ARCHIVES

Vol.

OF BIOCHEMISTRY

288, No. 1, July,

AND

BIOPHYSICS

pp. 131-140,

1991

Antibodies against the SV40 Large T Antigen Nuclear Localization Sequence Barbara

Wolff,

Laboratory

of Biochemistry

Received

December

Min

Kyun

Park, Emily

and Metabolism,

19, 1990, and in revised

form

Klima,

NIDDK

March

and John A. Hanover1

National

Inc.

An increasing body of evidence suggests that the translocation of large proteins (MW > 65,000) into the nucleus depends on the presence of one or more specific nuclear

1 To whom

correspondence

should

0003.9861/91$3.00 Copyright 0 1991 by Academic Press, Al1 rights of reproduction in any form

be addressed.

of Health, Bethesda, Maryland

20892

4,199l

Transport of large proteins into the nucleus requires both a nuclear localization signal (NLS) and exposure of that signal to components of the transport machinery. In this report, polyclonal and monoclonal antibodies were generated against the NLS of SV40 large T antigen. Several of these antibodies immunoprecipitated large T antigen produced by in vitro transcription-translation and recognized T antigen expressed in cultured cells. Binding of the antibodies to T antigen was quantified using an indirect radioimmunoassay and found to be specifically inhibited by peptides corresponding to the T antigen NLS. The ability of NLS-specific antibodies to recognize large T antigen suggests that the NLS is exposed on the surface of T antigen. When one of the NLS-specific monoclonal antibodies was introduced into the cytoplasm of cells expressing T antigen, the antibody remained cytoplasmic. These results suggested either that cytoplasmic components compete for binding to the NLS or that the antibody dissociates from T antigen during transport into the nucleus. When an antibody directed against an epitope distinct from the NLS was microinjetted into the cytoplasm of cells expressing large T antigen, both the antibody and antigen were transported into the nucleus. The observed stability of the antigenantibody complex strongly suggests protein unfolding is 0 1991 Academic not required for nuclear protein transport. Press,

Institutes

localization sequences (NLS)’ (l-5). Such amino acid sequences have been identified in a variety of proteins. In general, they are short stretches of highly basic amino acids that are preceded or followed by a structure breaking amino acid such as Pro or Thr. However, similar basic amino acid sequences also occur in a number of cytoplasmic proteins (5). The basic sequences in these cytoplasmic proteins might be buried inside the folded molecule or involved in a salt bridge. In nuclear proteins, the stretch of basic residues would be expected to be exposed on the surface of the molecule, making them accessible to elements of the nuclear transport machinery. The simian virus 40 large tumor antigen (SV40 T antigen) contains a nuclear localization signal which has been studied in great detail. This sequence (amino acids 126 and 132) is located in a very hydrophilic part of the molecule and would be expected to be exposed on its surface (6). Roberts et al. (7) inserted the SV40 large T antigen signal into different parts of the chicken pyruvate kinase molecule. Chicken pyruvate kinase was chosen since its crystal structure has been solved. Nuclear transport was not observed when the targeting sequence was inserted into a part of the pyruvate kinase molecule that was not exposed on the surface suggesting that exposure of the NLS is required for it to function. A number of antibodies against T antigen have been produced in order to analyze the function of various regions of the polypeptide chain. These include the state of aggregation of T antigen, phosphorylation, DNA binding, and complex formation with p53 (8-10). To determine

* Abbreviations used: NLS, nuclear localization signal; SV40 T antigen; simian virus 40 large tumor antigen; HPLC, high performance liquid chromatography; BSA, bovine serum albumin; DCCD, dicyclohexylcarbodiimide; ELISA, enzyme-linked immunosorbant assay; FCS, fetal calf serum; PBS, phosphate-buffered saline; DMEM, Dulbecco’s modified Eagle’s medium; FPLC, fast protein liquid chromatography; SDSPAGE, sodium dodecyl sulfate-polyacrilamide gel electrophoresis.

131 Inc. reserved

132

WOLFF

the accessibility of the nuclear transport signal in SV40 large T antigen we have taken a similar approach. In this report, we have prepared polyclonal and monoclonal antibodies against the nuclear localization signal of T antigen. Our results demonstrate that a variety of the antibodies specifically interact with T antigen produced by in vitro transcription and translation or expressed in tissue culture cells. A NLS-specific monoclonal antibody was found to remain in the cytoplasm following direct microinjection into that compartment. However, when an antibody against an epitope of T antigen other than the nuclear localization signal was microinjected, the antibody was cotransported with T antigen into the nucleus; the antigen-antibody complex survives the transport event. EXPERIMENTAL

