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Isolation of a mouse cDNA encoding mSTI1, a stress-inducible protein containing the TPR motif
Gene 194 (1997) 277–282
Isolation of a mouse cDNA encoding mSTI1, a stress-inducible protein containing the TPR motif Gregory L. Blatch a,*, Michael La¨ssle b, Bruce R. Zetter c, Vikas Kundra d a Department of Biochemistry, University of the, Witwatersrand, P O WITS 2050, South Africa b MIT, Department of Chemical Engineering and Center for Biomedical Engineering, Cambridge, MA 02139, USA c Department of Cell Biology and Surgery, Harvard Medical School, Children’s Hospital, Boston, MA 02115, USA d Brigham and Woman’s Hospital, Department of Radiology, Boston, MA 02115, USA Received 26 December 1996; accepted 11 March 1997; Received by J. Wild
Abstract We report the isolation and sequencing of the complete 2079-bp cDNA fragment encoding mSTI1, a murine stress-inducible protein. The predicted ORF encodes a protein of 543 amino acids (aa) and M 62 582. The predicted protein has significant r homology to stress-inducible proteins from humans (IEF SSP 3521), soybean (GMSTI ), yeast (STI1) and a parasite, Leishmania donovani (LSIP). All of these proteins contain 34-aa repeat motifs, termed tetratricopeptide repeats ( TPRs), that are proposed to be involved in intra- and intermolecular protein interactions. mSTI1 has ten potential TPR motifs, a putative nuclear localization signal (NLS ), six potential phosphorylation sites for casein kinase II and a central proline-rich region. Western analysis detected a protein of approx. 63 kDa in all the major mouse organs and in mouse, monkey and human cell lines. © 1997 Elsevier Science B.V. Keywords: STI1; IEF SSP 3521; p60; Hop; RF-hsp70; GMSTI; LSIP
1. Introduction Cells respond to cellular stress by modifying the expression, localization and post-translational modification of a set of proteins generally referred to as stressproteins (Georgopoulos and Welch, 1993). A novel stress-inducible protein, STI1, has been described for yeast where it is proposed to mediate the heat shock (HS) response of some Hsp70 genes (Nicolet and Craig, * Corresponding author. Tel. +27 11 7162204; Fax: +27 11 7164479; e-mail: [email protected] Abbreviations: aa, amino acid(s); Ab, antibody(ies); bp, base pair(s); cDNA, DNA complementary to RNA; E., Escherichia; EDTA, ethylene diamine tetra-acetic acid; GCG, Genetics Computer Group (Madison, WI, USA); GMSTI, Glycine max stress-inducible protein; GST, glutathione S-transferase; HS, heat shock; Hsp70, HS protein 70; Hsp90, HS protein 90; IEF SSP 3521, isoelectric focusing sample spot 3521; LSIP, Leishmania donovani stress-inducible protein; mSTI1, murine stress inducible protein 1; NLS, nuclear localization signal; nt, nucleotide(s); ORF, open reading frame; p, plasmid; PA, polyacrylamide; PAGE, PA-gel electrophoresis; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; SSPE, 0.18 M NaCl/0.01 M sodium phosphate/0.001 M EDTA pH 7.7; STI1, yeast stress-inducible protein 1; TBE, Tris-borate EDTA; TPR, tetratricopeptide repeats; ∞ (prime), denotes truncated gene at the indicated side 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 2 06 - 0
1989). The STI1 protein shows no homology to the Hsp70 family of proteins but shares 42% identity to IEF SSP 3521, a human transformation-sensitive protein ( Honore et al., 1992), 38% identity to GMSTI, a soybean (Glycine max) stress-inducible protein ( Torres et al., 1995), and 26% identity to LSIP, a stress-inducible protein of the parasite Leishmania donovani (Joshi et al., 1993). STI1, IEF SSP 3521 and GMSTI contain a number of 34-aa tetratricopeptide repeats ( TPR) proposed to be important for intra- and intermolecular protein interactions (Lamb et al., 1995). Both IEF SSP 3521 and STI1 have been identified as components of multiprotein chaperone complexes involving the HS proteins, Hsp70 and Hsp90 (Smith et al., 1993; Chang and Lindquist, 1994; Schumacher et al., 1994). IEF SSP 3521, also referred to as p60 or Hop, has been shown to bind to both Hsp90 and Hsp70, and to be important for the successful formation of Hsp90 heterocomplexes (Smith et al., 1993; Owens-Grillo et al., 1996; Chen et al., 1996). Sequencing of tryptic peptides of a 66-kDa rabbit protein suggest that it is the rabbit homologue of IEF SSP 3521 (Gross et al., 1996). This protein has been named RF-hsp70, and has been shown to promote the recycling of Hsp70 by catalyzing the dissociation of
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Hsp70-bound ADP in exchange for ATP (Gross and Hessefort, 1996). Recently, we identified a murine stressinducible protein, mSTI1, that interacts with Hsc70 and Hsp90 (La¨ssle et al., 1997). In this report, we show by cDNA sequence analysis that mSTI1 is homologous to STI1, IEF SSP 3521, GMSTI1 and LSIP and is a TPRcontaining protein.
