- Email: [email protected]
Murine Peritoneal Macrophages Induce a Novel 60-kDa Protein with Structural Similarity to a Tyrosine Kinase p56lck-Associated Protein in Response to Oxidative Stress
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
226, 456–460 (1996)
1377
Murine Peritoneal Macrophages Induce a Novel 60-kDa Protein with Structural Similarity to a Tyrosine Kinase p56lck-Associated Protein in Response to Oxidative Stress1 Tetsuro Ishii,*,2 Toru Yanagawa,* Tetsuya Kawane,*,3 Koichi Yuki,* Jun Seita,* Hiroshi Yoshida,† and Shiro Bannai* *Department of Biochemistry, Institute of Basic Medical Sciences, and †Department of Oral and Maxillofacial Surgery, Institute of Clinical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Received July 31, 1996
Using differential screening we have cloned a cDNA encoding a novel oxidative stress protein designated A170 from murine peritoneal macrophages. It has a Zn-finger domain, a PEST domain and several potential phosphorylation sites for kinases. Treatments with oxidative stress agents such as diethyl maleate and paraquat increased a 2.0-kilobase A170 mRNA about twofold in the macrophages after 12 hours in culture. However, H2O2 or glucose/glucose oxidase did not increase the level of the A170 mRNA. Using an A170specific antibody we have detected in the macrophages a 60-kDa protein that was induced 5 to 10 hours after the addition of the oxidative stress agents. A search of sequence databases revealed that the A170 protein is roughly 90% identical to a human protein that binds to the Src homology 2 domain of the Tcell-specific tyrosine kinase p56lck. These features suggest that the A170 protein plays a significant role in oxidative stress-responsive signal transduction in macrophages. q 1996 Academic Press, Inc.
Macrophages produce various reactive oxygen species such as superoxide (O20) and hydrogen peroxide (H2O2) in response to various extracellular stimuli. These reactive oxygen species contribute to the pathogenesis of various diseases such as inflammation and atherosclerosis. Upon exposure to those reactive oxygen species and subsequent toxic by-products, macrophages and other cells induce various stress proteins such as heme oxygenase (1), HSP70 (2, 3), and ferritin (4, 5) to protect themselves. We have previously cloned a 23-kDa protein termed MSP23 that is induced by various oxidative stress agents (6). This protein belongs to the peroxiredoxin family and has a thiol-specific antioxidant activity (7). Here we report the cloning and characterization of a new type of stress-inducible protein termed A170 from mouse macrophages. We show that oxidative stress agents specifically induce the 60-kDa A170 protein in the macrophages. Interestingly, a human lymphocyte protein closely related to the A170 protein has been cloned and characterized as a phosphotyrosineindependent ligand of the tyrosine kinase p56lck(Lck) (8, 9). It is also reported that the same protein has been found to be associated with a novel cytokine receptor induced by latent Epstein-barr virus infection (10). The structural similarity of the A170 protein to the human protein suggests that the A170 protein is not an antioxidant itself, but is rather a modulator of signal transduction to induce cellular responses under oxidative stress. 1
The nucleotide sequence of A170 cDNA has been deposited with the GenBank database, Accession No. U40930. To whom correspondence should be addressed. Fax: 81-298-53-3061. 3 Present address: Department of Biochemistry, Ohu University, School of Dentistry, 31-1 Misumido, Tomita, Koriyama, Fukushima 963, Japan. 2
456 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AID
BBRC 5347
/
6909$$$321
08-26-96 07:21:51
bbrca
AP: BBRC
Vol. 226, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS cDNA cloning and sequencing. Peritoneal macrophages were prepared from ddY female mice and cultured as described previously (11). We first prepared a poly(A)/ RNA fraction from cells that had previously been exposed to 100 mM diethyl maleate, a sulfhydryl reactive agent, for 12 h. We separated a 1.5-3.0 kb poly(A)/ RNA fraction by a sucrose density gradient centrifugation to remove 1.0 kb MSP23 mRNA, which is the major stress-induced transcript (6). We then synthesized cDNAs from the fraction using a cDNA synthesis system (Amersham) and Not I-oligo dT primer (Promega), and ligated into the Not I and EcoRI sites of lZAPII (Stratagene). The cDNA library was subjected to a plus/minus screening using two hybridization probes as described previously (6). We screened about 21104 phage plaques and selected 126 cDNA clones. The corresponding mRNAs were induced during culture with diethyl maleate. Among the selected clones, we characterized here a group of four cDNA clones having a common nucleotide sequence. Using a Sequenase Kit (U.S. Biochemical) and Exo III/Mung bean nuclease deletion system (Stratagene), we sequenced the 1720 nucleotides plus 48 poly(A) tail of the longest clone, #A170. Clone #A170 was, however, too short to contain a whole open reading frame. To determine nucleotide sequence at the 5*end region of A170 mRNA that was not included in the clone, we used a 5*RACE system (Gibco BRL) and a TA cloning kit (Invitrogen). We determined the sequence of 280 nucleotides using a autosequencer (ABI 373S). The synthetic primers used for the PCR reactions are as follows: 5*-TTATAGCGAGTTCCCACCAC-3* (GSP1), 5*CTCTTTAATGTAGATGCGGA-3* (GSP2), and 5*-GACATAGCCATTGTCAGCT-3* (GSP3). Sequence recording and analysis were performed using the Genetyx and Sequence Database (GenBank). Northern blot analysis. We added each stress agent to the culture medium 1 h after seeding macrophages and usually cultured for another 12 h. Total RNAs were prepared with RNeasy (QIAGEN) from the cells cultured for 13 h. Each well contained the RNA extracted from 21106 cells. The hybridizations were performed as described previously (6). To compare the levels of A170 mRNA using b-actin mRNA as an internal standard, the RNAs were first hybridized with an A170 cDNA probe and later rehybridized with the b-actin cDNA probe. The hybridized counts of 32P-labeled actin cDNAs were roughly one order of magnitude higher than those of the A170 cDNA. The bands were detected by autoradiography on Hyperfilm-MP (Amersham). Preparation of antisera and immunological analysis. We first constructed a fusion protein of the A170 protein with a maltose binding protein (New England Biolabs) as follows. The #A170 cDNA was cut with Pst I and Eco RI, and ligated with the vector pMAL-cRI. The nucleotide sequence of the ligated vector was confirmed. The fused MalA170 protein having an apparent molecular mass of 94 kDa contains the maltose binding protein and the 82-442nd amino acid residues of the A170 protein. The protein was extracted from the gel band and was injected into Japanese white rabbits to obtain antisera. To prepare the specimens for immunoblotting, the cells were solubilized with the SDS-sample buffer without a marker dye and 2-mercaptoethanol, boiled for 5 min and stored at 0207C until use. The protein content was determined by BCA protein assay reagent (Pierce). Fifteen mg of cell proteins were used in each well for the immunoblotting. The dye and 2-mercaptoethanol were added to the specimens, and the mixture was then boiled just before SDS-PAGE. To detect immunoreactive proteins, we used horseradish peroxidase-conjugated antirabbit IgG and blotting reagents (ECL, Amersham).
RESULTS AND DISCUSSION
cDNA screening. Using a differential plaque hybridization method (6), we selected cDNA clones that were derived from diethyl maleate-induced transcripts as described in materials and methods. In this paper, we characterize a cDNA that encodes a protein of 442 amino acid residues, designated here A170 protein (Fig. 1). It has a Zn-finger domain containing three conserved Cys-X2-Cys motifs and a conserved Asp-Tyr-Asp-Leu motif (ZZ fingers) (12). It has a PEST domain composed of two sequences with remarkably high scores; 267-RLTPTTPESSSTGTEDK-283 with PEST scoreÅ26.5, and 283-KSNTQPSSCSSEVSK-297 with PEST scoreÅ16.3. These PEST scores are higher than those found in c-fos and c-myc proteins (13). A protein with PEST sequences is assumed to have a short half-life in the cells (13). The protein also contains several consensus sequences for phosphorylation by serine/threonine kinases: eleven sites for casein kinase II (S/TXXD/EX), four for protein kinase C (S/TXK/R) and one for MAP kinase (PXS/TP). The A170 protein has no structural homologies to any known antioxidative enzymes or proteins. A recent homology search in a data base detected two nucleotide sequences of human cDNAs (GenBank accession numbers U46751 & U41806). Each sequence encodes almost the same protein that is roughly 90% identical to the A170 protein (Fig. 1). This 62-kDa human protein of 440 amino acids has recently been cloned (8) and characterized as an Lck associated protein (9) or as 457
AID
BBRC 5347
/
6909$$$321
08-26-96 07:21:51
bbrca
AP: BBRC
Vol. 226, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Structure of the A170 protein. The deduced amino acid sequence of the A170 protein is compared with that of the human Lck associated p62 protein (8). Shaded amino acids in the p62 protein denote differences with A170. The full-length A170 cDNA sequence (2,000 bases:GenBank Accession No. U40930) has one open reading frame that predicts the above polypeptide of 442 amino acids. Translations assumed to initiate at the ATG methionine codon denoted 1, as its context (corresponding base sequence: GCGGTTATGG) conforms well to the translation initiation site consensus sequence (19). Underlined residues denote positions of a Zn-finger domain (single) and a PEST domain (double), respectively.
