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RA70 Is a Src Kinase-Associated Protein Expressed Ubiquitously
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
252, 738 –742 (1998)
RC989637
RA70 Is a Src Kinase-Associated Protein Expressed Ubiquitously Yoriko Kouroku,* Akiko Soyama,* Eriko Fujita,* Koko Urase,* Toshifumi Tsukahara,† and Takashi Momoi*,1 *Division of Development and Differentiation, †Department of Neuromuscular Research, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187-8502, Japan
Received October 2, 1998
RA70, which is expressed during neuronal differentiation of P19 EC, is highly homologous to human src kinase-associated phosphoprotein (SKAP55). Here we isolated human full-length RA70 cDNA. Unlike SKAP55, which is specifically expressed in thymus and T cells, RA70 was expressed ubiquitously in various tissues including lung, skeletal muscle, and spleen, and in various cell lines including human monocytic leukemia (U937) cells, but RA70 was undetectable in thymus and T cell lymphoma (Jurkat) cells. RA70 as well as SKAP55 proved to be a protein with molecular weight 55 kDa associated with SH2 domain of Fyn. Interaction between RA70 and src family kinases, Fyn, Hck and Lyn, was detected during monocytes/ macrophage-differentiation of U937 cells. Thus, like SKAP55, RA70 is an adaptor protein of the src family kinases. RA70 may play an essential role in the src signaling pathway in various cells. © 1998 Academic Press
Src family kinases, nonreceptor protein tyrosine kinases such as Fyn, Lyn, Hck, and Lck, are involved in the signal transduction of both tyrosine kinase receptors and non-tyrosine kinase receptors in various cells (1-11); Fyn, Lyn and Lck through their src homology 2 (SH2) domain, are associated with Fc receptors and multiple T cell receptor-CD3 complexes and are involved in the proliferation signals of T cells and B cells (1-7). Fyn is also associated with platelet-derived growth factor receptor and epidermal growth factor receptor with tyrosine kinase activity (8,9). Hck is a downstream signal of leukocyte inhibitory factor receptor in embryonal stem cells (10). Very recently, Fyn was shown to be associated with Trk B, receptor for neurotrophin, and to be involved in the neurotrophin signal transduction pathways in rat cortical neurons (11). 1 To whom correspondence should be addressed. Fax: 81-042-3461754. E-mail: [email protected].
0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
These src family kinases are also involved in the differentiation of various cells (12-13). Fyn and Lyn are expressed during monocytes/macrophage-differentiation (12). Fyn-deficient mice show impaired hippocampal development and impaired long-term potentiation (13). However, little is known about the downstream of the src signaling pathway. P19 EC cells differentiate into neuronal cells by culturing with retinoic acid (RA) in the aggregate form (14). A RA-inducible gene, termed RA70, which we isolated during investigation of the gene specifically expressed during neuronal differentiation of P19 EC cells using differential hybridization method, is highly homologous to human src kinase-associated phosphoprotein (SKAP55) (submitted elsewhere). SKAP55, which is expressed preferentially in thymus and human T cell lymphoma (Jurkat) cells, binds selectively in vitro to SH2 domains of Lck and Fyn. SKAP55 is an adaptor protein likely involved in Fyn-mediated signaling in human T-lymphocytes (15). However, SKAP55 alone is not sufficient to play a role in the src signaling pathway required in the various cells. Many src-specific adaptor proteins are assumed to be necessary for the src signaling pathway. Csk, C-terminal src kinase, which regulates the src kinase activity, plays a role in the regulation of cell adhesion during neuronal differentiation of P19 EC cells induced by RA (16), suggesting that RA70 plays a role as an adaptor protein of src family kinases during neuronal differentiation. However, since little is known about src family kinases expressed during neuronal differentiation of P19 EC cells, we could not identify the src family kinases specifically associated with RA70. To make clear the function of RA70 as an adaptor protein of src family kinases and its biological significance, we intended to examine the interaction between RA70 and src family kinases in human cell lines, in which src family kinase signals are well studied. Unlike SKAP55, RA70 was expressed ubiquitously in various human tissues and cell lines including U937 cells,