PROCEDURES

Materials. The peptides Pro-Lys-Lys-Lys-Arg-Lys-Val-Tyr and Pro-Lys-Lys-Lys-Arg-Lys-Val-Tyr-Cys were synthesized by Peninsula (Belmont, CA) and purified on reverse phase HPLC using an Altex Model 322 HPLC system with a Whatman ODS-3 column, 50 cm X 9.4 mm, and a linear gradient of O-60% acetonitrile in 0.1% trifluoroacetic acid. The other peptides mentioned in the text, which were used for differential screening of the antibodies were also synthesized by Penninsula, and used without further purification. Bovine serum albumin (BSA) was from Sigma (St. Louis, MO), and dicyclohexylcarbodiimide (DCCD) from Eastman-Kodak (Rochester, NY). Cyanogen bromideactivated Sepharose 4B was provided by Pharmacia (Piscataway, NJ). Rhodamine- and alkaline phosphatase-labeled goat-anti-mouse IgG + M were purchased from Jackson (Avondale, PA), as well as unlabeled rabbit-anti-mouse IgG and rabbit-anti-mouse IgM. lz51-labeled goat-antimouse IgG and [35S]methionine were from New England Nuclear (Boston, MA) and materials for in uitro transcription and translation were obtained from Promega (Madison, WI). TIB117 and TIB115 were obtained from the hybridoma collection of the American Type Culture Collection (Rockville, MD). Production and afinity purification of polyclonal antibodies. The peptide Pro-Lys-Lys-Lys-Arg-Lys-Val-Tyr was coupled to BSA using DCCD according to (11). Two rabbits were immunized subcutaneously with 500 pg of peptide conjugate. Rabbit bleeds were checked for reactivity in the ELISA assay (see below), and bleeds reactive at a dilution of 1:104 or more were pooled and used for affinity purification. The peptide Pro-Lys-Lys-Lys-Arg-Lys-Val-Tyr was coupled to cyanogen bromide-activated Sepharose 4B and free reactive groups were blocked with 1 M ethanolamine according to the manufacturer’s manual. A 50% ammonium sulfate cut of the antiserum, redissolved in borat.e buffer, pH 8.0 (0.1 M boric acid, 0.5 M NaCl), was applied to the column, washed extensively with borate buffer, pH 8, and, subsequently, acetate buffer pH 4.8 (0.1 M acetate, 0.5 M NaCl) to remove IgM antibodies. IgG antibodies were eluted with acetate buffer, pH 2.5 (0.2 M borate buffer, 0.5 M NaCI), and the fractions were immediately neutralized with 1 M TrisHCl, pH 8 (1 ml Tris for 2 ml acetate buffer). The fract,ions which showed the strongest reactivity in an enzyme-linked immunosorbent assay (ELISA) were pooled and stored at -20°C. By this purification procedure, anti-peptide reactivity was enriched approximately 900-fold, and reactivity with BSA was eliminated. Production of monoclonal antibodies: Immunization. A BALB/c female mouse 6-weeks-old was initially immunized by intraperitoneal injection of water in oil emulsion of 500 Fg of the peptide Cys-Pro-LysLys-Lys-Arg-Lys-Val coupled to bovine serum albumin in complete Freund’s adjuvant (GIBCO). Four weeks later, 500 fig of the peptide conjugate was injected intravenously. Three days after final injection, the mouse was anesthetized with ether and sacrificed. The spleen was