2. Experimental and discussion 2.1. cDNA Isolation and sequence analysis Antibody screening of a mouse M27 Lewis lung carcinoma cDNA expression library resulted in the isolation of a 1400-bp 5∞-truncated cDNA fragment (pSK1400; Fig. 1). Using PCR amplification, a cDNA fragment was isolated (pCR1300; Fig. 1) that overlapped with pSK1400 and contained the 5∞ end of the cDNA. The complete cDNA was constructed and sequenced in its entirety on both strands (pSK2000; Fig. 1). The complete sequence consists of 2079 bp with a 14-bp long poly(A) tail and contains a complete open reading frame (ORF ) (Fig. 2). The ORF begins with an AUG start codon at position 56–58 and consists of 1629 bp. Two stop codons are located at nt 1685–1690 and the 3∞-untranslated region is 403 bp with a putative polyadenylation signal AAUAAA located 22 bp upstream from the poly(A) tail. The size of the cDNA approximated the size of the transcript detected by
Northern analysis of total RNA from both M27 and NIH3T3 cells (2.3 kb; Fig. 3). 2.2. Identity to stress-inducible TPR-containing proteins The predicted ORF encodes a protein of 543 aa (62 582 Da). Amino acid sequence alignments revealed that our predicted protein has significant aa identity to the stress-inducible proteins, IEF SSP 3521 (97%; Honore et al., 1992), GMSTI (44%; Torres et al., 1995), STI1 (42%; Nicolet and Craig, 1989) and LSIP (32%, Joshi et al., 1993). All of these proteins contain the 34-aa TPR motif ( Fig. 4; Lamb et al., 1995). Our protein has 10 putative TPR motifs in an identical arrangement to that found in IEF SSP 3521, however, some of the TPR motifs are overlapping so that only six out of the ten may be functional (Fig. 4). These TPR motifs may be important for the proper folding of mSTI1 and for its interactions with other proteins. Multiple sequence alignment of our protein with the human, soybean, yeast and parasite stress-inducible proteins revealed several conserved blocks ( Fig. 4), the largest being a perfectly conserved b-sheet-forming (Chou and Fasman, 1974) NHVLYSNRSA, situated at the end of the first TPR motif. This sequence data suggests that our protein, IEF SSP 3521, GMSTI, STI1 and LSIP are the mouse, human, soybean, yeast and parasite homologues, respectively, of a common ancestral protein. We propose a more consistent nomenclature by naming our protein mSTI1 and the human, soybean, yeast and parasite
Fig. 1. Restriction map of the insert DNA of pSK2000 and its derivatives pSK1400 and pCR1300. Insert restriction sites are indicated by thick vertical lines while vector restriction sites used for subcloning are indicated by thin vertical lines. Methods: A mouse M27 Lewis lung carcinoma lZAPII cDNA library (Brightman et al., 1995) was screened using Ab to a partially purified actin capping activity from Dictyostelium (Eddy et al., 1993). This screening resulted in the isolation of plasmid pSK1400 containing a 5∞-truncated cDNA fragment in the vector pBluescript SK(−) (Stratagene, La Jolla, CA). The 5∞ end of the cDNA, cloned into plasmid pCR1300, was obtained by PCR amplification using the M27 lZAPII cDNA library (9.2×1012 PFU ) as a template and primer 1 (5∞-tcacagtccttgagtgcc; nucleotides 1291–1308 of cDNA for mSTI1) and primer 2 (5∞-gtaaaacgacggccagt; the M13–20 vector primer). PCR was performed in a 100 ml volume containing 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl , 0.2 mM each of dGTP, dATP, dTTP and dCTP, 0.5 units of Taq DNA polymerase (Boehringer-Mannheim, Indianapolis, IN, USA), 2 300 pmol of each primer and 5 ml of cDNA template. The optimal conditions for amplification using these primers on cDNA was found to be 35 cycles of denaturation for 1 min at 94°C, of annealing for 1.5 min at 65°C, and of polymerization for 2.5 min at 72°C. The amplification cycle was preceded by a "hot start" of 4.5 min at 94°C to denature the cDNA template and 5 min at 80°C when the Taq DNA polymerase was added. The amplification cycle was ended by a polymerization step for 10 min at 72°C. PCR fragments of approx. 1300 bp were cloned directly into the pCRII vector (Invitrogen, San Diego, CA, USA) to generate pCR1300. Three identical PCR fragments representing three separate PCR reactions were cloned and their identity confirmed by Southern analysis (Sambrook et al., 1989) and DNA sequencing ( Tabor and Richardson, 1987). The fulllength cDNA in plasmid pSK2000 was constructed by ligating the 670-bp EcoRV–BglII fragment of pCR1300 to the 4350-bp SmaI–BglII fragment of pSK1400.