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but not in thymus and T cell line. Since functions of src family kinases are well known in U937 cells (12), we examined the biological roles of RA70 during monocytes/macrophage-differentiation of U937 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Herein we demonstrate that, like SKAP55, RA70 interacts with Fyn SH2 domain and is associated with Fyn, Hck and Lyn during monocytes/macrophage-differentiation. RA70 is another adaptor protein in src-mediated signaling in non-T cells. MATERIALS AND METHODS Reverse transcription-polymerase chain reaction (RT-PCR) products using consensus primers of RA70 and SKAP55. Total RNA was prepared from untreated and RA-treated P19 EC cells, U937 cells, and Jurkat cells according to the guanidium thiocyanate method (17). Complementary DNAs were synthesized from total RNAs (1 mg) by a reverse transcriptase (Stratagene, LaJolla, CA) and were subjected to PCR as described in the manual from Perkin-Elmer (Branchburg, NJ) using primers degenerate for the consensus of mouse RA70 cDNA fragment and human SKAP55: forward primer; 59-GA(A/G)TGGCAGAA(A/G)CG(G/A)TGGTGT-39 and reverse primer; 59-GCA(G/A)TCCCA(C/T)AG(A/G)CCCTGGTA-39. The gene was amplified as follows: 1 cycle at 95°C for 2 min, 30 cycles at 95°C for 1 min and 60°C for 2 min, and 1 cycle at 60°C for 7 min. The RT-PCR products were cloned into the pGEM-T vector (Promega, Madison, WI) by TA cloning and sequenced with a fully automated DNA sequencer ALFII (Pharmacia, Milwaukee, WI). Northern blot hybridization. Nylon filters with mRNAs from multiple human tissues and cell lines (Clontech Laboratories, Inc., CA) were prehybridized and then hybridized with 32P-labeled RA70 cDNA fragment, which was labeled using the random primer method (Amersham, Buckingamshare, UK). After overnight hybridization in buffer containing 5 x SSC (1x SSC; 0.15 M NaCl, 0.015 M NaCitrate, pH 7.0) and 50% formamide at 42°C, the filters were washed twice with 2 x SSC, 0.1% sodium dodecyl sulfate (SDS) for 30 min at 42°C, followed by two washes with 0.1 x SSC, 0.1% SDS for 30 min each at 42°C. The filters were air dried and autoradiographed at 280°C for 7 days. Isolation of full length human RA70 cDNA. Full length human RA70 cDNA was obtained by screening a cDNA library from human fetal lung (Stratagene, LaJolla, CA) using an human RA70 cDNA fragment as a probe. Both strands were sequenced. Transfection. RA70 was amplified by PCR from cDNA of the isolated human RA70: forward primer; 59-GAGAATTCAATGCCCAACCCCAGCTGTA-39 and reverse primer; 59-GACTCGAGTCAAATATCATACATCTCC-39. The condition was 1 cycle at 95°C for 2 min, 20 cycles at 95°C for 1 min and 60°C for 2 min, and 1 cycle at 60°C for 7 min. The PCR product of RA70 was cloned into the pGEM-T vector by TA cloning and then cloned in-frame into the EcoR I site of the FLAG expression vectors with CMV promoter (Kodak, New Haven, CT). Restorations of reading frames were confirmed by DNA sequencing. FLAG-RA70 was transfected into Cos cells according to the calcium-phosphate method (18). Preparation of antiserum against RA70. The cDNA encoding C-terminal region of human RA70 was amplified by RT-PCR using the following primers under the condition as described above: forward primer; 59-GAGCATATGAAAAGCACTGATTACGCTA-39 and reverse primer; 59-GACTCGAGTCAAATATCATACATCTCC-39. The PCR products were then subcloned in-frame into the Nde I and Xho I site of the Histag-pET15b vector (Novagen, Madison, WI) and transformed into DE3 cells. Restorations of reading frames were confirmed by DNA sequencing. The Histag-fusion protein of
C-terminal region of human RA70 was produced by culturing transformed DE3 cells in the presence of 2 mM isopropylthiogalactopyranoside (IPTG; Wako Pure Chemical In., Osaka, Japan). The Histag-fusion protein was extracted with 6 M urea and purified from cell extracts by Nickel-affinity column chromatography. The antiserum against RA70 was raised by injecting the fusion protein with Freund’s complete adjuvant into rabbits. The antiserum was used for experiments after anti-Histag antibody was absorbed with Histag peptides-conjugated Sepharose. Immunoblot analysis. The cell pellets were suspended in the lysis buffer ( 50 mM Tris-HCl, pH7.4, 150 mM NaCl, 1 mM Na3VO4, 10 mM NaF, 1 mg/ml leupeptin ) and sonicated on ice. After centrifugation at 10,000 x g for 10 min, the cell extracts (20 mg protein) were subjected to SDS polyacrylamide gel (10%) electrophoresis (19). Proteins of the gels were electrophoretically transferred to nitrocellulose filters (S & S, Dassel, Germany) (20). After filters were incubated with monoclonal anti-FLAG antibody (Kodak, New Haven, CT), monoclonal anti-Fyn, anti-Lck antibodies (Santa Cruz Biot., Santa Cruz, CA), monoclonal anti-Hck, anti-Lyn antibodies (Transduction Lab., Lexington, KY), and anti-RA70 antibodies, respectively, reactivities were detected by alkaline phosphatase-conjugated goat antimouse or anti-rabbit immunoglobulin (IgG) (Promega, Madison, WI), respectively and nitro blue tetrazolium and 5-bromo-4-chloro-3indolyl-1-phosphate. Interaction between RA70 and Fyn-SH2 peptide. The cell extracts (200 mg protein) of U937 cells and Jurkat cells were incubated with Fyn-SH2 peptides-conjugated agarose (Santa Cruz Biot.) or protein A and G-conjugated agarose (Santa Cruz Biot.) in the presence or absence of 1% Triton X-100 at 4°C overnight and centrifuged. The precipitates were subjected to immunoblot analysis using anti-RA70. Interaction between RA70 and src kinases. The cell pellets of TPA (Sigma, St. Louis, MO)-treated and untreated U937 cells were sonicated with the lysis buffer containing 0.1% Triton X-100 on ice and centrifuged. The cell extracts (200 mg protein) were incubated with anti-Fyn, anti-Hck, anti-Lyn, and anti-Lck antibodies at 4 °C overnight, respectively, and immunocomplexes were precipitated with protein A and G-conjugated agarose. Immunoprecipitates were subjected to SDS-polyacrylamide gel (10%) electrophoresis and immunoblot analysis using anti-RA70.