a cytokine receptor, EBI3, associated B-cell protein (10). Although both sequences are identical at the C-terminal region, they are significantly different around the PEST domain including the potential phosphorylation sites for MAP kinase: PTTP in A170 versus PVSP in p62 protein. Therefore, the A170 protein appears to be in the same family as the 60 to 62-kDa human protein and may have a regulatory function similar to that of the human protein. Northern blotting. Using the A170 cDNA fragment as a probe, we detected a 2.0 kb A170 mRNA in the macrophages by northern blotting (Fig. 2A). This size is almost the same as that of b-actin mRNA used as an internal standard to calculate the relative levels of A170 mRNA. When the cells were exposed to 100 mM diethyl maleate for 12 h, the level of A170 mRNA was 1.8{0.3 (nÅ4) times the control level. This result confirmed the validity of the differential screening. Paraquat and menadion, O20 generating agents, evidently increased the transcript by 2.8 and 1.4 times the control level, respectively (Fig. 2B lanes 1, 8). Sodium arsenite and cadmium chloride also increased the transcript by 2.8 and 4.0 times, respectively (Fig. 2B lanes 2, 3). However, hydrogen peroxide (H2O2) and glucose oxidase that produced H2O2 from glucose did not increase the level of transcript after 12 h in culture (Fig. 2B lanes 6, 7). There was no marked induction of the A170 mRNA between 3 and 10 h after the addition of the stress agents (not shown). Heat shock treatments (437C for 30 min) did not induce the transcript in the cells (not shown). Induction of a 60-kDa A170 protein in the macrophages. Using affinity purified rabbit antiA170 immunoglobulin, we detected a 60-kDa protein in the macrophages by immunoblotting (Fig. 458
AID
BBRC 5347
/
6909$$$321
08-26-96 07:21:51
bbrca
AP: BBRC
Vol. 226, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Northern blot analysis of A170 mRNA. A. Induction of A170 mRNA by 100 mM diethyl maleate after 12 h. RNA size markers are 23S (2.9 kb) and 16S (1.5 kb) E.coli rRNAs. B. Effects of various stress agents on the level of A170 mRNA. Lane 1, 100 mM paraquat; lane 2, 2.5 mM sodium arsenite; lane 3, 10 mM cadmium chloride; lane 4, control, after 1h in culture; lane 5, control, after 13h in culture; lane 6, 100 mM H2O2 ; lane 7, 5 mU/ml glucose oxidase; lane 8, 5 mM menadione. Relative levels of A170 mRNA are calculated from each ratio to b-actin mRNA. Standard (1.0) is lane 5.
3A). We also observed that the antibody coprecipitated the metabolically labeled 60-kDa protein in the macrophage lysate (not shown). We assumed this 60-kDa protein to have been the A170 protein shown in Figure 1, although the calculated molecular mass of A170 is about 48 kDa. This type of anomalous electrophoretic migration has been observed for proteins rich in proline and glutamate residues (14-18). The level of the 60-kDa immunoreactive protein in the fresh macrophages was quite low. However, during culture in control medium the 60-kDa protein gradually increased after 5 h and reached a plateau in 13 h (Fig. 3A, B). Addition of paraquat to the culture medium markedly enhanced the induction of the 60-kDa protein. Diethyl maleate, menadione,
FIG. 3. Immunoblot analysis of the time-dependent induction of the 60-kDa A170 protein in the macrophages. A. Effects of 100 mM paraquat on the induction of A170 protein. B. Quantitative analysis of the effect of 100 mM paraquat on the induction of A170 protein by densitometry. The ordinate denotes relative amounts of A170 protein. The arrow shows the time of paraquat addition. 459
AID
BBRC 5347
/
6909$$$321
08-26-96 07:21:51
bbrca
AP: BBRC
Vol. 226, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. Effects of stress agents on the induction of the 60-kDa A170 protein. Lane 1, control; lane 2, 100 mM diethyl maleate; lane 3, 5 mM menadione; lane 4, 100 mM paraquat; lane 5, 10 mM cadmium chloride; lane 6, 2.5 mM sodium arsenite. The cells were incubated for 7 h with each agent.