Fig. 2. Nucleotide sequence of the mSTI1 cDNA insert in pSK2000 (GenBank accession No. U27830) and deduced aa sequence. The boxed aa residues represent potential casein kinase II phosphorylation sites, while thin and thick underlining represent a polyproline stretch and a potential NLS, respectively. Methods: Sequencing of both DNA strands was done by the chain termination method of Tabor and Richardson (1987) using the modified T7 DNA polymerase (Sequenase; US Biochemical, Cleveland, OH, USA). Sequence analysis was carried out using the GCG sequence analysis software package version 7.
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single protein of approx. 63 kDa ( Fig. 5). This protein most likely corresponds to mSTI1 since its size correlates well with the predicted molecular mass for mSTI1. mSTI1 was detected in all the major mouse organs and in some cases at levels that approximated the levels detected in mouse M27 Lewis lung carcinoma cells ( Fig. 5). In addition to detecting mSTI1 in various different mouse cell lines, Ab to mSTI1 detected a protein of 63 kDa in monkey and human cell lines, but not in bovine cell lines (data not shown).
3. Conclusions
Fig. 3. Northern analysis of mSTI1 transcript length in mouse M27 Lewis lung carcinoma cells ( lane 1) and mouse NIH3T3 fibroblast cells ( lane 2). Methods: Total cellular RNA was isolated from cells by the guanidinium thiocyanate extraction method of Chomczynski and Sacchi (1987). RNA (20 mg) was resolved by 1% agarose gel electrophoresis under denaturing conditions (2.2 M formaldehyde) and blotted onto a Hybond N+ membrane (Amersham, Arlington Heights, IL, USA) by the capillary method (Sambrook et al., 1989). Northern hybridization was carried out using a 32P-labeled (Random Primed DNA Labeling kit, Boehringer-Mannheim) 750-bp BglII–BstXI cDNA fragment as a probe. Hybridization was performed at 65°C under low stringency conditions (5×SSPE, 5×Denhardt’s solution, 0.5% SDS ). Blots were washed with high stringency (65°C, 0.2×SSPE, 0.1% SDS ).
counterparts as hSTI1, gmSTI1, ySTI1 and lSTI1, respectively. We have identified a number of other interesting motifs in mSTI1. There is a putative bipartite nuclear localization signal (NLS; Dingwall and Laskey, 1991) situated between aa 222 and 239 (Fig. 2). This potential NLS is present in a similar position in all the mSTI1 homologues except for STI1 and LSIP. There are data that suggests IEF SSP 3521 (Honore et al., 1992) and mSTI1 (La¨ssle et al., 1997) may be found in the nucleus, making this potential NLS a target for further study. There are six potential casein kinase II phosphorylation sites in mSTI1 ( Fig. 2; S/T-X-X-D/E; Krebs et al., 1988), and recently we showed by in vitro phosphorylation experiments that Ser189 is a substrate for casein kinase II (La¨ssle et al., 1997). A Pro-rich region including seven consecutive Pro residues was found at position 199–219 (Fig. 2). If this centrally positioned polyproline stretch can form an extended polyproline II helix, it could serve as a linker region between two potentially globular TRP-containing domains in mSTI1 (see Fig. 4). 2.3. Western analysis of cultured cells and organ tissue Western analysis of mouse cells using Ab made against a recombinant GST–mSTI1 fusion protein detected a
(1) We report the isolation of a cDNA fragment encoding a murine stress-inducible protein, mSTI1. (2) The size of the full-length cDNA (2079 bp) approximated the size of the transcript detected by Northern analysis of total RNA from mouse cells (2300 bp). (3) The deduced aa sequence for mSTI1 predicted a protein of 62 582 Da that contained ten potential TPR motifs, a putative nuclear localization signal, six potential phosphorylation sites for casein kinase II and a central Pro rich region. (4) Western analysis detected mSTI1 in all the major mouse organs and in mouse cell lines, and its homologue in monkey and human cell lines.