RESULTS AND DISCUSSIONS Expression of RA70 in Various Tissues and Various Cell Lines We isolated human RA70 cDNA fragment by RT-PCR using consensus primers of mouse RA70 and human SKAP55. RT-PCR fragments (537-bp and 519-bp) with similar size of mouse RA70 cDNA fragment were detected in Jurkat cells and U937 cells, respectively (Figure 1A). To distinguish RT-PCR fragments, RT-PCR products were digested with Spe I, an unique site in SKAP55 fragment. PCR fragment (537-bp) of Jurkat cells was cleaved into 199-bp and 338-bp, but PCR fragment (519bp) of U937 cells was not cleaved (Figure 1A). DNA sequence analysis confirmed that 537-bp PCR fragment of Jurkat cells was SKAP55 and 519-bp PCR fragment of U937 cells was human homologue of RA70. Northern blot analysis revealed that RA70 was preferentially expressed in HeLa cells, chronic myelogenous leukemia cells (K-562), and colorectal adenocarcinoma cells (SW480) (Figure 1B). RA70 was also expressed in human promyelocytic leukemia cells
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FIG. 1. Expression of RA70 in human cell lines and tissues. (A) The expression of RA70 and SKAP55 by the RT-PCR analysis. Lane 1 and 2, RT-PCR products of the untreated and RA-treated (for 2 days) P19 EC cells, respectively; lane 3 and 4, RT-PCR products of U937 cells undigested and digested with Spe I, respectively; lane 5 and 6, RT-PCR products of Jurkat cells undigested and digested with Spe I, respectively. A thick black arrow indicates the mouse RA70 cDNA fragment (519-bp) detected in the RA-treated P19 EC cells. Thin black arrows indicate cDNA fragments (537-bp and 519-bp) Jurkat cells and U937 cells with similar size of the mouse RA70 cDNA fragment, respectively. Thick white arrows indicate RT-PCR products cleaved by Spe I. Arrowheads indicate the RT-PCR products unrelated to RA70 and SKAP55. M; DNA size marker. (B) Northern blot hybridization of RA70 in human cell lines and various tissues. Lane 1, HL-60; lane 2, HeLa; lane 3, K-562; lane 4, MOLT-4; lane 5, Raji; lane 6, SW480; lane 7, A549; lane 8, G361; lane 9, heart; lane 10, brain; lane 11, placenta; lane 12, lung; lane 13, liver; lane 14, skeletal muscle; lane 15, kidney; lane 16, spleen; lane 17, thymus; lane 18, prostate; lane 19, testis; lane 20, ovary; lane 21, small intestine; lane 22, colon; and lane 23, peripheral blood leukocytes. Thick arrows indicate RA70 signals.
(HL60), lung adenocarcinoma cells (A549), and melanoma cells (G361), but RA70 was not detected in lymphoblastic leukemia cells (MOLT-4) and Burkitt’s lymphoma cells (Raji). Strong RA70 signals were detected in the human lung, skeletal muscle, kidney, peripheral blood leukocytes, and spleen tissues; weak signals were detected in the brain, placenta, heart, prostate, testis, ovary, and colon tissues; and signals were undetectable in the thymus, liver, and small intestine (Figure 1B).
Isolation of RA70 A full-length human RA70, which was isolated from human fetal lung cDNA library, encoded a 359-amino acid protein. The deduced human RA70 protein was homologous to human SKAP55 sequences in the NCBI database (Figure 2). Sequence comparison indicated that human RA70 shared 43% overall amino acid identity and 6% similarity with human SKAP55. RA70 residues 115 to 214 and 302 to 359 showed high homol-
FIG. 2. Deduced amino acid sequence of human RA70. Comparison of amino acid sequence of human RA70 and SKAP55. Shadow boxes and open boxes indicate identical amino acids and similar amino acids, respectively. Single and double underlines indicate pleckstrin domain and SH3 domain in SKAP55, respectively. Closed circles, closed and open triangles indicate putative phosphorylation sites of protein kinase C or casein kinase, cyclic AMP dependent kinase, and tyrosine kinase, respectively. Asterisks indicate putative N-glycosylation sites. Nucleotide sequence data of human RA70 have been deposited with DDBJ Data Libraries under Accession No. ABO14486. 740
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FIG. 3. Identification of RA70 protein. (A) Detection of molecular mass of FLAG-tagged RA70 expressed in the transfected Cos cells. Cytosolic proteins were extracted from Cos cells transfected with vector alone (lane 1), and FLAG-tagged RA70 (lane 2). The cytosolic extracts (20 mg protein) were analyzed by SDS-polyacrylamide gel and immunoblotting using anti-RA70 and anti-FLAG. A band of 55 kDa was detected by anti-FLAG and anti-RA70. (B) RA70 protein in U937 cells. Anti-RA70 reacted a band of 55 kDa in U937 cells, but not in Jurkat cells. Lane 1, U937 cells; lane 2, Jurkat cells. U and J, U937 cells and Jurkat cells, respectively. M, rainbow molecular marker of proteins. Thick arrows indicate a 55 kDa band reacting with anti-RA70 and thin arrow indicates a 55 kDa band reacting with anti-FLAG.
ogy with the SKAP55 pleckstrin domain (residues 106 to 205, 56% identity and 13% similarity) and the C-terminal SH3 domain (residues 300 to 356, 63% identity and 7% similarity). In place of the putative tyrosine phosphorylation site (EDIYEVL) in SKAP55, which is predicted to bind to isolated SH2 domains of src kinases with high affinity based on its homology with EXXYXXL (21), RA70 contained a DEIYEEL sequence and several putative phosphorylation sites. A part of the 39-nucleotide sequence of human RA70 was found in a human PAC clone (DJ1139P01 from 7p15p21; accession number 003999, human genome database), indicating that the human RA70 gene resides on chromosome 7.
