©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning of Rodent p72
EVIDENCE OF ALTERNATIVE mRNA SPLICING (*)

R. Bruce Rowley (§) , Joseph B. Bolen , Joseph Fargnoli

From the (1) Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Northern blot analysis of polyadenylated RNA prepared from RBL-2H3 cells revealed the presence of three distinct mRNAs encoding p72, 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.


INTRODUCTION

One of the primary responses observed following antigen-induced cross-linking of the high affinity IgE receptor (FcRI)() 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 FcRI 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.

Recent evidence suggests that engagement of the FcRI 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 FcRI receptor from activated cells (12, 13) . While these results clearly suggest a role for both of these kinases in mediating signal transduction through the FcRI, the exact nature of their roles remains unclear.

In order to gain a better understanding of the role of p72 in FcRI-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.


MATERIALS AND METHODS

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) .


RESULTS

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.

Sequencing of the original clones also indicated that cDNAs encoding two different isoforms of the p72 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.

Recent evidence indicates the possibility that a family of p72-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).

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 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.

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 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.




DISCUSSION

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 FcRI is p72.

The results of the Northern blot analysis presented in Fig. 1prompted the cloning of cDNAs encoding p72, with the intention of identifying the molecular differences underlying the presence of three apparent messages encoding p72 in RBL-2H3 cells.

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 message.

Herein we report the molecular cloning of two cDNAs encoding the rat homolog of p72 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.

The 23-amino acid insert appears to have little effect upon p72 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).

It should be noted that, during completion of this work, potential alternative splicing of p72 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) .

It is interesting that a comparison of the amino acid sequences of the rat, porcine, and human p72 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.

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 family member protein-tyrosine kinases with other proteins in a tissue-specific manner.

It is interesting that the rat p72 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.


FOOTNOTES

*
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) U21683 (SykA) and U21684 (SykB).

§
To whom correspondence and reprint requests should be addressed: H4114, Dept. of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000. Tel.: 609-252-5497; Fax: 609-252-6051.

The abbreviations used are: FcRI, high affinity IgE receptor; SH, src homology; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; kb, kilobase(s).


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Benhamou, M., Gutkind, J. S, Robbins, K. C., and Siraganian, R. P. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5327-5330 [Abstract]
  2. Connolly, P. A., Farrell, C. A., Merenda, J. M., Conklyn, M. J., and Showell, H. J.(1991) Biochem. Biophys. Res. Commun. 177, 192-201 [Medline] [Order article via Infotrieve]
  3. Yu, K.-T., Lyall, R., Jariwala, N., Zilberstein, A., and Haimovich, J. (1991) J. Biol. Chem. 266, 22564-22568 [Abstract/Free Full Text]
  4. Li, W., Deanin, G. G., Margolis, B., Schlessinger, J., and Oliver, J. M.(1992) Mol. Cell. Biol. 12, 3176-3182 [Abstract]
  5. Kawakami, T., Inagaki, N., Takei, M., Fukamachi, H., Coggeshall, K. M., Ishizaka, K., and Ishizaka, T.(1992) J. Immunol. 148, 3513-3519 [Abstract/Free Full Text]
  6. Paolini, R., Jouvin, M. H., and Kinet, J.-P.(1991) Nature 353, 855-858 [CrossRef][Medline] [Order article via Infotrieve]
  7. Shimizu, A., Tepler, I., Benfey, P. N., Berenstein, E. H., Siraganian, R. P., and Leder, P.(1988) Proc. Natl. Acad Sci. U. S. A. 85, 1907-1911 [Abstract]
  8. Kinet, J.-P., Blank, U., Ra, C., White, K., Metzger, H., and Kochan, J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6483-6487 [Abstract]
  9. Kuster, H., Thompson, H., and Kinet, J.-P.(1990) J. Biol Chem. 265, 6448-6452 [Abstract/Free Full Text]
  10. Blank, U., Ra, C., Miller, L., White, K., Metzger, H., and Kinet, J.-P. (1989) Nature 337, 187-189 [CrossRef][Medline] [Order article via Infotrieve]
  11. Alber, G., Miller, L., Jelsema, C. L., Varin-Blank, N., and Metzger, H. (1991) J. Biol. Chem. 266, 22613-22620 [Abstract/Free Full Text]
  12. Eiseman, E., and Bolen, J.(1992) Nature 355, 78-80 [CrossRef][Medline] [Order article via Infotrieve]
  13. Hutchcroft, J. E., Geahlen, R. L., Deanin, G. G., and Oliver, J. M. (1992) Proc. Natl. Acad. Sci. U. S.A. 89, 9107-9111 [Abstract]
  14. Barsumian, E. L., Isersky, C., Petrino, M. G., and Siraganian, R. P. (1981) Eur. J. Immunol. 11, 317-323 [Medline] [Order article via Infotrieve]
  15. Taniguchi, T., Kobayashi, T., Kondo, J., Takahashi, K., Nakamura, H., Suzuki, J., Nagai, K., Yamada, T., Nakamura, S., and Yamamura, H. (1991) J. Biol. Chem. 266, 15790-15796 [Abstract/Free Full Text]
  16. Sanger, F., Nicklen, S., and Coulson, A. R.(1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  17. Veillette, A., Bookman, M. A., Horak, E. M., and Bolen, J. B.(1988) Cell 55, 301-308 [Medline] [Order article via Infotrieve]
  18. Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U. K. (1977) J. Biol. Chem. 252, 1102-1106 [Abstract]
  19. Chan, A. C., Iwashima, M., Turck, C. W., and Weiss, A.(1992) Cell 71, 649-662 [Medline] [Order article via Infotrieve]
  20. Burkhardt, A. L., Brunswick, M., Bolen, J. B., and Mond, J. J.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7410-7414 [Abstract]
  21. Law, C.-L., Sidorenko, S. P., Chandran, K. A., Draves, K. E., Chan, A. C., Weiss, A., Edelhoff, S., Disteche, C. M., and Clark, E. A.(1994) J. Biol Chem. 269, 12310-12319 [Abstract/Free Full Text]
  22. Yi, T., Bolen, J. B., and Ihle, J. N.(1991) Mol Cell. Biol. 11, 2391-2398 [Medline] [Order article via Infotrieve]
  23. Stanley, E., Ralph, S., McEwan, S., Boulet, I., Holtzman, D. A., Lock, P., and Dunn, A. R.(1991), Mol. Cell. Biol. 11, 3399-3406 [Medline] [Order article via Infotrieve]
  24. Cooke, M. P., and Perlmutter, R. M.(1989) New Biol. 1, 66-74 [Medline] [Order article via Infotrieve]
  25. Levy, J. B., Dorai, T., Wang, L.-H., and Brugge, J.(1987) Mol. Cell. Biol. 7, 4142-4145 [Medline] [Order article via Infotrieve]
  26. Martinez, R. B., Mathey-Prevot, B., Bernards, A., and Baltimore, D. (1987) Science 237, 411-415 [Medline] [Order article via Infotrieve]
  27. Pyper, J. M., and Bolen, J. B.(1990) Mol. Cell. Biol. 10, 2035-2040 [Medline] [Order article via Infotrieve]
  28. Yagi, S., Suzuki, K., Hasegawa, A., Okumura, K., and Ra, C.(1994) Biochem. Biophys. Res. Commun. 200, 28-34 [CrossRef][Medline] [Order article via Infotrieve]
  29. Maniatis, T., Fritsch, E. F., and Sambrook, J.(1984) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.