References Brightman, S.E., Blatch, G.L., Zetter, B.R., 1995. Isolation of a mouse cDNA encoding MTJ1, a new murine member of the DnaJ family of proteins. Gene 153, 249–254. Chang, H.-C.J., Lindquist, S., 1994. Conservation of Hsp90 macromolecular complexes in Saccharomyces cerevisiae. J. Biol. Chem. 269, 24983–24988. Chen, S., Prapapanich, V., Rimerman, R.A., Honore, B., Smith, D.F., 1996. Interactions of p60, a mediator of progesterone receptor assembly, with heat shock proteins Hsp90 and Hsp70. Mol. Endocrinol. 10, 682–693. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem. 162, 156–159. Chou, F.Y., Fasman, G.D., 1974. Prediction of protein conformation. Biochemistry 13, 122–145. Dingwall, C., Laskey, R.A., 1991. Nuclear targeting sequences–a consensus? Trends Biochem. Sci. 16, 478–481. Eddy, R.J., Sauterer, R.A., Condeelis, J.S., 1993. Aginactin, an agonist-regulated F-actin capping activity is associated with an Hsc70 in Dictyostelium. J. Biol. Chem. 268, 23267–23274. Georgopoulos, C., Welch, W.J., 1993. Role of the major heat shock proteins as molecular chaperones. Annu. Rev. Cell Biol. 9, 601–634. Gross, M., Hessefort, S., 1996. Purification and characterization of a 66-kDa protein from rabbit reticulocyte lysate which promotes the recycling of Hsp70. J. Biol. Chem. 271, 16833–16841. Gross, M., Hessefort, S., Olin, A., Reddy, G., 1996. Extensive sequencing of tryptic peptides of a rabbit reticulocyte 66-kDa protein that
Fig. 4. Multiple sequence alignment of the mSTI1 aa sequence with the aa sequences of stress-inducible protein homologues; IEF SSP 3521 from humans (hSTI1; Honore et al., 1992), STI1 from yeast (ySTI1; Nicolet and Craig, 1989), GMSTI from soybean (gmSTI1; Torres et al., 1995) and LSIP from a parasite ( lSTI1; Joshi et al., 1993). Sequence alignments were carried out using the GCG sequence analysis software package version 7 and the accompanying databases. Identical or conservatively substituted aa (four out of five) are shown in uppercase and used to generate a consensus sequence below the alignment. The positions of six potential TPR motifs are indicated by underlining.
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Fig. 5. Western analysis to detect the presence of mSTI1 protein in mouse organs including lung (Lu), liver (Li), heart (H ), skeletal muscle (Sk), spleen (Sp), kidney ( Ki) and brain (B), and mouse M27 Lewis lung carcinoma cells (M27). Methods: For Western analysis, cell lysates were prepared in lysis buffer (20 mM Tris pH 8.0; 137 mM NaCl; 1% Nonidet P-40; 1 mM phenylmethylsulphonyl fluoride; 0.15 U/ml aprotinin; 1 mM sodium vanadate) and any insoluble cell debris was removed by a 20 min centrifugation at 10 000×g. SDS–PAGE was performed according to Laemmli (1970) using 10 mg of protein. After SDS–PAGE, polypeptides were transferred to nitrocellulose. mSTI1 protein was detected with an enhanced chemiluminescence system (ECL, Amersham) using Ab raised to a C-terminal peptide of mSTI1 (aa 370–381) or a GST fusion protein comprising the 334 C-terminal aa of mSTI1 (La¨ssle et al., 1997).
promotes recycling of Hsp70: homology to stress-related proteins. J. Biol. Chem. 271, 16842–16849. Honore, B., Leffers, H., Madsen, P., Rasmussen, H.H., Vandekerckhove, J., Celis, J.E., 1992. Molecular cloning and expression of a transformation-sensitive human protein containing the TRP motif and sharing identity to the stress-inducible yeast protein STI1. J. Biol. Chem. 267, 8485–8491. Joshi, M., Dwyer, D.M., Nakhasi, H.L., 1993. Cloning and characterization of differentially expressed genes from in vitro-grown ‘‘amastigotes’’ of Leishmania donovani. Mol. Biol. Parasitol. 58, 345–354.