FIG. 4. Interaction between RA70 and Fyn-SH2 peptide. Interaction between RA70 and Fyn-SH2 was examined in U937 cells. Cytosolic proteins of U937 cells (lane 1 to 4) and Jurkat cells (lane 5 to 8) were incubated with Fyn-SH2 peptide-conjugated agarose (lane 1, 3, 5, and 7) or protein A and G-conjugated agarose (lane 2, 4, 6, and 8) in the presence (lane 3, 4, 7, and 8) or absence (lane 1, 2, 5, and 6) of 1% Triton X-100 at 4°C overnight. The precipitates were subjected to the immunoblot analysis using anti-RA70.
Interaction between RA70 and Src Kinases Since RA70 was shown to be highly homologous to SKAP55 (Figure 2), RA70 was expected to have a similar function as SKAP55. RA70 (55 kDa) in U937 cell extracts was precipitated by Fyn-SH2 peptide-conjugated agarose, but not by protein A and G-conjugated agarose (Figure 4). RA70 was not detected in the precipitate of Jurkat cell extracts. The interaction between RA70 and Fyn-SH2 peptides was decreased by 1% Triton X-100, but still stable. These results suggest that RA70, along with SKAP55, is strongly bound with Fyn through interaction with its SH2 domain. Since src kinases are expressed during monocytes/ macrophage differentiation (12), we examined the interaction between the RA70 and src kinases during monocytes/macrophage differentiation of U937 cells in-
The Protein Encoded by RA70 Although a calculated molecular mass of RA70 is 41.2 kDa, transfection of FLAG-tagged RA70 caused expression of a 55 kDa protein, with which antiFLAG and anti-RA70 antibodies reacted (Figure 3A). Consistent with the results of the RT-PCR analysis, the 55 kDa band was detected in U937 cells, but not in Jurkat cells by immunoblot analysis using antiRA70 (Figure 3B). These results suggest that molecular weight of RA70 shown by SDS-polyacrylamide gel electrophoresis is 55 kDa. Human SKAP55, which has a calculated molecular mass of 41.3 kDa, is also shown to be 55 kDa by SDS-polyacrylamide gel electrophoresis (15). Like SKAP55, mobility of RA70 protein in the SDS-polyacrylamide gel may be influenced by conformational effects and/or posttranslational modifications.
FIG. 5. The expression of RA70 and src kinases and interaction between RA70 and src kinases during monocytes/macrophage differentiation of U937 cells induced by TPA. U937 cells were treated with TPA (10 ng/ml) for 0 h (lane 1), 8 h (lane 2), 12 h (lane 3), and 24 h (lane 4). The expression of RA70 and src kinases were detected by immunoblot analysis. Interaction between RA70 and src kinases was detected by immunoprecipitation with immunoblot analysis. RA70 was detected in the immunoprecipitates by anti-Fyn, anti-Hck, antiLyn, and anti-Lck antibodies by immunoblot analysis using antiRA70.
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duced by TPA. Expression of RA70 and src kinases, Fyn, Hck, and Lyn, was detected during differentiation, but the expression of Lck was undetectable level. The interaction between the src kinases and RA70 was examined using immunoprecipitation with immunoblotting (Figure 5). RA70 was detected in the immunoprecipitates by antiFyn, anti-Hck, and anti-Lyn antibodies during differentiation, but not in the immunoprecipitates by anti-Lck antibodies; RA70 was detected in the immunoprecipitates by anti-Hck of U937 cells treated with TPA for 8, 12, and 24 h and was faintly detected only in the immunoprecipitates by anti-Fyn of the cells treated with TPA for 24 h. Thus RA70 was more preferentially associated with Hck or Fyn in the TPA-treated cells than the untreated cells. RA70 may play a role in the monocytes/macrophage differentiation through interaction with Hck and/or Fyn. RA70 is an adaptor protein of src kinases and may play essential roles in the src signaling pathway in non-T cells. ACKNOWLEDGMENTS This work was supported by a grant from the Human Science Foundation for HIV and Grants-in-Aid for Scientific Research on Priority Areas (No. 09254104) from the Ministry of Education, Science and Culture of Japan.
REFERENCES 1. Samelson, L. E., Philips, A. F., Loung, E. T., and Klasuner, R. D. (1990) Proc. Natl. Acad. Sci. USA 87, 4358 – 4362. 2. Cooke, J. P., Abraham, K. M., Forbush, K. A., and Perlmutter, R. M. (1991) Cell 65, 281–291. 3. Gassmann, M., Guttinger, M., Amrein, K. E., and Burn, P. (1992) Eur. J. Immunol. 22, 283–286. 4. Sugie, K., Kawakami, T., Maeda, Y., Kawabe, T., Uchida, A., and Yodoi, J. (1991) Proc. Natl. Acad. Sci. USA 88, 9123–9135.