cadmium chloride and sodium arsenite respectively induced the 60-kDa protein in the cells (Fig. 4). The induction of A170 mRNA by these agents may partly contribute to the induction of the protein. However, the gradual induction of the A170 protein in the cells in normal culture did not depend on the increase in the level of A170 mRNA (Fig. 2B). We need further experiments to elucidate the precise mechanism of the induction of this protein. Possible role of the A170 protein in stress response. Recent reports on the A170-related 62-kDa protein (8-10) suggest that the A170 protein also has a significant role in signal transduction. The amino terminal domain of the human p62 protein seems to bind to the Lck Src homology 2 domain (8). The SH2 domain is indispensable for the proper role of Lck in T-cell signal transduction. Since Lck is not highly expressed in the macrophages, the A170 protein may bind to a tyrosine kinase other than Lck and modulate the signal transduction. Further studies of the A170 protein and related proteins will uncover the molecular mechanism that determines how oxidative stress affects tyrosine kinase and cytokine signaling and induces metabolic changes within immune cells. ACKNOWLEDGMENTS This work was supported by Grants in Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture in Japan.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Taketani, S., Sato, H., Yoshinaga, T., Tokunaga, R., Ishii, T., and Bannai, S. (1990) J. Biochem. 108, 28–32. Spitz, D. R., Dewey, W. C., and Li, G. C. (1987) J. Cell. Physiol. 131, 364–373. Polla, B. S., Healy, A. M., Wojno, W. C., and Krane, S. M. (1987) Am. J. Physiol. 252, C640–C649. Sato, H., Yamaguchi, M., and Bannai, S. (1994) Biochem. Biophys. Res. Comm. 201, 38–44. Pizurki, L., and Polla, B. S. (1994) J. Cellular Physiology 161, 169–177. Ishii, T., Yamada, M., Sato, H., Matsue, M., Taketani, S., Nakayama, K., Sugita, Y., and Bannai, S. (1993) J. Biol. Chem. 268, 18633–18636. Ishii, T., Kawane, T., Taketani, S., and Bannai, S. (1995) Biochem. Biophys. Res. Comm. 216, 970–975. Joung, I., Strominger, J. L., and Shin, J. (1996) Proc. Natl. Acad. Sci. USA 93, 5991–5995. Park, I., Chung, J., Walsh, C. T., Strominger, J. L., and Shin, J. (1995) Proc. Natl. Acad. Sci. USA 92, 12338– 12342. Devergne, O., Hummel, M., Koeppen, H., Beau, M. L., Nathanson, E. C., Kieff, E., and Birkenbach, M. (1996) J. Virology 70, 1143–1153. Sato, H., Ishii, T., Sugita, Y., Tateishi, N., and Bannai, S. (1993) Biochim. Biophys. Acta 1148, 127–132. Ponting, C. P., Blake, D. J., Davies, K. E., Kendrick-Jones, J., and Winder, S. J. (1996) TIBS 21, 11–13. Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364–368. Persson, H., Hennighausen, L., Taub, R., Degrado, W., and Leder, P. (1984) Science 225, 687–693. Hann, S. R., and Eisenman, R. N. (1984) Mol. Cell. Biol. 4, 2486–2497. Soeda, E., Arrand, J., Smolar, N., Walsh, J., and Griffin, B. (1980) Nature 283, 445–453. Verma, J. (1984) Nature 308, 317. Dezelee, S., Bras, F., Contamine, D., Lopez-Ferber, M., Segretain, D., and Teninges, D. (1989) EMBO J. 8, 3437–3446. Kozak, M. (1991) J. Biol. Chem. 266, 19867–19870. 460
AID
BBRC 5347
/
6909$$$321
08-26-96 07:21:51
bbrca
AP: BBRC
226, 456–460 (1996)
1377
Murine Peritoneal Macrophages Induce a Novel 60-kDa Protein with Structural Similarity to a Tyrosine Kinase p56lck-Associated Protein in Response to Oxidative Stress1 Tetsuro Ishii,*,2 Toru Yanagawa,* Tetsuya Kawane,*,3 Koichi Yuki,* Jun Seita,* Hiroshi Yoshida,† and Shiro Bannai* *Department of Biochemistry, Institute of Basic Medical Sciences, and †Department of Oral and Maxillofacial Surgery, Institute of Clinical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Received July 31, 1996
Using differential screening we have cloned a cDNA encoding a novel oxidative stress protein designated A170 from murine peritoneal macrophages. It has a Zn-finger domain, a PEST domain and several potential phosphorylation sites for kinases. Treatments with oxidative stress agents such as diethyl maleate and paraquat increased a 2.0-kilobase A170 mRNA about twofold in the macrophages after 12 hours in culture. However, H2O2 or glucose/glucose oxidase did not increase the level of the A170 mRNA. Using an A170specific antibody we have detected in the macrophages a 60-kDa protein that was induced 5 to 10 hours after the addition of the oxidative stress agents. A search of sequence databases revealed that the A170 protein is roughly 90% identical to a human protein that binds to the Src homology 2 domain of the Tcell-specific tyrosine kinase p56lck. These features suggest that the A170 protein plays a significant role in oxidative stress-responsive signal transduction in macrophages. q 1996 Academic Press, Inc.