Krebs, E.G., Eisenman, R.N., Kuenzel, E.A., Litchfield, D.W., Lozeman, F.J., Lu¨scher, B., Sommercorn, J., 1988. Casein kinase II as a potentially important enzyme concerned with signal transduction. Cold Spring Harbor Symp. Quant. Biol. 53, 77–84. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680–685. Lamb, J.R., Tugendreich, S., Hieter, P., 1995. Tetratrico peptide repeat interactions: to TPR or not to TPR. Trends Biochem. Sci. 20, 257–259. La¨ssle, M., Blatch, G.L., Takatori, T., Kundra, V., Zetter, B.R., 1997. Stress-inducible, murine protein mSTI1: characterization of binding domains for heat shock proteins and in vitro phosphorylation by different kinases. J. Biol. Chem. 272, 1876–1884. Nicolet, C.M., Craig, E.A., 1989. Isolation and characterization of STI1, a stress-inducible gene from Saccharomyces cerevisiae. Mol. Cell. Biol. 9, 3638–3646. Owens-Grillo, J.K., Czar, M.J., Hutchison, K.A., Hoffmann, K., Perdew, G.H., Pratt, W.B., 1996. A model of protein targeting mediated by immunophilins and other proteins that bind to hsp90 via tetratricopeptide repeat domains. J. Biol. Chem. 271, 13468–13475. Sambrook, J., Fritsch, E.F., Maniatis T., 1989. Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schumacher, R.J., Hurst, R., Sullivan, W.P., McMahon, N.J., Toft, D.O., Matts, R.L., 1994. ATP-dependent chaperoning activity of reticulocyte lysate. J. Biol. Chem. 269, 9493–9499. Smith, D.F., Sullivan, W.P., Marion, T.N., Zaitsu, K., Madden, B., McCormick, D.J., Toft, D.O., 1993. Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70. Mol. Cell. Biol. 13, 869–876. Tabor, S., Richardson, C.C., 1987. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 84, 4767–4771. Torres, J.H., Chatellard, P., Stutz, E., 1995. Isolation and characterization of gmsti, a stress-inducible gene from soybean (Glycine max) coding for a protein belonging to the TPR (tetratricopeptide repeats) family. Plant Mol. Biol. 27, 1221–1226.
Isolation of a mouse cDNA encoding mSTI1, a stress-inducible protein containing the TPR motif Gregory L. Blatch a,*, Michael La¨ssle b, Bruce R. Zetter c, Vikas Kundra d a Department of Biochemistry, University of the, Witwatersrand, P O WITS 2050, South Africa b MIT, Department of Chemical Engineering and Center for Biomedical Engineering, Cambridge, MA 02139, USA c Department of Cell Biology and Surgery, Harvard Medical School, Children’s Hospital, Boston, MA 02115, USA d Brigham and Woman’s Hospital, Department of Radiology, Boston, MA 02115, USA Received 26 December 1996; accepted 11 March 1997; Received by J. Wild
Abstract We report the isolation and sequencing of the complete 2079-bp cDNA fragment encoding mSTI1, a murine stress-inducible protein. The predicted ORF encodes a protein of 543 amino acids (aa) and M 62 582. The predicted protein has significant r homology to stress-inducible proteins from humans (IEF SSP 3521), soybean (GMSTI ), yeast (STI1) and a parasite, Leishmania donovani (LSIP). All of these proteins contain 34-aa repeat motifs, termed tetratricopeptide repeats ( TPRs), that are proposed to be involved in intra- and intermolecular protein interactions. mSTI1 has ten potential TPR motifs, a putative nuclear localization signal (NLS ), six potential phosphorylation sites for casein kinase II and a central proline-rich region. Western analysis detected a protein of approx. 63 kDa in all the major mouse organs and in mouse, monkey and human cell lines. © 1997 Elsevier Science B.V. Keywords: STI1; IEF SSP 3521; p60; Hop; RF-hsp70; GMSTI; LSIP