5. Campbell, M., and Sefton, B. M. (1992) Mol. Cell. Biol. 12, 2315–2321. 6. Yellen-Shaw, A. J., and Monoe, J. G. (1992) J. Exp. Med. 176, 129 –137. 7. Ghazizadeh, S., Bolen, J. B., and Fleit, H. B. (1994) J. Biol. Chem. 269, 8878 – 8884. 8. Kypta, R. M., Goldberg, Y., Ulug, E. T., and Courtneidge, S. A. (1990) Cell 62, 481– 492. 9. Margolis, B., Silvnnoinen, O., Comoglio, F., Roonpraunt, C., Skolnik, E., Ullrich, A., and Schlessinger, J. (1992) Proc. Natl. Acad. Sci. USA 89, 8894 – 8898. 10. Ernst, M., Gearing, D. P., and Dunn, A. R. (1994) EMBO J. 13, 1574 –1584. 11. Iwasaki, Y., Gay, B., Wada, K., and Koizumi, S. (1998) J. Neurochem. 71, 106 –111. 12. Katagiri, K., Katagiri, T., Koyama, Y., Morikawa, M., Yamamoto, T., and Yoshida, T. (1991) J. Immunol. 146, 701–707. 13. Grant, S. G., O’Dell, T. J., Karl, K. A., Stein, P. L., Soriano, P., and Kandel, E. R. (1992) Science 258, 1903–1910. 14. McBurney, M. W., Jones-Villeneuve, E. M. V., Edwards, M. K. S., and Anderson, P. J. (1982) Nature 299, 165–167. 15. Marie-Cardine, A., Bruyns, E., Eckerskorn, C., Kirchgessner, H., Meuer, S. C., and Schraven, B. (1997) J. Biol. Chem. 272, 16077– 16080. 16. Takayama, Y., Nada, S., Nagai, K., and Okada, M. (1997) FEBS Lett. 406, 11–16. 17. Sambrook, J., Frritch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, Cold Spring Harbor, NY. 18. Graham, F. L., and Van der Eb, A. J. (1973) Virology 52, 456 – 467. 19. Lammli, U. K. (1970) Nature (Lond.) 227, 680 – 685. 20. Towbin, H., Staeheling, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350 – 4354. 21. Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72, 767–778.
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RC989637
RA70 Is a Src Kinase-Associated Protein Expressed Ubiquitously Yoriko Kouroku,* Akiko Soyama,* Eriko Fujita,* Koko Urase,* Toshifumi Tsukahara,† and Takashi Momoi*,1 *Division of Development and Differentiation, †Department of Neuromuscular Research, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187-8502, Japan
Received October 2, 1998
RA70, which is expressed during neuronal differentiation of P19 EC, is highly homologous to human src kinase-associated phosphoprotein (SKAP55). Here we isolated human full-length RA70 cDNA. Unlike SKAP55, which is specifically expressed in thymus and T cells, RA70 was expressed ubiquitously in various tissues including lung, skeletal muscle, and spleen, and in various cell lines including human monocytic leukemia (U937) cells, but RA70 was undetectable in thymus and T cell lymphoma (Jurkat) cells. RA70 as well as SKAP55 proved to be a protein with molecular weight 55 kDa associated with SH2 domain of Fyn. Interaction between RA70 and src family kinases, Fyn, Hck and Lyn, was detected during monocytes/ macrophage-differentiation of U937 cells. Thus, like SKAP55, RA70 is an adaptor protein of the src family kinases. RA70 may play an essential role in the src signaling pathway in various cells. © 1998 Academic Press
Src family kinases, nonreceptor protein tyrosine kinases such as Fyn, Lyn, Hck, and Lck, are involved in the signal transduction of both tyrosine kinase receptors and non-tyrosine kinase receptors in various cells (1-11); Fyn, Lyn and Lck through their src homology 2 (SH2) domain, are associated with Fc receptors and multiple T cell receptor-CD3 complexes and are involved in the proliferation signals of T cells and B cells (1-7). Fyn is also associated with platelet-derived growth factor receptor and epidermal growth factor receptor with tyrosine kinase activity (8,9). Hck is a downstream signal of leukocyte inhibitory factor receptor in embryonal stem cells (10). Very recently, Fyn was shown to be associated with Trk B, receptor for neurotrophin, and to be involved in the neurotrophin signal transduction pathways in rat cortical neurons (11). 1 To whom correspondence should be addressed. Fax: 81-042-3461754. E-mail: [email protected].
0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
These src family kinases are also involved in the differentiation of various cells (12-13). Fyn and Lyn are expressed during monocytes/macrophage-differentiation (12). Fyn-deficient mice show impaired hippocampal development and impaired long-term potentiation (13). However, little is known about the downstream of the src signaling pathway. P19 EC cells differentiate into neuronal cells by culturing with retinoic acid (RA) in the aggregate form (14). A RA-inducible gene, termed RA70, which we isolated during investigation of the gene specifically expressed during neuronal differentiation of P19 EC cells using differential hybridization method, is highly homologous to human src kinase-associated phosphoprotein (SKAP55) (submitted elsewhere). SKAP55, which is expressed preferentially in thymus and human T cell lymphoma (Jurkat) cells, binds selectively in vitro to SH2 domains of Lck and Fyn. SKAP55 is an adaptor protein likely involved in Fyn-mediated signaling in human T-lymphocytes (15). However, SKAP55 alone is not sufficient to play a role in the src signaling pathway required in the various cells. Many src-specific adaptor proteins are assumed to be necessary for the src signaling pathway. Csk, C-terminal src kinase, which regulates the src kinase activity, plays a role in the regulation of cell adhesion during neuronal differentiation of P19 EC cells induced by RA (16), suggesting that RA70 plays a role as an adaptor protein of src family kinases during neuronal differentiation. However, since little is known about src family kinases expressed during neuronal differentiation of P19 EC cells, we could not identify the src family kinases specifically associated with RA70. To make clear the function of RA70 as an adaptor protein of src family kinases and its biological significance, we intended to examine the interaction between RA70 and src family kinases in human cell lines, in which src family kinase signals are well studied. Unlike SKAP55, RA70 was expressed ubiquitously in various human tissues and cell lines including U937 cells,
