©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Splicing Isoforms of Rat Ash/Grb2
ISOLATION AND CHARACTERIZATION OF THE cDNA AND GENOMIC DNA CLONES AND IMPLICATIONS FOR THE PHYSIOLOGICAL ROLES OF THE ISOFORMS (*)

Kazutada Watanabe (1), Tsunehiro Fukuchi (1), Hiroko Hosoya (1), Takuji Shirasawa (2), Koozi Matuoka (3), Hiroaki Miki (4), Tadaomi Takenawa (4)(§)

From the (1) Department of Experimental Biology, (2) Department of Molecular Pathology, and (3) Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, 35-2, Sakaecho, Itabashi-ku, Tokyo 173, Japan and the (4) Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We obtained three types of cDNA clones homologous to Ash/Grb2(Ash-l) cDNA from rats. One of these clones, Ash-, 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.


INTRODUCTION

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() 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.


MATERIALS AND METHODS

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 Avalue 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 1(SH3) and GST-Phosphatidylinositol 3-kinase 85-kDa Subunit (SH3)

The cDNA encoding phospholipase C 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.

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/HO (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).


RESULTS

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 andG-Ash2, encoded Ash/Grb2(Ash-l). The amino- and carboxyl-terminal regions were encoded in G-Ash1 andG-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.




DISCUSSION

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) 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.

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.

  
Table: Nucleotide sequences at exon-intron boundaries

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.



FOOTNOTES

*
This work was supported in part by grants for Science Research and for Cancer Research from the Ministry of Education, Science and Culture, Japan, and for Aging and Health from the Ministry of Health and Welfare, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/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-).

§
To whom correspondence and reprint requests should be addressed: Dept. of Molecular Oncology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108 Japan. Tel.: 81-3-5449-5510; Fax: 81-3-5449-5417.

The abbreviations used are: SH, Src homology; PDGF, platelet-derived growth factor; Sos, son of sevenless; PCR, polymerase chain reaction; GST, glutathione S-transferase; BrdUrd, 5-bromo-2-deoxyuridine; PI, phosphatidylinositol; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis.

K. Watanabe, T. Fukuchi, H. Hosoya, T. Shirasawa, K. Matuoka, H. Miki, and T. Takenawa, unpublished data.


ACKNOWLEDGEMENTS

We are greatly indebted to Yoko Kagawa and Kyoko Takahashi for their excellent technical assistance.


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