1. Introduction Cells respond to cellular stress by modifying the expression, localization and post-translational modification of a set of proteins generally referred to as stressproteins (Georgopoulos and Welch, 1993). A novel stress-inducible protein, STI1, has been described for yeast where it is proposed to mediate the heat shock (HS) response of some Hsp70 genes (Nicolet and Craig, * Corresponding author. Tel. +27 11 7162204; Fax: +27 11 7164479; e-mail: [email protected] Abbreviations: aa, amino acid(s); Ab, antibody(ies); bp, base pair(s); cDNA, DNA complementary to RNA; E., Escherichia; EDTA, ethylene diamine tetra-acetic acid; GCG, Genetics Computer Group (Madison, WI, USA); GMSTI, Glycine max stress-inducible protein; GST, glutathione S-transferase; HS, heat shock; Hsp70, HS protein 70; Hsp90, HS protein 90; IEF SSP 3521, isoelectric focusing sample spot 3521; LSIP, Leishmania donovani stress-inducible protein; mSTI1, murine stress inducible protein 1; NLS, nuclear localization signal; nt, nucleotide(s); ORF, open reading frame; p, plasmid; PA, polyacrylamide; PAGE, PA-gel electrophoresis; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; SSPE, 0.18 M NaCl/0.01 M sodium phosphate/0.001 M EDTA pH 7.7; STI1, yeast stress-inducible protein 1; TBE, Tris-borate EDTA; TPR, tetratricopeptide repeats; ∞ (prime), denotes truncated gene at the indicated side 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 2 06 - 0
1989). The STI1 protein shows no homology to the Hsp70 family of proteins but shares 42% identity to IEF SSP 3521, a human transformation-sensitive protein ( Honore et al., 1992), 38% identity to GMSTI, a soybean (Glycine max) stress-inducible protein ( Torres et al., 1995), and 26% identity to LSIP, a stress-inducible protein of the parasite Leishmania donovani (Joshi et al., 1993). STI1, IEF SSP 3521 and GMSTI contain a number of 34-aa tetratricopeptide repeats ( TPR) proposed to be important for intra- and intermolecular protein interactions (Lamb et al., 1995). Both IEF SSP 3521 and STI1 have been identified as components of multiprotein chaperone complexes involving the HS proteins, Hsp70 and Hsp90 (Smith et al., 1993; Chang and Lindquist, 1994; Schumacher et al., 1994). IEF SSP 3521, also referred to as p60 or Hop, has been shown to bind to both Hsp90 and Hsp70, and to be important for the successful formation of Hsp90 heterocomplexes (Smith et al., 1993; Owens-Grillo et al., 1996; Chen et al., 1996). Sequencing of tryptic peptides of a 66-kDa rabbit protein suggest that it is the rabbit homologue of IEF SSP 3521 (Gross et al., 1996). This protein has been named RF-hsp70, and has been shown to promote the recycling of Hsp70 by catalyzing the dissociation of
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Hsp70-bound ADP in exchange for ATP (Gross and Hessefort, 1996). Recently, we identified a murine stressinducible protein, mSTI1, that interacts with Hsc70 and Hsp90 (La¨ssle et al., 1997). In this report, we show by cDNA sequence analysis that mSTI1 is homologous to STI1, IEF SSP 3521, GMSTI1 and LSIP and is a TPRcontaining protein.
2. Experimental and discussion 2.1. cDNA Isolation and sequence analysis Antibody screening of a mouse M27 Lewis lung carcinoma cDNA expression library resulted in the isolation of a 1400-bp 5∞-truncated cDNA fragment (pSK1400; Fig. 1). Using PCR amplification, a cDNA fragment was isolated (pCR1300; Fig. 1) that overlapped with pSK1400 and contained the 5∞ end of the cDNA. The complete cDNA was constructed and sequenced in its entirety on both strands (pSK2000; Fig. 1). The complete sequence consists of 2079 bp with a 14-bp long poly(A) tail and contains a complete open reading frame (ORF ) (Fig. 2). The ORF begins with an AUG start codon at position 56–58 and consists of 1629 bp. Two stop codons are located at nt 1685–1690 and the 3∞-untranslated region is 403 bp with a putative polyadenylation signal AAUAAA located 22 bp upstream from the poly(A) tail. The size of the cDNA approximated the size of the transcript detected by
Northern analysis of total RNA from both M27 and NIH3T3 cells (2.3 kb; Fig. 3). 