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but not in thymus and T cell line. Since functions of src family kinases are well known in U937 cells (12), we examined the biological roles of RA70 during monocytes/macrophage-differentiation of U937 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Herein we demonstrate that, like SKAP55, RA70 interacts with Fyn SH2 domain and is associated with Fyn, Hck and Lyn during monocytes/macrophage-differentiation. RA70 is another adaptor protein in src-mediated signaling in non-T cells. MATERIALS AND METHODS Reverse transcription-polymerase chain reaction (RT-PCR) products using consensus primers of RA70 and SKAP55. Total RNA was prepared from untreated and RA-treated P19 EC cells, U937 cells, and Jurkat cells according to the guanidium thiocyanate method (17). Complementary DNAs were synthesized from total RNAs (1 mg) by a reverse transcriptase (Stratagene, LaJolla, CA) and were subjected to PCR as described in the manual from Perkin-Elmer (Branchburg, NJ) using primers degenerate for the consensus of mouse RA70 cDNA fragment and human SKAP55: forward primer; 59-GA(A/G)TGGCAGAA(A/G)CG(G/A)TGGTGT-39 and reverse primer; 59-GCA(G/A)TCCCA(C/T)AG(A/G)CCCTGGTA-39. The gene was amplified as follows: 1 cycle at 95°C for 2 min, 30 cycles at 95°C for 1 min and 60°C for 2 min, and 1 cycle at 60°C for 7 min. The RT-PCR products were cloned into the pGEM-T vector (Promega, Madison, WI) by TA cloning and sequenced with a fully automated DNA sequencer ALFII (Pharmacia, Milwaukee, WI). Northern blot hybridization. Nylon filters with mRNAs from multiple human tissues and cell lines (Clontech Laboratories, Inc., CA) were prehybridized and then hybridized with 32P-labeled RA70 cDNA fragment, which was labeled using the random primer method (Amersham, Buckingamshare, UK). After overnight hybridization in buffer containing 5 x SSC (1x SSC; 0.15 M NaCl, 0.015 M NaCitrate, pH 7.0) and 50% formamide at 42°C, the filters were washed twice with 2 x SSC, 0.1% sodium dodecyl sulfate (SDS) for 30 min at 42°C, followed by two washes with 0.1 x SSC, 0.1% SDS for 30 min each at 42°C. The filters were air dried and autoradiographed at 280°C for 7 days. Isolation of full length human RA70 cDNA. Full length human RA70 cDNA was obtained by screening a cDNA library from human fetal lung (Stratagene, LaJolla, CA) using an human RA70 cDNA fragment as a probe. Both strands were sequenced. Transfection. RA70 was amplified by PCR from cDNA of the isolated human RA70: forward primer; 59-GAGAATTCAATGCCCAACCCCAGCTGTA-39 and reverse primer; 59-GACTCGAGTCAAATATCATACATCTCC-39. The condition was 1 cycle at 95°C for 2 min, 20 cycles at 95°C for 1 min and 60°C for 2 min, and 1 cycle at 60°C for 7 min. The PCR product of RA70 was cloned into the pGEM-T vector by TA cloning and then cloned in-frame into the EcoR I site of the FLAG expression vectors with CMV promoter (Kodak, New Haven, CT). Restorations of reading frames were confirmed by DNA sequencing. FLAG-RA70 was transfected into Cos cells according to the calcium-phosphate method (18). Preparation of antiserum against RA70. The cDNA encoding C-terminal region of human RA70 was amplified by RT-PCR using the following primers under the condition as described above: forward primer; 59-GAGCATATGAAAAGCACTGATTACGCTA-39 and reverse primer; 59-GACTCGAGTCAAATATCATACATCTCC-39. The PCR products were then subcloned in-frame into the Nde I and Xho I site of the Histag-pET15b vector (Novagen, Madison, WI) and transformed into DE3 cells. Restorations of reading frames were confirmed by DNA sequencing. The Histag-fusion protein of
C-terminal region of human RA70 was produced by culturing transformed DE3 cells in the presence of 2 mM isopropylthiogalactopyranoside (IPTG; Wako Pure Chemical In., Osaka, Japan). The Histag-fusion protein was extracted with 6 M urea and purified from cell extracts by Nickel-affinity column chromatography. The antiserum against RA70 was raised by injecting the fusion protein with Freund’s complete adjuvant into rabbits. The antiserum was used for experiments after anti-Histag antibody was absorbed with Histag peptides-conjugated Sepharose. Immunoblot analysis. The cell pellets were suspended in the lysis buffer ( 50 mM Tris-HCl, pH7.4, 150 mM NaCl, 1 mM Na3VO4, 10 mM NaF, 1 mg/ml leupeptin ) and sonicated on ice. After centrifugation at 10,000 x g for 10 min, the cell extracts (20 mg protein) were subjected to SDS polyacrylamide gel (10%) electrophoresis (19). Proteins of the gels were electrophoretically transferred to nitrocellulose filters (S & S, Dassel, Germany) (20). After filters were incubated with monoclonal anti-FLAG antibody (Kodak, New Haven, CT), monoclonal anti-Fyn, anti-Lck antibodies (Santa Cruz Biot., Santa Cruz, CA), monoclonal anti-Hck, anti-Lyn antibodies (Transduction Lab., Lexington, KY), and anti-RA70 antibodies, respectively, reactivities were detected by alkaline phosphatase-conjugated goat antimouse or anti-rabbit immunoglobulin (IgG) (Promega, Madison, WI), respectively and nitro blue tetrazolium and 5-bromo-4-chloro-3indolyl-1-phosphate. Interaction between RA70 and Fyn-SH2 peptide. The cell extracts (200 mg protein) of U937 cells and Jurkat cells were incubated with Fyn-SH2 peptides-conjugated agarose (Santa Cruz Biot.) or protein A and G-conjugated agarose (Santa Cruz Biot.) in the presence or absence of 1% Triton X-100 at 4°C overnight and centrifuged. The precipitates were subjected to immunoblot analysis using anti-RA70. Interaction between RA70 and src kinases. The cell pellets of TPA (Sigma, St. Louis, MO)-treated and untreated U937 cells were sonicated with the lysis buffer containing 0.1% Triton X-100 on ice and centrifuged. The cell extracts (200 mg protein) were incubated with anti-Fyn, anti-Hck, anti-Lyn, and anti-Lck antibodies at 4 °C overnight, respectively, and immunocomplexes were precipitated with protein A and G-conjugated agarose. Immunoprecipitates were subjected to SDS-polyacrylamide gel (10%) electrophoresis and immunoblot analysis using anti-RA70.
