Splicing Isoforms of Rat Ash/Grb2.

Abstract

We obtained three types of cDNA clones homologous to Ash/Grb2(Ash-l) cDNA from rats. One of these clones, Ash-ψ, 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. We obtained three types of cDNA clones homologous to Ash/Grb2(Ash-l) cDNA from rats. One of these clones, Ash-ψ, 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. INTRODUCTIONA novel gene isolated by Clark et al., 1992Clark S.G. Stern M.J. Horvitz H.R. Nature. 1992; 356: 340-344Crossref PubMed Scopus (460) Google Scholar 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 SH21( 1The abbreviations used are: SHSrc homologyPDGFplatelet-derived growth factorSosson of sevenlessPCRpolymerase chain reactionGSTglutathione S-transferaseBrdUrd5-bromo-2-deoxyuridinePIphosphatidylinositolbpbase pair(s)PAGEpolyacrylamide gel electrophoresis.) 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., 1992Matuoka K. Shibata M. Yamakawa A. Takenawa T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9015-9019Crossref PubMed Scopus (137) Google Scholar 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., 1992Lowenstein E.J. Daly R.J. Batzer A.G. Li W. Margolis B. Lammers R. Ullrich A. Skolnik E.Y. Bar-Sagi H. Schlessinger J. Cell. 1992; 70: 431-442Abstract Full Text PDF PubMed Scopus (1331) Google Scholar 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 λgt11 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., 1993Egan S.E. Giddings B.W. Brooks M.W. Buday L. Siezland A.M. Weinberg R.A. Nature. 1993; 363: 45-51Crossref PubMed Scopus (1003) Google Scholar; Rozakis-Adcock et al., 1993Rozakis-Adcock M. Fernley R. Wade J. Pawson T. Bowtell Nature. 1993; 363: 83-85Crossref PubMed Scopus (835) Google Scholar; Li et al., 1993Li N. Batzer A. Daly R. Yajnik V. Skolnik E. Chardin P. Bar S.D. Margolis B. Schlessinger J. Nature. 1993; 363: 85-88Crossref PubMed Scopus (797) Google Scholar; Simon et al., 1993Simon M.A. Dodson G.S. Rubin G.M. Cell. 1993; 73: 169-177Abstract Full Text PDF PubMed Scopus (364) Google Scholar; Olivier et al., 1993Olivier J.P. Raabe T. Henkemeyer M. Dickson B. Mbamalu G. Margolis B. Schlessinger J. Hafen E. Pawson T. Cell. 1993; 73: 179-191Abstract Full Text PDF PubMed Scopus (447) Google Scholar; Chardin et al., 1993Chardin P. Camonis J.H. Gale N.W. van A.L. Schlessinger J. Wigler M.H. Bar S.D. Science. 1993; 260: 1338-1343Crossref PubMed Scopus (648) Google Scholar). 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., 1993Matuoka K. Shibasaki F. Shibata M. Takenawa T. EMBO J. 1993; 12: 3467-3473Crossref PubMed Scopus (100) Google Scholar), controlled by Rac proteins (Ridley and Hall, 1992Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3797) Google Scholar; Ridley et al., 1992Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3049) Google Scholar). In addition, dynamin, which stimulates GTPase activity by binding to microtubules, also binds to Ash-l through the SH3 domains (Gout et al., 1993Gout I. Dhand R. Hiles I.D. Fry M.J. Panayotou G. Das P. Truong O. Totty N.F. Hsuan J. Booker G.W. Campbell I.D. Waterfield M.D. Cell. 1993; 75: 25-36Abstract Full Text PDF PubMed Scopus (482) Google Scholar; Herskovits et al., 1993Herskovits J.S. Shpetner H.S. Burgess C.C. Vallee R.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11468-11472Crossref PubMed Scopus (126) Google Scholar; Miki et al., 1994Miki H. Miura K. Matuoka K. Nakata T. Hirokawa N. Orita S. Kaibuchi K. Takai Y. Takenawa T. J. Biol. Chem. 1994; 269: 5489-5492Abstract Full Text PDF PubMed Google Scholar). 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 of the isoforms as revealed by genomic of the Ash-l-encoding gene, and the of these isoforms in mitogenic of rat cDNA in was screened with Ash-l cDNA. for the of to and of DNA were the as the et al., T. J. A Scholar). was in 5 5 at The were three in at and at was at using with an of to was to the The were in of the DNA was as et al., K. M. S. Y. K. N. T. T. 1992; PubMed Scopus Google of Ash-l phages of a rat genomic et al., K. M. S. Y. K. N. T. T. 1992; PubMed Scopus Google Scholar), were screened with Ash-l and Ash-m The of to and were the as those used in the cDNA the cDNA were with of to and at in the region. The DNA was with and to various of and R. PubMed Scopus Google Scholar). were using DNA by the chain method et al., F. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar; et al., J. R. used to were and The were isolated by the method of et al., R.J. PubMed Scopus Google was using The used to Ash-l and Ash-m mRNAs in the were in and The to the are by in The was as of for for 5 and for 5 by of for for and for and by for The were by gel The used to Ash-s mRNAs were and The were by hybridization with the and of of and of Ash-l and coding of Ash-s and were by The was with a at the Ash-m coding were in the as Ash-s that the from the carboxyl-terminal region of Ash-l was utilized as a for Ash-s and were between the and of in and between the in the and an in the region of the The were between the and of a DNA coding the carboxyl-terminal SH3 domains of Ash-l and Ash-m were using oligonucleotides of Ash-l with the and of Ash-l with the as The and were with and into the and of were in at On the the were to with and was the was between and was induced by to the 5 the were and in a and The were and at for The were to a proteins were with and of and cDNA encoding was with at nucleotide and at nucleotide The cDNA was into the The was into the E. for protein were the as was as an the amino to et al., F. K. Y. Takenawa T. J. 1994; PubMed Scopus Google with in pheochromocytoma PC12 cells were with by in for the cells to nerve growth factor for The cells were with in and by and at for at The cell obtained were mixed with of binding proteins of and at The were in and and in was were obtained using for The protein of dynamin and Sos were by using antibody et al., 1994Miki H. Miura K. Matuoka K. Nakata T. Hirokawa N. Orita S. Kaibuchi K. Takai Y. Takenawa T. J. Biol. Chem. 1994; 269: 5489-5492Abstract Full Text PDF PubMed Google and Sos antibody into Balb/c 3T3 and from the were against were to 3T3 cells were in with the cells were of in and and for at proteins or were into the of cells using an and One the cells were to 5 and in the of for the were in and the cells were in for at The of cells DNA was using a cell and of cDNA from and of the phages with Ash/Grb2(Ash-l) cDNA as a probe which to the probe were these cDNA clones, three not to the from the region of the Ash-l nucleotide sequence in sequence that two clones were identical with other for a in and 93% identity in nucleotide sequence with Ash-l cDNA the clones not of the of stop We that these Ash-ψ, were from unusual transcribed The Ash-s had a nucleotide sequence identical with Ash-l cDNA in the first SH3 domain a and the coding sequence was terminated at the end of the SH3 domain by a stop which the sequence from that of Ash-l cDNA. the Ash-s is of a single SH3 domain identical with that of Ash-l. In to search for other we which had signals in the first we polymerase chain the using the and and for Ash-l cDNA. We detected three clones Since these clones were identical with we 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 to 14 amino acid residues between and 170 of Ash-l, which comprise the amino-terminal region of the second SH3 domain to the of the and two isoforms are Ash-l, and and of the Ash-l-encoding and of to that the three isoforms, Ash-l, from a single gene by alternative we isolated genomic clones of the rat Ash-l-encoding gene and gene by and nucleotide sequencing with genomic of a rat genomic DNA genomic clones to the cDNA probe. These clones were into three types by the with One of to genomic DNA encoding T. H. T. K. H. and T. The other and encoded The and carboxyl-terminal were encoded in and and not the nucleotide sequences of the exons in these two clones for the sequence of Ash-l cDNA. Ash-l gene has a of more than 16 kilobases and consists of exons and The nucleotide sequences at the show that with at the end and with at the end This to the and R. P. PubMed Scopus Google Scholar). The genomic of the gene revealed that the two isoforms, Ash-s and are generated from a single gene by unusual alternative splicings. Ash-s mRNAs are of the first two exons and a of the second of the A splicing for the was not in Ash-s Thus, a sequence is in the transcribed region of the Since a stop codon at the boundary of the gene, a of the is as a sequence in Ash-s Ash-s consists of the first SH3 domain of Ash-l. On the other hand, splicing for Ash-m mRNAs at the sequence which is the of at as for Ash-l mRNAs and The first 42 nucleotide residues of the of Ash-l are as a of the in Thus, the of Ash-m the Ash-l and 14 amino acid residues encoding the amino-terminal of the second SH3 domain are defective in and of the and are shown and coding and The the isoforms are to the sequences the splicing The nucleotide sequences are of the the genomic cloned in in the first and the second SH3 domains. in the sequences at sequences at genomic using the region from the in the which is a and and the in the gene of for the of in which two were likely to be in the identical The generated from and for these we that Ash-l and Ash-m were transcribed from a single of with cDNA probe in genomic DNA with those in cloned The probe was a of cDNA