2.2. Identity to stress-inducible TPR-containing proteins The predicted ORF encodes a protein of 543 aa (62 582 Da). Amino acid sequence alignments revealed that our predicted protein has significant aa identity to the stress-inducible proteins, IEF SSP 3521 (97%; Honore et al., 1992), GMSTI (44%; Torres et al., 1995), STI1 (42%; Nicolet and Craig, 1989) and LSIP (32%, Joshi et al., 1993). All of these proteins contain the 34-aa TPR motif ( Fig. 4; Lamb et al., 1995). Our protein has 10 putative TPR motifs in an identical arrangement to that found in IEF SSP 3521, however, some of the TPR motifs are overlapping so that only six out of the ten may be functional (Fig. 4). These TPR motifs may be important for the proper folding of mSTI1 and for its interactions with other proteins. Multiple sequence alignment of our protein with the human, soybean, yeast and parasite stress-inducible proteins revealed several conserved blocks ( Fig. 4), the largest being a perfectly conserved b-sheet-forming (Chou and Fasman, 1974) NHVLYSNRSA, situated at the end of the first TPR motif. This sequence data suggests that our protein, IEF SSP 3521, GMSTI, STI1 and LSIP are the mouse, human, soybean, yeast and parasite homologues, respectively, of a common ancestral protein. We propose a more consistent nomenclature by naming our protein mSTI1 and the human, soybean, yeast and parasite
Fig. 1. Restriction map of the insert DNA of pSK2000 and its derivatives pSK1400 and pCR1300. Insert restriction sites are indicated by thick vertical lines while vector restriction sites used for subcloning are indicated by thin vertical lines. Methods: A mouse M27 Lewis lung carcinoma lZAPII cDNA library (Brightman et al., 1995) was screened using Ab to a partially purified actin capping activity from Dictyostelium (Eddy et al., 1993). This screening resulted in the isolation of plasmid pSK1400 containing a 5∞-truncated cDNA fragment in the vector pBluescript SK(−) (Stratagene, La Jolla, CA). The 5∞ end of the cDNA, cloned into plasmid pCR1300, was obtained by PCR amplification using the M27 lZAPII cDNA library (9.2×1012 PFU ) as a template and primer 1 (5∞-tcacagtccttgagtgcc; nucleotides 1291–1308 of cDNA for mSTI1) and primer 2 (5∞-gtaaaacgacggccagt; the M13–20 vector primer). PCR was performed in a 100 ml volume containing 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl , 0.2 mM each of dGTP, dATP, dTTP and dCTP, 0.5 units of Taq DNA polymerase (Boehringer-Mannheim, Indianapolis, IN, USA), 2 300 pmol of each primer and 5 ml of cDNA template. The optimal conditions for amplification using these primers on cDNA was found to be 35 cycles of denaturation for 1 min at 94°C, of annealing for 1.5 min at 65°C, and of polymerization for 2.5 min at 72°C. The amplification cycle was preceded by a "hot start" of 4.5 min at 94°C to denature the cDNA template and 5 min at 80°C when the Taq DNA polymerase was added. The amplification cycle was ended by a polymerization step for 10 min at 72°C. PCR fragments of approx. 1300 bp were cloned directly into the pCRII vector (Invitrogen, San Diego, CA, USA) to generate pCR1300. Three identical PCR fragments representing three separate PCR reactions were cloned and their identity confirmed by Southern analysis (Sambrook et al., 1989) and DNA sequencing ( Tabor and Richardson, 1987). The fulllength cDNA in plasmid pSK2000 was constructed by ligating the 670-bp EcoRV–BglII fragment of pCR1300 to the 4350-bp SmaI–BglII fragment of pSK1400.
Fig. 2. Nucleotide sequence of the mSTI1 cDNA insert in pSK2000 (GenBank accession No. U27830) and deduced aa sequence. The boxed aa residues represent potential casein kinase II phosphorylation sites, while thin and thick underlining represent a polyproline stretch and a potential NLS, respectively. Methods: Sequencing of both DNA strands was done by the chain termination method of Tabor and Richardson (1987) using the modified T7 DNA polymerase (Sequenase; US Biochemical, Cleveland, OH, USA). Sequence analysis was carried out using the GCG sequence analysis software package version 7.
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single protein of approx. 63 kDa ( Fig. 5). This protein most likely corresponds to mSTI1 since its size correlates well with the predicted molecular mass for mSTI1. mSTI1 was detected in all the major mouse organs and in some cases at levels that approximated the levels detected in mouse M27 Lewis lung carcinoma cells ( Fig. 5). In addition to detecting mSTI1 in various different mouse cell lines, Ab to mSTI1 detected a protein of 63 kDa in monkey and human cell lines, but not in bovine cell lines (data not shown).