RESULTS AND DISCUSSIONS Expression of RA70 in Various Tissues and Various Cell Lines We isolated human RA70 cDNA fragment by RT-PCR using consensus primers of mouse RA70 and human SKAP55. RT-PCR fragments (537-bp and 519-bp) with similar size of mouse RA70 cDNA fragment were detected in Jurkat cells and U937 cells, respectively (Figure 1A). To distinguish RT-PCR fragments, RT-PCR products were digested with Spe I, an unique site in SKAP55 fragment. PCR fragment (537-bp) of Jurkat cells was cleaved into 199-bp and 338-bp, but PCR fragment (519bp) of U937 cells was not cleaved (Figure 1A). DNA sequence analysis confirmed that 537-bp PCR fragment of Jurkat cells was SKAP55 and 519-bp PCR fragment of U937 cells was human homologue of RA70. Northern blot analysis revealed that RA70 was preferentially expressed in HeLa cells, chronic myelogenous leukemia cells (K-562), and colorectal adenocarcinoma cells (SW480) (Figure 1B). RA70 was also expressed in human promyelocytic leukemia cells
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FIG. 1. Expression of RA70 in human cell lines and tissues. (A) The expression of RA70 and SKAP55 by the RT-PCR analysis. Lane 1 and 2, RT-PCR products of the untreated and RA-treated (for 2 days) P19 EC cells, respectively; lane 3 and 4, RT-PCR products of U937 cells undigested and digested with Spe I, respectively; lane 5 and 6, RT-PCR products of Jurkat cells undigested and digested with Spe I, respectively. A thick black arrow indicates the mouse RA70 cDNA fragment (519-bp) detected in the RA-treated P19 EC cells. Thin black arrows indicate cDNA fragments (537-bp and 519-bp) Jurkat cells and U937 cells with similar size of the mouse RA70 cDNA fragment, respectively. Thick white arrows indicate RT-PCR products cleaved by Spe I. Arrowheads indicate the RT-PCR products unrelated to RA70 and SKAP55. M; DNA size marker. (B) Northern blot hybridization of RA70 in human cell lines and various tissues. Lane 1, HL-60; lane 2, HeLa; lane 3, K-562; lane 4, MOLT-4; lane 5, Raji; lane 6, SW480; lane 7, A549; lane 8, G361; lane 9, heart; lane 10, brain; lane 11, placenta; lane 12, lung; lane 13, liver; lane 14, skeletal muscle; lane 15, kidney; lane 16, spleen; lane 17, thymus; lane 18, prostate; lane 19, testis; lane 20, ovary; lane 21, small intestine; lane 22, colon; and lane 23, peripheral blood leukocytes. Thick arrows indicate RA70 signals.
(HL60), lung adenocarcinoma cells (A549), and melanoma cells (G361), but RA70 was not detected in lymphoblastic leukemia cells (MOLT-4) and Burkitt’s lymphoma cells (Raji). Strong RA70 signals were detected in the human lung, skeletal muscle, kidney, peripheral blood leukocytes, and spleen tissues; weak signals were detected in the brain, placenta, heart, prostate, testis, ovary, and colon tissues; and signals were undetectable in the thymus, liver, and small intestine (Figure 1B).
Isolation of RA70 A full-length human RA70, which was isolated from human fetal lung cDNA library, encoded a 359-amino acid protein. The deduced human RA70 protein was homologous to human SKAP55 sequences in the NCBI database (Figure 2). Sequence comparison indicated that human RA70 shared 43% overall amino acid identity and 6% similarity with human SKAP55. RA70 residues 115 to 214 and 302 to 359 showed high homol-
FIG. 2. Deduced amino acid sequence of human RA70. Comparison of amino acid sequence of human RA70 and SKAP55. Shadow boxes and open boxes indicate identical amino acids and similar amino acids, respectively. Single and double underlines indicate pleckstrin domain and SH3 domain in SKAP55, respectively. Closed circles, closed and open triangles indicate putative phosphorylation sites of protein kinase C or casein kinase, cyclic AMP dependent kinase, and tyrosine kinase, respectively. Asterisks indicate putative N-glycosylation sites. Nucleotide sequence data of human RA70 have been deposited with DDBJ Data Libraries under Accession No. ABO14486. 740
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FIG. 3. Identification of RA70 protein. (A) Detection of molecular mass of FLAG-tagged RA70 expressed in the transfected Cos cells. Cytosolic proteins were extracted from Cos cells transfected with vector alone (lane 1), and FLAG-tagged RA70 (lane 2). The cytosolic extracts (20 mg protein) were analyzed by SDS-polyacrylamide gel and immunoblotting using anti-RA70 and anti-FLAG. A band of 55 kDa was detected by anti-FLAG and anti-RA70. (B) RA70 protein in U937 cells. Anti-RA70 reacted a band of 55 kDa in U937 cells, but not in Jurkat cells. Lane 1, U937 cells; lane 2, Jurkat cells. U and J, U937 cells and Jurkat cells, respectively. M, rainbow molecular marker of proteins. Thick arrows indicate a 55 kDa band reacting with anti-RA70 and thin arrow indicates a 55 kDa band reacting with anti-FLAG.
