNAs which hybridized to the
probe were purified. Among these cDNA clones, three did not hybridize
to the oligonucleotide derived from the 3`-coding region of the Ash-l
nucleotide sequence (oligo-4 in Fig. 1a). Nucleotide
sequence analysis showed that two clones were identical with each other
except for a difference in size and shared 93% identity in nucleotide
sequence with Ash-l cDNA (Fig. 1a). However, the clones
do not have long open reading frames because of the frequent appearance
of stop codons. We concluded, therefore, that these cDNAs, designated
Ash-
, were derived from unusual transcribed genes. The third
clone, Ash-s cDNA, had a nucleotide sequence identical with Ash-l cDNA
in the first SH3 domain (Fig. 1, a and b).
However, the coding sequence was terminated at the end of the SH3
domain by a stop codon, after which the sequence differed from that of
Ash-l cDNA. Accordingly, the Ash-s isoform is solely comprised of a
single SH3 domain identical with that of Ash-l. In order to search for
other isoform types, we purified 47 additional phage DNAs which had
given positive signals in the first library screening. Then we
performed polymerase chain reaction experiments on the DNAs using the
oligonucleotide primers (oligo-2 and -5) described under
``Materials and Methods,'' this gave 506-bp fragments for
Ash-l cDNA. We detected three clones generating about 450-bp fragments.
Since these clones were identical with one another, we designated them
as Ash-m cDNA. The coding sequence of Ash-m cDNA is 42 nucleotides
shorter than that of Ash-l cDNA. The defective region of Ash-m
corresponds to 14 amino acid residues between 157 and 170 of Ash-l,
which comprise the amino-terminal region of the second SH3 domain
(Fig. 1b). According to the length of the cDNAs,
Ash/Grb2 and two isoforms are named Ash-l, Ash-m, and Ash-s.
Cloning and Organization of the Ash-l-encoding Gene and
Generation of Ash Isoforms
In order to confirm that the three
isoforms, including Ash-l, arise from a single gene by alternative
splicing, we isolated genomic clones of the rat Ash-l-encoding gene and
analyzed gene organization by restriction mapping and nucleotide
sequencing together with genomic Southern blot analysis. Screening of a
rat genomic DNA library yielded 37 genomic clones hybridizing to the
Ash cDNA probe. These clones were classified into three types by
digesting the recombinant phage DNAs with restriction enzymes. One of
these, G-Ash-
corresponded to genomic DNA encoding
Ash-
.
(
)
The other two,
G-Ash1
and
G-Ash2, encoded Ash/Grb2(Ash-l). The amino- and
carboxyl-terminal regions were encoded in
G-Ash1 and
G-Ash2,
respectively. Although
G-Ash1 and
G-Ash2 do not overlap, the
nucleotide sequences of the exons in these two clones account for the
entire sequence of Ash-l cDNA. Ash-l gene has a length of more than 16
kilobases and consists of six exons and five introns (Fig. 2).
The nucleotide sequences at the exon-intron boundaries show that all
introns start with GT at the 5` end and terminate with AG at the 3` end
(). This conforms to the ``GT-AG rule''
(Breathnach and Chambon, 1981). The genomic cloning of the gene
revealed that the two isoforms, Ash-s and -m, are generated from a
single gene by unusual alternative splicings. Ash-s mRNAs are comprised
of the first two exons and a part of the second intron of the gene. A
splicing donor site for the third exon was not recognized in Ash-s
transcripts. Thus, a poly(A) sequence is attached in the transcribed
region of the intron. Since a stop codon appears in-frame at the
exon-intron boundary of the gene, a part of the third intron is
recognized as a 3`-flanking sequence in Ash-s mRNAs. Thus Ash-s
consists solely of the first SH3 domain of Ash-l. On the other hand,
splicing for Ash-m mRNAs occurred at the sequence
5`-GACTTTGACCCCCAGG-3`, which is located within the sixth exon, instead
of at 5`-CTCTTCTCTCTTGCAGC-3` as for Ash-l mRNAs ( Fig. 2and
). The first 42 nucleotide residues of the sixth exon of
Ash-l are recognized as a part of the fifth intron in Ash-m. Thus, the
sixth exon of Ash-m starts inside the Ash-l exon, and 14 amino acid
residues encoding the amino-terminal of the second SH3 domain are
defective in Ash-m.
Figure 2:
Restriction map and organization of the
Ash isoform-encoding gene. Exons and introns are shown below.
Closed and open boxes indicate coding and noncoding
exons, respectively. The exon-intron boundaries generating the Ash
isoforms are enlarged to indicate the partial sequences around the
splicing sites. The boxed nucleotide sequences are parts of
the exons. Long black arrows represent the genomic regions
cloned in phages. Shaded bars indicate regions in the
first and the second SH3 domains. (A)n represents poly(A) in
the transcripts.