Macrophages produce various reactive oxygen species such as superoxide (O20) and hydrogen peroxide (H2O2) in response to various extracellular stimuli. These reactive oxygen species contribute to the pathogenesis of various diseases such as inflammation and atherosclerosis. Upon exposure to those reactive oxygen species and subsequent toxic by-products, macrophages and other cells induce various stress proteins such as heme oxygenase (1), HSP70 (2, 3), and ferritin (4, 5) to protect themselves. We have previously cloned a 23-kDa protein termed MSP23 that is induced by various oxidative stress agents (6). This protein belongs to the peroxiredoxin family and has a thiol-specific antioxidant activity (7). Here we report the cloning and characterization of a new type of stress-inducible protein termed A170 from mouse macrophages. We show that oxidative stress agents specifically induce the 60-kDa A170 protein in the macrophages. Interestingly, a human lymphocyte protein closely related to the A170 protein has been cloned and characterized as a phosphotyrosineindependent ligand of the tyrosine kinase p56lck(Lck) (8, 9). It is also reported that the same protein has been found to be associated with a novel cytokine receptor induced by latent Epstein-barr virus infection (10). The structural similarity of the A170 protein to the human protein suggests that the A170 protein is not an antioxidant itself, but is rather a modulator of signal transduction to induce cellular responses under oxidative stress. 1
The nucleotide sequence of A170 cDNA has been deposited with the GenBank database, Accession No. U40930. To whom correspondence should be addressed. Fax: 81-298-53-3061. 3 Present address: Department of Biochemistry, Ohu University, School of Dentistry, 31-1 Misumido, Tomita, Koriyama, Fukushima 963, Japan. 2
456 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AID
BBRC 5347
/
6909$$$321
08-26-96 07:21:51
bbrca
AP: BBRC
Vol. 226, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS cDNA cloning and sequencing. Peritoneal macrophages were prepared from ddY female mice and cultured as described previously (11). We first prepared a poly(A)/ RNA fraction from cells that had previously been exposed to 100 mM diethyl maleate, a sulfhydryl reactive agent, for 12 h. We separated a 1.5-3.0 kb poly(A)/ RNA fraction by a sucrose density gradient centrifugation to remove 1.0 kb MSP23 mRNA, which is the major stress-induced transcript (6). We then synthesized cDNAs from the fraction using a cDNA synthesis system (Amersham) and Not I-oligo dT primer (Promega), and ligated into the Not I and EcoRI sites of lZAPII (Stratagene). The cDNA library was subjected to a plus/minus screening using two hybridization probes as described previously (6). We screened about 21104 phage plaques and selected 126 cDNA clones. The corresponding mRNAs were induced during culture with diethyl maleate. Among the selected clones, we characterized here a group of four cDNA clones having a common nucleotide sequence. Using a Sequenase Kit (U.S. Biochemical) and Exo III/Mung bean nuclease deletion system (Stratagene), we sequenced the 1720 nucleotides plus 48 poly(A) tail of the longest clone, #A170. Clone #A170 was, however, too short to contain a whole open reading frame. To determine nucleotide sequence at the 5*end region of A170 mRNA that was not included in the clone, we used a 5*RACE system (Gibco BRL) and a TA cloning kit (Invitrogen). We determined the sequence of 280 nucleotides using a autosequencer (ABI 373S). The synthetic primers used for the PCR reactions are as follows: 5*-TTATAGCGAGTTCCCACCAC-3* (GSP1), 5*CTCTTTAATGTAGATGCGGA-3* (GSP2), and 5*-GACATAGCCATTGTCAGCT-3* (GSP3). Sequence recording and analysis were performed