which is in the of genomic and cloned DNA were with various and to gel genomic of with and with and and 5 and of Ash-m and and Ash-s mRNAs were detected in various tissues by the reverse-transcribed polymerase chain reaction. The between Ash-m and Ash-l mRNAs tissues of Ash-m than that of Ash-l were in and contrast, Ash-m mRNAs were in and On the other hand, Ash-s mRNAs were detected in and but in and of Ash-l, and reverse-transcribed for Ash-l and Ash-m from various tissues were with reverse-transcribed for Ash-s from various tissues were with hybridization as and to Ash-m and the binding of isoforms, the of proteins were and binding were In binding using the proteins of isoforms demonstrated that Ash-m not with proteins as dynamin and Sos in PC12 cell to Ash-l The of dynamin and Sos were identified by and the binding of Ash-m to Sos was On the other hand, Ash-s formed a complex with dynamin and Sos in PC12 cell lysates, binding to other proteins and proteins not as that of Ash-l. These results were by the cells with nerve growth factor. Ash-s is exclusively of a single SH3 domain. the binding of Ash-s to Sos and dynamin was with that of SH3 domains from and shown in the SH3 domains from and bound less to or dynamin in cell than to the Ash-s SH3 domain associated with The binding of Ash-s to dynamin in PC12 cell was more than that to In contrast, Ash-s bound to Sos more than to dynamin in Balb/c 3T3 cell These results that signaling molecules bound to Ash-s it only the of of isoforms to the proteins in cell lysates. binding of isoforms to Sos and dynamin in PC12 cell lysates. PC12 cells were with in the and of nerve growth factor were to protein and bound proteins were to binding of various SH3 domains to Sos and The SH3 domains from and were as proteins in E. from Balb/c cells and PC12 cells were to SH3 of the of the carboxyl-terminal SH3 domain of Ash-m a of Therefore, the SH3 different proteins from those bound to carboxyl-terminal SH3 of Ash-l. the we 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 shown in carboxyl-terminal SH3 of Ash-m not Sos or it was to be to proteins and proteins shown by Since Ash-m was to be in we also proteins bound to carboxyl-terminal Ash-m SH3 using The protein binding to carboxyl-terminal Ash-m SH3 was different from that of Ash-l and proteins shown by were bound to carboxyl-terminal Ash-m These that Ash-m generates signals different from those of proteins to carboxyl-terminal SH3 domains of Ash-m and Ash-l. or were to protein proteins were by and with SH3 domain of SH3 domain of SH3 domain of Sos and of Ash-l, and Ash-s DNA the of Ash-l, and Ash-s DNA the proteins were into Balb/c 3T3 with various proteins or the cells were with The cells were with and with with to a in the of cells for and was by the proteins On the other hand, injection of Ash-l protein a in in cells from to the not of Ash-s or the inhibited the of with the by Ash-m was less effective than by to Ash-l, Ash-m Ash-s the of These results that Ash-m and Ash-s act as negative regulators in cells and the signaling of Ash-l to DNA of Ash-l, and Ash-s DNA Ash-l, and protein were into Balb/c 3T3 protein was into more than One the cells were with with and for The cells were with and cells were are shown as S.E. in we demonstrated that are at two types of splicing isoforms of Ash/Grb2(Ash-l) as well as unusual transcribed These isoforms are generated by unusual as demonstrated by the isolation and of the Ash-l-encoding The splicing of Ash-m from that of Ash-l in the The Ash-l-encoding gene has the sequence in the which is to the consensus sequence of the splicing PubMed Scopus Google Scholar). This sequence is utilized in Ash-m as a splicing Ash-l which to the consensus sequence and Thus, of the of Ash-l is as the and in Ash-m On the other hand, the splicing for Ash-s from that for Ash-l at the Ash-s a at the signal in the of Ash-l, at the of Ash-l. Thus, the are not alternative is defective in the second SH3 domain. Ash-l associates with Sos and dynamin by of SH3 the of the two SH3 domains is not results show that the binding of Ash-m to proteins Sos and dynamin is than that of Ash-l. Ash-m is not to with Sos In contrast, Ash-s binds to Sos and dynamin the that Ash-s is almost exclusively of the first SH3 domain of Ash-l. the second SH3 of Ash-l is not essential for binding to dynamin and the first SH3 domain to an in by the of the defective SH3 domain in Ash-m for the of association with has been that the two SH3 domains in Ash-l are not and different functions in SH3 domain the gene activity in et al., 1992Clark S.G. Stern M.J. Horvitz H.R. Nature. 