We carried out genomic Southern blot analysis
using the 3` region from the SplI site in the Ash cDNA, which
is both a carboxyl terminus-encoding and 3`-flanking region, and
constitutes the last exon in the Ash gene (Fig. 3). Each lane
yielded pairs of bands except for the lane of BamHI digestion,
in which two fragments were likely to be in the identical mobility. The
restriction fragments generated from G-Ash2 and
G-Ash-
accounted for these bands. Accordingly, we concluded that Ash-l and
Ash-m were transcribed from a single gene.
Figure 3:
Comparison of restriction fragments
hybridizing with Ash cDNA probe in genomic DNA with those in cloned
DNAs. The probe was a 523-bp fragment of Ash cDNA which is in the last
exon of Ash gene. Rat genomic DNAs (10 µg) and cloned DNA (10 ng)
were digested with various restriction enzymes and subjected to 1%
agarose gel electrophoresis. a, genomic Southern blot
analysis. Lanes: 1, BglII; 2,
EcoRI; 3, HindIII; 4,
SacI; 5, BamHI; 6, PstI.
b, Southern blot analysis of G-Ash2 (lanes with odd
numbers) and
G-Ash-
(lanes with even numbers).
Lanes: 1 and 2, EcoRI; 3 and 4, SacI; 5 and 6,
PstI.
Tissue Distribution of Ash-m and -s mRNAs
Ash-m
and Ash-s mRNAs were detected in various tissues by the
reverse-transcribed polymerase chain reaction. The ratio between Ash-m
and Ash-l mRNAs varied among tissues (Fig. 4a). Higher
contents of Ash-m mRNA than that of Ash-l were observed in lung,
stomach, and skeletal muscle. By contrast, Ash-m mRNAs were barely
detectable in cerebrum, spleen, kidney, and testis. On the other hand,
Ash-s mRNAs were detected abundantly in spleen and kidney but rarely
expressed in stomach and skeletal muscle (Fig. 4b).
Figure 4:
Tissue distribution of Ash-l, Ash-m, and
Ash-s. a, reverse-transcribed PCR products for Ash-l and Ash-m
from various tissues were observed with ethidium bromide.
Lanes: 1, cerebrum; 2, thymus; 3,
spleen; 4, heart; 5, lung; 6, liver;
7, kidney; 8, stomach; 9, small intestine;
10, large intestine; 11, skeletal muscle;
12, testis. b, reverse-transcribed PCR products for
Ash-s from various tissues were analyzed with Southern blot
hybridization as described under ``Materials and
Methods.''
Binding Proteins to Ash-m and -s Proteins
To
compare the binding ability of Ash isoforms, the concentrations of
GST-fusion proteins were adjusted and binding assays were carried out.
In vitro binding experiments using the GST-fusion proteins of
Ash isoforms demonstrated that Ash-m does not associate effectively
with proteins such as dynamin and Sos in PC12 cell lysates compared to
Ash-l (Fig. 5a). The bands of dynamin and Sos were
identified by anti-dynamin and anti-Sos antibodies. Especially, the
binding ability of Ash-m to Sos was very low. On the other hand, Ash-s
formed a significant complex with dynamin and Sos in PC12 cell lysates,
although binding to other proteins including 55-kDa, 130-kDa, and
180-kDa proteins appeared not so marked as that of Ash-l. These results
were unaffected by treating the cells with nerve growth factor. Ash-s
is composed exclusively of a single SH3 domain. Hence, the binding
ability of Ash-s to Sos and dynamin was compared with that of SH3
domains from phospholipase C -1 and phosphatidylinositol 3-kinase
(PI-3-kinase). As shown in Fig. 5b, the SH3 domains from
phospholipase C and PI-3-kinase bound less effectively to SOS or
dynamin in cell lysates than to Ash-s, while the Ash-s SH3 domain
associated with both molecules. The binding of Ash-s to dynamin in PC12
cell lysates was more marked than that to Sos. In contrast, Ash-s bound
to Sos more than to dynamin in Balb/c 3T3 cell lysates
(Fig. 5b). These results suggest that signaling
molecules bound to Ash-s differ among cells, although it only reflects
the existing amounts of proteins.
Figure 5:
Binding specificity of Ash isoforms to the
proteins in cell lysates. a, binding of Ash isoforms to Sos
and dynamin in PC12 cell lysates. PC12 cells were metabolically labeled
with [S]methionine. Cell lysates harvested in
the presence and absence of nerve growth factor (NGF) were
applied to GST-fusion protein columns, and bound proteins were
subjected to SDS-PAGE. b, binding of various SH3 domains to
Sos and dynamin. The SH3 domains from Ash-s, phospholipase C, and
PI-3-kinase were prepared as GST-fusion proteins in E. coli. Lysates from [
S]methionine-labeled Balb/c
cells and PC12 cells were used.