using the Genetyx and Sequence Database (GenBank). Northern blot analysis. We added each stress agent to the culture medium 1 h after seeding macrophages and usually cultured for another 12 h. Total RNAs were prepared with RNeasy (QIAGEN) from the cells cultured for 13 h. Each well contained the RNA extracted from 21106 cells. The hybridizations were performed as described previously (6). To compare the levels of A170 mRNA using b-actin mRNA as an internal standard, the RNAs were first hybridized with an A170 cDNA probe and later rehybridized with the b-actin cDNA probe. The hybridized counts of 32P-labeled actin cDNAs were roughly one order of magnitude higher than those of the A170 cDNA. The bands were detected by autoradiography on Hyperfilm-MP (Amersham). Preparation of antisera and immunological analysis. We first constructed a fusion protein of the A170 protein with a maltose binding protein (New England Biolabs) as follows. The #A170 cDNA was cut with Pst I and Eco RI, and ligated with the vector pMAL-cRI. The nucleotide sequence of the ligated vector was confirmed. The fused MalA170 protein having an apparent molecular mass of 94 kDa contains the maltose binding protein and the 82-442nd amino acid residues of the A170 protein. The protein was extracted from the gel band and was injected into Japanese white rabbits to obtain antisera. To prepare the specimens for immunoblotting, the cells were solubilized with the SDS-sample buffer without a marker dye and 2-mercaptoethanol, boiled for 5 min and stored at 0207C until use. The protein content was determined by BCA protein assay reagent (Pierce). Fifteen mg of cell proteins were used in each well for the immunoblotting. The dye and 2-mercaptoethanol were added to the specimens, and the mixture was then boiled just before SDS-PAGE. To detect immunoreactive proteins, we used horseradish peroxidase-conjugated antirabbit IgG and blotting reagents (ECL, Amersham).
RESULTS AND DISCUSSION
cDNA screening. Using a differential plaque hybridization method (6), we selected cDNA clones that were derived from diethyl maleate-induced transcripts as described in materials and methods. In this paper, we characterize a cDNA that encodes a protein of 442 amino acid residues, designated here A170 protein (Fig. 1). It has a Zn-finger domain containing three conserved Cys-X2-Cys motifs and a conserved Asp-Tyr-Asp-Leu motif (ZZ fingers) (12). It has a PEST domain composed of two sequences with remarkably high scores; 267-RLTPTTPESSSTGTEDK-283 with PEST scoreÅ26.5, and 283-KSNTQPSSCSSEVSK-297 with PEST scoreÅ16.3. These PEST scores are higher than those found in c-fos and c-myc proteins (13). A protein with PEST sequences is assumed to have a short half-life in the cells (13). The protein also contains several consensus sequences for phosphorylation by serine/threonine kinases: eleven sites for casein kinase II (S/TXXD/EX), four for protein kinase C (S/TXK/R) and one for MAP kinase (PXS/TP). The A170 protein has no structural homologies to any known antioxidative enzymes or proteins. A recent homology search in a data base detected two nucleotide sequences of human cDNAs (GenBank accession numbers U46751 & U41806). Each sequence encodes almost the same protein that is roughly 90% identical to the A170 protein (Fig. 1). This 62-kDa human protein of 440 amino acids has recently been cloned (8) and characterized as an Lck associated protein (9) or as 457
AID
BBRC 5347
/
6909$$$321
08-26-96 07:21:51
bbrca
AP: BBRC
Vol. 226, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Structure of the A170 protein. The deduced amino acid sequence of the A170 protein is compared with that of the human Lck associated p62 protein (8). Shaded amino acids in the p62 protein denote differences with A170. The full-length A170 cDNA sequence (2,000 bases:GenBank Accession No. U40930) has one open reading frame that predicts the above polypeptide of 442 amino acids. Translations assumed to initiate at the ATG methionine codon denoted 1, as its context (corresponding base sequence: GCGGTTATGG) conforms well to the translation initiation site consensus sequence (19). Underlined residues denote positions of a Zn-finger domain (single) and a PEST domain (double), respectively.