3. Conclusions
Fig. 3. Northern analysis of mSTI1 transcript length in mouse M27 Lewis lung carcinoma cells ( lane 1) and mouse NIH3T3 fibroblast cells ( lane 2). Methods: Total cellular RNA was isolated from cells by the guanidinium thiocyanate extraction method of Chomczynski and Sacchi (1987). RNA (20 mg) was resolved by 1% agarose gel electrophoresis under denaturing conditions (2.2 M formaldehyde) and blotted onto a Hybond N+ membrane (Amersham, Arlington Heights, IL, USA) by the capillary method (Sambrook et al., 1989). Northern hybridization was carried out using a 32P-labeled (Random Primed DNA Labeling kit, Boehringer-Mannheim) 750-bp BglII–BstXI cDNA fragment as a probe. Hybridization was performed at 65°C under low stringency conditions (5×SSPE, 5×Denhardt’s solution, 0.5% SDS ). Blots were washed with high stringency (65°C, 0.2×SSPE, 0.1% SDS ).
counterparts as hSTI1, gmSTI1, ySTI1 and lSTI1, respectively. We have identified a number of other interesting motifs in mSTI1. There is a putative bipartite nuclear localization signal (NLS; Dingwall and Laskey, 1991) situated between aa 222 and 239 (Fig. 2). This potential NLS is present in a similar position in all the mSTI1 homologues except for STI1 and LSIP. There are data that suggests IEF SSP 3521 (Honore et al., 1992) and mSTI1 (La¨ssle et al., 1997) may be found in the nucleus, making this potential NLS a target for further study. There are six potential casein kinase II phosphorylation sites in mSTI1 ( Fig. 2; S/T-X-X-D/E; Krebs et al., 1988), and recently we showed by in vitro phosphorylation experiments that Ser189 is a substrate for casein kinase II (La¨ssle et al., 1997). A Pro-rich region including seven consecutive Pro residues was found at position 199–219 (Fig. 2). If this centrally positioned polyproline stretch can form an extended polyproline II helix, it could serve as a linker region between two potentially globular TRP-containing domains in mSTI1 (see Fig. 4). 2.3. Western analysis of cultured cells and organ tissue Western analysis of mouse cells using Ab made against a recombinant GST–mSTI1 fusion protein detected a
(1) We report the isolation of a cDNA fragment encoding a murine stress-inducible protein, mSTI1. (2) The size of the full-length cDNA (2079 bp) approximated the size of the transcript detected by Northern analysis of total RNA from mouse cells (2300 bp). (3) The deduced aa sequence for mSTI1 predicted a protein of 62 582 Da that contained ten potential TPR motifs, a putative nuclear localization signal, six potential phosphorylation sites for casein kinase II and a central Pro rich region. (4) Western analysis detected mSTI1 in all the major mouse organs and in mouse cell lines, and its homologue in monkey and human cell lines.
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Fig. 4. Multiple sequence alignment of the mSTI1 aa sequence with the aa sequences of stress-inducible protein homologues; IEF SSP 3521 from humans (hSTI1; Honore et al., 1992), STI1 from yeast (ySTI1; Nicolet and Craig, 1989), GMSTI from soybean (gmSTI1; Torres et al., 1995) and LSIP from a parasite ( lSTI1; Joshi et al., 1993). Sequence alignments were carried out using the GCG sequence analysis software package version 7 and the accompanying databases. Identical or conservatively substituted aa (four out of five) are shown in uppercase and used to generate a consensus sequence below the alignment. The positions of six potential TPR motifs are indicated by underlining.
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Fig. 5. Western analysis to detect the presence of mSTI1 protein in mouse organs including lung (Lu), liver (Li), heart (H ), skeletal muscle (Sk), spleen (Sp), kidney ( Ki) and brain (B), and mouse M27 Lewis lung carcinoma cells (M27). Methods: For Western analysis, cell lysates were prepared in lysis buffer (20 mM Tris pH 8.0; 137 mM NaCl; 1% Nonidet P-40; 1 mM phenylmethylsulphonyl fluoride; 0.15 U/ml aprotinin; 1 mM sodium vanadate) and any insoluble cell debris was removed by a 20 min centrifugation at 10 000×g. SDS–PAGE was performed according to Laemmli (1970) using 10 mg of protein. After SDS–PAGE, polypeptides were transferred to nitrocellulose. mSTI1 protein was detected with an enhanced chemiluminescence system (ECL, Amersham) using Ab raised to a C-terminal peptide of mSTI1 (aa 370–381) or a GST fusion protein comprising the 334 C-terminal aa of mSTI1 (La¨ssle et al., 1997).
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