1992; 356: 340-344Crossref PubMed Scopus (460) Google Scholar). We are the two for the of Since Ash-m not with Sos of defective SH3 the signals through Ash-m be to dynamin and other proteins the signals cell functions dynamin is the the of the signals be in the by the between the of Ash-m and Ash-l. possibility is that the second SH3 domain in Ash-m generates signals differing from those of Ash-l. The amino acid residues of the region are to those of the region in The newly amino acid sequence by the is to the amino of SH3 as in et al., G. J.P. PubMed Scopus Google Scholar), it from SH3 domains et al., S. H. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). the second SH3 domain of Ash-m is the signals be to other by newly formed SH3 domain by splicing binds different proteins from those bound to the carboxyl-terminal SH3 domain of for it associates with Sos and The of with proteins in PC12 cell from with Balb/c 3T3 cell Since a single SH3 domain it binds to tyrosine kinase receptors has other domain to to signals. Ash-s Ash-l by a different of Ash-l splicing has been et al., I. F. I. J. M. B. Science. 1994; PubMed Scopus Google Scholar). has two SH3 domains but has a from SH2 domain. was that of into 3T3 cells of Ash-m or Ash-s not cell Therefore, Ash-m and Ash-s act as from as We findings a to be used in the of signals from tyrosine kinase receptors to molecules downstream of Ash-l. INTRODUCTIONA novel gene isolated by Clark et al., 1992Clark S.G. Stern M.J. Horvitz H.R. Nature. 1992; 356: 340-344Crossref PubMed Scopus (460) Google Scholar 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 SH21( 1The abbreviations used are: SHSrc homologyPDGFplatelet-derived growth factorSosson of sevenlessPCRpolymerase chain reactionGSTglutathione S-transferaseBrdUrd5-bromo-2-deoxyuridinePIphosphatidylinositolbpbase pair(s)PAGEpolyacrylamide gel electrophoresis.) 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., 1992Matuoka K. Shibata M. Yamakawa A. Takenawa T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9015-9019Crossref PubMed Scopus (137) Google Scholar 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., 1992Lowenstein E.J. Daly R.J. Batzer A.G. Li W. Margolis B. Lammers R. Ullrich A. Skolnik E.Y. Bar-Sagi H. Schlessinger J. Cell. 1992; 70: 431-442Abstract Full Text PDF PubMed Scopus (1331) Google Scholar 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 λgt11 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., 1993Egan S.E. Giddings B.W. Brooks M.W. Buday L. Siezland A.M. Weinberg R.A. Nature. 1993; 363: 45-51Crossref PubMed Scopus (1003) Google Scholar; Rozakis-Adcock et al., 1993Rozakis-Adcock M. Fernley R. Wade J. Pawson T. Bowtell Nature. 1993; 363: 83-85Crossref PubMed Scopus (835) Google Scholar; Li et al., 1993Li N. Batzer A. Daly R. Yajnik V. Skolnik E. Chardin P. Bar S.D. Margolis B. Schlessinger J. Nature. 1993; 363: 85-88Crossref PubMed Scopus (797) Google Scholar; Simon et al., 1993Simon M.A. Dodson G.S. Rubin G.M. Cell. 1993; 73: 169-177Abstract Full Text PDF PubMed Scopus (364) Google Scholar; Olivier et al., 1993Olivier J.P. Raabe T. Henkemeyer M. Dickson B. Mbamalu G. Margolis B. Schlessinger J. Hafen E. Pawson T. Cell. 1993; 73: 179-191Abstract Full Text PDF PubMed Scopus (447) Google Scholar; Chardin et al., 1993Chardin P. Camonis J.H. Gale N.W. van A.L. Schlessinger J. Wigler M.H. Bar S.D. Science. 1993; 260: 1338-1343Crossref PubMed Scopus (648) Google Scholar). 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., 1993Matuoka K. Shibasaki F. Shibata M. Takenawa T. EMBO J. 1993; 12: 3467-3473Crossref PubMed Scopus (100) Google Scholar), controlled by Rac proteins (Ridley and Hall, 1992Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3797) Google Scholar; Ridley et al., 1992Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3049) Google Scholar). In addition, dynamin, which stimulates GTPase activity by binding to microtubules, also binds to Ash-l through the SH3 domains (Gout et al., 1993Gout I. Dhand R. Hiles I.D. Fry M.J. Panayotou G. Das P. Truong O. Totty N.F. Hsuan J. Booker G.W. Campbell I.D. Waterfield M.D. Cell. 1993; 75: 25-36Abstract Full Text PDF PubMed Scopus (482) Google Scholar; Herskovits et al., 1993Herskovits J.S. Shpetner H.S. Burgess C.C. Vallee R.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11468-11472Crossref PubMed Scopus (126) Google Scholar; Miki et al., 1994Miki H. Miura K. Matuoka K. Nakata T. Hirokawa N. Orita S. Kaibuchi K. Takai Y. Takenawa T. J. Biol. Chem. 1994; 269: 5489-5492Abstract Full Text PDF PubMed Google Scholar). 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 of the isoforms as revealed by genomic of the Ash-l-encoding gene, and the of these isoforms in mitogenic signaling.