a cytokine receptor, EBI3, associated B-cell protein (10). Although both sequences are identical at the C-terminal region, they are significantly different around the PEST domain including the potential phosphorylation sites for MAP kinase: PTTP in A170 versus PVSP in p62 protein. Therefore, the A170 protein appears to be in the same family as the 60 to 62-kDa human protein and may have a regulatory function similar to that of the human protein. Northern blotting. Using the A170 cDNA fragment as a probe, we detected a 2.0 kb A170 mRNA in the macrophages by northern blotting (Fig. 2A). This size is almost the same as that of b-actin mRNA used as an internal standard to calculate the relative levels of A170 mRNA. When the cells were exposed to 100 mM diethyl maleate for 12 h, the level of A170 mRNA was 1.8{0.3 (nÅ4) times the control level. This result confirmed the validity of the differential screening. Paraquat and menadion, O20 generating agents, evidently increased the transcript by 2.8 and 1.4 times the control level, respectively (Fig. 2B lanes 1, 8). Sodium arsenite and cadmium chloride also increased the transcript by 2.8 and 4.0 times, respectively (Fig. 2B lanes 2, 3). However, hydrogen peroxide (H2O2) and glucose oxidase that produced H2O2 from glucose did not increase the level of transcript after 12 h in culture (Fig. 2B lanes 6, 7). There was no marked induction of the A170 mRNA between 3 and 10 h after the addition of the stress agents (not shown). Heat shock treatments (437C for 30 min) did not induce the transcript in the cells (not shown). Induction of a 60-kDa A170 protein in the macrophages. Using affinity purified rabbit antiA170 immunoglobulin, we detected a 60-kDa protein in the macrophages by immunoblotting (Fig. 458
AID
BBRC 5347
/
6909$$$321
08-26-96 07:21:51
bbrca
AP: BBRC
Vol. 226, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Northern blot analysis of A170 mRNA. A. Induction of A170 mRNA by 100 mM diethyl maleate after 12 h. RNA size markers are 23S (2.9 kb) and 16S (1.5 kb) E.coli rRNAs. B. Effects of various stress agents on the level of A170 mRNA. Lane 1, 100 mM paraquat; lane 2, 2.5 mM sodium arsenite; lane 3, 10 mM cadmium chloride; lane 4, control, after 1h in culture; lane 5, control, after 13h in culture; lane 6, 100 mM H2O2 ; lane 7, 5 mU/ml glucose oxidase; lane 8, 5 mM menadione. Relative levels of A170 mRNA are calculated from each ratio to b-actin mRNA. Standard (1.0) is lane 5.
3A). We also observed that the antibody coprecipitated the metabolically labeled 60-kDa protein in the macrophage lysate (not shown). We assumed this 60-kDa protein to have been the A170 protein shown in Figure 1, although the calculated molecular mass of A170 is about 48 kDa. This type of anomalous electrophoretic migration has been observed for proteins rich in proline and glutamate residues (14-18). The level of the 60-kDa immunoreactive protein in the fresh macrophages was quite low. However, during culture in control medium the 60-kDa protein gradually increased after 5 h and reached a plateau in 13 h (Fig. 3A, B). Addition of paraquat to the culture medium markedly enhanced the induction of the 60-kDa protein. Diethyl maleate, menadione,
FIG. 3. Immunoblot analysis of the time-dependent induction of the 60-kDa A170 protein in the macrophages. A. Effects of 100 mM paraquat on the induction of A170 protein. B. Quantitative analysis of the effect of 100 mM paraquat on the induction of A170 protein by densitometry. The ordinate denotes relative amounts of A170 protein. The arrow shows the time of paraquat addition. 459
AID
BBRC 5347
/
6909$$$321
08-26-96 07:21:51
bbrca
AP: BBRC
Vol. 226, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. Effects of stress agents on the induction of the 60-kDa A170 protein. Lane 1, control; lane 2, 100 mM diethyl maleate; lane 3, 5 mM menadione; lane 4, 100 mM paraquat; lane 5, 10 mM cadmium chloride; lane 6, 2.5 mM sodium arsenite. The cells were incubated for 7 h with each agent.