Binding Proteins to Carboxyl-terminal SH3 Domain of
Ash-m
As the result of splicing, the carboxyl-terminal SH3
domain of Ash-m forms a new type of SH3. Therefore, the new SH3 may
bind different proteins from those bound to carboxyl-terminal SH3 of
Ash-l. To examine the possibility, we compared the proteins bound to
carboxyl-terminal SH3 of Ash-m to those bound to amino-terminal and
carboxyl-terminal SH3 domains of Ash-l using bovine brain. As shown in
Fig. 6a, carboxyl-terminal SH3 of Ash-m did not bind Sos
or dynamin. However, it was found to be able to bind several new
proteins (60-kDa and 170-kDa proteins shown by arrows). Since
Ash-m was found to be abundantly expressed in skeletal muscle, we also
examined proteins bound to carboxyl-terminal Ash-m SH3 using chicken
skeletal muscle. The protein binding pattern to carboxyl-terminal Ash-m
SH3 was different from that of Ash-l (Fig. 6b). Proteins
(90-kDa and 170-kDa proteins shown by arrows) were
specifically bound to carboxyl-terminal Ash-m SH3. These data suggest
that Ash-m generates new signals different from those of Ash-l.
Figure 6:
Different proteins bind to
carboxyl-terminal SH3 domains of Ash-m and Ash-l. Bovine brains
(100,000 g supernatant fractions) (a) or
chicken skeletal muscle supernatant fractions (b) were applied
to GST-fusion protein columns. Bound proteins were analyzed by SDS-PAGE
and stained with Coomassie Blue. Lane 1, GST-amino-terminal
SH3 domain of Ash-l; lane 2, GST-carboxyl-terminal SH3 domain
of Ash-l; lane 3, GST-carboxyl-terminal SH3 domain of Ash-m.
&cjs0800; indicates Sos and &cjs0801; indicates
dynamin.
Effect of Ash-l, Ash-m, and Ash-s on PDGF-induced DNA
Synthesis
To examine the effect of Ash-l, Ash-m, and Ash-s on
DNA synthesis, the proteins were microinjected into Balb/c 3T3 cells.
After microinjection with various Ash proteins or control GST protein,
the cells were stimulated with PDGF. The cells were then labeled with
bromodeoxyuridine (BrdUrd), fixed, and immunostained with anti-BrdUrd
antibody. Stimulation with PDGF led to a marked increase in the
proportion of cells staining positive for BrdUrd incorporation, and
this was unaffected by the control GST proteins (Fig. 7). On the
other hand, injection of Ash-l protein caused a further increase in
BrdUrd incorporation in PDGF-stimulated cells from 56% to 70%, although
the basal incorporation did not increase without PDGF. Injection of
Ash-s or Ash-m, on the contrary, inhibited the enhanced incorporation
of BrdUrd with PDGF. But the inhibition by Ash-m was less effective
than by Ash-s. Similar to Ash-l, neither Ash-m nor Ash-s affected the
basal incorporation of BrdUrd without PDGF. These results suggest that
Ash-m and Ash-s act as negative regulators in cells and inhibit the
signaling of Ash-l to DNA synthesis.
Figure 7:
Effect of Ash-l, Ash-m, and Ash-s on
PDGF-induced DNA synthesis. Ash-l, Ash-m, Ash-s, and GST protein were
microinjected into serum-starved Balb/c 3T3 cells. Each protein was
injected into more than 100 cells. One hour later, the cells were
stimulated with PDGF (5 units/ml), labeled with bromodeoxyuridine, and
incubated for 20 h. The cells were stained with anti-bromodeoxyuridine
antibody, and positive cells were counted. Results are shown as mean
± S.E. in four separate experiments.
N(C/T)AGG-3`) of the
splicing acceptor (Mount, 1982). This sequence is utilized in Ash-m as
a splicing acceptor, while Ash-l uses 5`-CTCTCTCTCTTGCAGC-3`, which
conforms to the consensus sequence ( Fig. 2and ).
Thus, part of the sixth exon of Ash-l is recognized as the fifth intron
and spliced out in Ash-m transcripts. On the other hand, the splicing
for Ash-s differs from that for Ash-l at the third exon. Ash-s
transcripts have a poly(A) attached at the polyadenylation signal in
the third intron of Ash-l, without being spliced at the donor site of
Ash-l. Thus, the splicings described here are not simple alternative
splicings.
Table:
Nucleotide sequences at exon-intron
boundaries
/EMBL Data Bank with accession number(s) D49846 (rat
mRNA for Ash-m), D49847 (rat mRNA for Ash-s), and D49848 (rat mRNA for
Ash-
).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.