ogy with the SKAP55 pleckstrin domain (residues 106 to 205, 56% identity and 13% similarity) and the C-terminal SH3 domain (residues 300 to 356, 63% identity and 7% similarity). In place of the putative tyrosine phosphorylation site (EDIYEVL) in SKAP55, which is predicted to bind to isolated SH2 domains of src kinases with high affinity based on its homology with EXXYXXL (21), RA70 contained a DEIYEEL sequence and several putative phosphorylation sites. A part of the 39-nucleotide sequence of human RA70 was found in a human PAC clone (DJ1139P01 from 7p15p21; accession number 003999, human genome database), indicating that the human RA70 gene resides on chromosome 7.
FIG. 4. Interaction between RA70 and Fyn-SH2 peptide. Interaction between RA70 and Fyn-SH2 was examined in U937 cells. Cytosolic proteins of U937 cells (lane 1 to 4) and Jurkat cells (lane 5 to 8) were incubated with Fyn-SH2 peptide-conjugated agarose (lane 1, 3, 5, and 7) or protein A and G-conjugated agarose (lane 2, 4, 6, and 8) in the presence (lane 3, 4, 7, and 8) or absence (lane 1, 2, 5, and 6) of 1% Triton X-100 at 4°C overnight. The precipitates were subjected to the immunoblot analysis using anti-RA70.
Interaction between RA70 and Src Kinases Since RA70 was shown to be highly homologous to SKAP55 (Figure 2), RA70 was expected to have a similar function as SKAP55. RA70 (55 kDa) in U937 cell extracts was precipitated by Fyn-SH2 peptide-conjugated agarose, but not by protein A and G-conjugated agarose (Figure 4). RA70 was not detected in the precipitate of Jurkat cell extracts. The interaction between RA70 and Fyn-SH2 peptides was decreased by 1% Triton X-100, but still stable. These results suggest that RA70, along with SKAP55, is strongly bound with Fyn through interaction with its SH2 domain. Since src kinases are expressed during monocytes/ macrophage differentiation (12), we examined the interaction between the RA70 and src kinases during monocytes/macrophage differentiation of U937 cells in-
The Protein Encoded by RA70 Although a calculated molecular mass of RA70 is 41.2 kDa, transfection of FLAG-tagged RA70 caused expression of a 55 kDa protein, with which antiFLAG and anti-RA70 antibodies reacted (Figure 3A). Consistent with the results of the RT-PCR analysis, the 55 kDa band was detected in U937 cells, but not in Jurkat cells by immunoblot analysis using antiRA70 (Figure 3B). These results suggest that molecular weight of RA70 shown by SDS-polyacrylamide gel electrophoresis is 55 kDa. Human SKAP55, which has a calculated molecular mass of 41.3 kDa, is also shown to be 55 kDa by SDS-polyacrylamide gel electrophoresis (15). Like SKAP55, mobility of RA70 protein in the SDS-polyacrylamide gel may be influenced by conformational effects and/or posttranslational modifications.
FIG. 5. The expression of RA70 and src kinases and interaction between RA70 and src kinases during monocytes/macrophage differentiation of U937 cells induced by TPA. U937 cells were treated with TPA (10 ng/ml) for 0 h (lane 1), 8 h (lane 2), 12 h (lane 3), and 24 h (lane 4). The expression of RA70 and src kinases were detected by immunoblot analysis. Interaction between RA70 and src kinases was detected by immunoprecipitation with immunoblot analysis. RA70 was detected in the immunoprecipitates by anti-Fyn, anti-Hck, antiLyn, and anti-Lck antibodies by immunoblot analysis using antiRA70.
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duced by TPA. Expression of RA70 and src kinases, Fyn, Hck, and Lyn, was detected during differentiation, but the expression of Lck was undetectable level. The interaction between the src kinases and RA70 was examined using immunoprecipitation with immunoblotting (Figure 5). RA70 was detected in the immunoprecipitates by antiFyn, anti-Hck, and anti-Lyn antibodies during differentiation, but not in the immunoprecipitates by anti-Lck antibodies; RA70 was detected in the immunoprecipitates by anti-Hck of U937 cells treated with TPA for 8, 12, and 24 h and was faintly detected only in the immunoprecipitates by anti-Fyn of the cells treated with TPA for 24 h. Thus RA70 was more preferentially associated with Hck or Fyn in the TPA-treated cells than the untreated cells. RA70 may play a role in the monocytes/macrophage differentiation through interaction with Hck and/or Fyn. RA70 is an adaptor protein of src kinases and may play essential roles in the src signaling pathway in non-T cells. ACKNOWLEDGMENTS This work was supported by a grant from the Human Science Foundation for HIV and Grants-in-Aid for Scientific Research on Priority Areas (No. 09254104) from the Ministry of Education, Science and Culture of Japan.
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