cadmium chloride and sodium arsenite respectively induced the 60-kDa protein in the cells (Fig. 4). The induction of A170 mRNA by these agents may partly contribute to the induction of the protein. However, the gradual induction of the A170 protein in the cells in normal culture did not depend on the increase in the level of A170 mRNA (Fig. 2B). We need further experiments to elucidate the precise mechanism of the induction of this protein. Possible role of the A170 protein in stress response. Recent reports on the A170-related 62-kDa protein (8-10) suggest that the A170 protein also has a significant role in signal transduction. The amino terminal domain of the human p62 protein seems to bind to the Lck Src homology 2 domain (8). The SH2 domain is indispensable for the proper role of Lck in T-cell signal transduction. Since Lck is not highly expressed in the macrophages, the A170 protein may bind to a tyrosine kinase other than Lck and modulate the signal transduction. Further studies of the A170 protein and related proteins will uncover the molecular mechanism that determines how oxidative stress affects tyrosine kinase and cytokine signaling and induces metabolic changes within immune cells. ACKNOWLEDGMENTS This work was supported by Grants in Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture in Japan.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Taketani, S., Sato, H., Yoshinaga, T., Tokunaga, R., Ishii, T., and Bannai, S. (1990) J. Biochem. 108, 28–32. Spitz, D. R., Dewey, W. C., and Li, G. C. (1987) J. Cell. Physiol. 131, 364–373. Polla, B. S., Healy, A. M., Wojno, W. C., and Krane, S. M. (1987) Am. J. Physiol. 252, C640–C649. Sato, H., Yamaguchi, M., and Bannai, S. (1994) Biochem. Biophys. Res. Comm. 201, 38–44. Pizurki, L., and Polla, B. S. (1994) J. Cellular Physiology 161, 169–177. Ishii, T., Yamada, M., Sato, H., Matsue, M., Taketani, S., Nakayama, K., Sugita, Y., and Bannai, S. (1993) J. Biol. Chem. 268, 18633–18636. Ishii, T., Kawane, T., Taketani, S., and Bannai, S. (1995) Biochem. Biophys. Res. Comm. 216, 970–975. Joung, I., Strominger, J. L., and Shin, J. (1996) Proc. Natl. Acad. Sci. USA 93, 5991–5995. Park, I., Chung, J., Walsh, C. T., Strominger, J. L., and Shin, J. (1995) Proc. Natl. Acad. Sci. USA 92, 12338– 12342. Devergne, O., Hummel, M., Koeppen, H., Beau, M. L., Nathanson, E. C., Kieff, E., and Birkenbach, M. (1996) J. Virology 70, 1143–1153. Sato, H., Ishii, T., Sugita, Y., Tateishi, N., and Bannai, S. (1993) Biochim. Biophys. Acta 1148, 127–132. Ponting, C. P., Blake, D. J., Davies, K. E., Kendrick-Jones, J., and Winder, S. J. (1996) TIBS 21, 11–13. Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364–368. Persson, H., Hennighausen, L., Taub, R., Degrado, W., and Leder, P. (1984) Science 225, 687–693. Hann, S. R., and Eisenman, R. N. (1984) Mol. Cell. Biol. 4, 2486–2497. Soeda, E., Arrand, J., Smolar, N., Walsh, J., and Griffin, B. (1980) Nature 283, 445–453. Verma, J. (1984) Nature 308, 317. Dezelee, S., Bras, F., Contamine, D., Lopez-Ferber, M., Segretain, D., and Teninges, D. (1989) EMBO J. 8, 3437–3446. Kozak, M. (1991) J. Biol. Chem. 266, 19867–19870. 460
AID
BBRC 5347
/
6909$$$321
08-26-96 07:21:51
bbrca
AP: BBRC