ET AL. removed aseptically from the mouse and vessels and connective tissue were removed with forceps in RPM1 medium. The spleen was teased with forceps and smashed onto a stainless steel mesh using the rubber part of the inner piston of a l-ml disposable plastic syringe. This was performed in a loo-mm plastic culture dish containing 10 ml of RPM1 media. The single spleen cell suspension was washed with RPM1 medium and the number of spleen cells were counted using a hematocytometer. Cell culture and fusion. RPM1 medium (GIBCO) was supplemented and used for with 1 mM sodium pyruvate and 1.5 mM LASglutamine the growth of X63.Ag8.653 mouse myeloma (American Type Cell Collection) and hyhridomas in a 6% CO? atmosphere. The myeloma was maintained in 15% FCS (Hyclone)-RPM1 medium containing 13 mM 8-azaguanine and frozen in a liquid nitrogen tank. One week before fusion myeloma cells were cultured in 15% FCS-RPM1 medium and harvested in midlogarithmic phase for fusion. Briefly, myeloma cells and immunized spleen cells were washed in RPM1 medium, combined, and centrifuged. The cell mixture was added with 2 ml of polyethylene glycol 4000 (50% solution in PBS; GIBCO) for 1 min and stirred for another 1 min. The fusion mixture was diluted stepwise with 18 ml of RPM1 medium over a period of 5-6 min and subjected to centrifugation at 2OOg for 10 min. After discarding the supernatant, the pellet was suspended in 15% FCS-RPM1 medium supplemented with HAT supplement (hypoxanthine, 10 mM, aminopterin, 40 pM, and thymidine, 1.6 mM; (GIBCO) and plated with 0.1 ml of the suspension into 96.well tissue culture plate (Costar and Falcon) which contained normal spleen cells (1065/well) as feeder layer. One week later, the medium was changed to 15% FCS-RPM1 medium supplemented with HT supplement (GIBCO). Approximately 2 weeks after fusion, the wells were screened microscopically for hybridoma growth and screened for reactivity with the peptide. ELISA screening assay. The peptide Cys-Tyr-Pro-Lys-Lys-LysArg-Lys-Val dissolved in PBS at a concentration of 50 pg/ml of this solution was used to coat 96.well plates (50 rl/well). After air drying, the plates were washed with phosphate-buffered saline containing Ca’, Mg’, and 0.1% tween 20 (PBS-tween). Hybridoma supernatants were added to the washed plates and incubated at room temperature for 2 h. The wells were then washed with PBS-tween and goat antimouse IgG and IgM alkaline phosphatase (Jackson Laboratories) was added to a concentration of 0.5 fig/ml in a buffer containing PBS and 5% horse serum. After 1 h incubation at room temperature, the wells were washed with PBS-tween (3X) and finally with 0.1 M diethanolamine. p-Nitrophenylphosphate was added to the wells at a concentration of 1 mg/ml in 1 M diethanolamine. Following color development, the positive wells were detected calorimetrically using a Titertek 96-well plate reader. Hybridoma maintenance and monoclonal antibody subclass determination. Hyhridomas secreting antibody were cloned by limited dilution (0.5, 1, and 5 cells per well) in 96-well tissue culture plates (Costar) and screened by ELISA assay described as above. Positive cloned hyhridomas were expanded and stored in liquid nitrogen. Monoclonal antibody subclass was identified by the Ouchterlony immunodiffusion technique using a monoclonal typing kit (Miles Scientific). Monoclonal subclasses were as follows: MAb 041-IgM, MAb 193-IgM, MAb 043.IgGl, MAb 163.IgGl, MAb 201-IgGl, MAb 152.IgG3. Microinjection. COS-1 or SV-40.infected CV-1 cells were plat.ed at 50% confluence on 35-mm plastic dishes in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum. In other experiments, NIH 3T3 cells, SVT2 cells expressing T antigen, or NIH3T3 CT cells expressing a cytoplasmic variant of T antigen were used. For microinjection, glass capillary pipets, a Zeiss inverted microscope equipped with fluorescence optics, and an Eppendorf microinjector (Model 5242) were used. Antibodies were diluted to a concentration of 1 mg/ml with phosphate-buffered saline prior to injection. Fluoresceinamine was added to the solution to a final concentration of 5 mg/ml. Injection was monitored by following the fluorescein fluorescence of the injected solution.

SV40 LARGE

133

T ANTIGEN

Molecular Weight Markers

Molecular Weight Markers

94kD-

-T-Antigen

- 94kD

-

68kD-

-68kD

43kD25kD-

-43kD -25kD - 18kD

18kD-

1

2

3

FIG. 1. SDS-PAGE of in vitro translated SV40 T antigen precipitated with polyclonal antibodies to the T antigen NLS. Transcript encoding SV40 large T antigen was translated in vitro as described under Experimental Procedures. The translation product was analyzed by SDS-PAGE in the panel at the left. This amount of translation product was then immunoprecipitated using 2 pg (lane 1) or 5 pg (lane 2) of aflinity-purified NLS polyclonal antibodies and the immunoprecipitated material was analyzed by SDS-PAGE and autoradiography. Control rabbit IgG (5 fig; lane 3) did not immunoadsorb T antigen from the translation mixture.

SVT2 cells, a mouse fibroblast cell Immunofluorescence microscopy. line expressing nuclear SV40 large T antigen and NIH 3T3 mouse fibroblasts were purchased from the American Type Culture Collection (Rockville, MD). 3T3,r cells which express cytoplasmic T antigen with Asn in the place of Lys’*s were a generous gift from J. S. Butel (Baylor University, Houston, TX). Cells were seeded at low density in 35.mm petri dishes (Costar), fixed in 90% acetone, and incubated with monoclonal antibodies for 2 h at room temperature. Incubation with rhodamine-labeled goat anti-mouse IgG + M was carried out for 2 h at room temperature. After mounting the cells in Bacto FA mounting fluid (Difco, Detroit, MI), they were examined under a Zeiss inverted microscope equipped with fluorescence optics. For detection of microinjected antibodies, subclass-specific second antibodies were used. To detect TIB 117, a rhodamine goat anti-mouse IgG 2a antisera (Southern Biotechnologies) was employed. Monoclonal antibody MAb 152 was detected using fluorescein goat anti-mouse IgG 3 (Southern Biotechnologies).

TABLE Relative

Cells were seeded into the wells of Antibody-binding to fined cells. 12.well plates (Costar) and after they were grown to confluence they were fixed in 90% acetone. They were then incubated with either antiSV40 T antigen antibodies (purified by FPLC) or hybridoma supernatants diluted in phosphate-buffered saline (PBS), pH 7.4, containing 1 mg/ml BSA. After 2 h at room temperature, the cells were washed four times with PBS and incubated with *? goat anti-mouse IgG (- 1 &i/ ml; sp act 8.3 lCi/pg). After another 2 h at room temperature, cells were washed four times with PBS and lysed in 500 ~1 0.5 N NaOH/well at 37°C for 30 min. The lysate was counted in a gamma counter (Packard). In the competition experiments, antibodies were preincubated with peptides for 1 h at room temperature.

I

Reactivity of Monoclonal to Deletion Peptides

Antibodies

Antihody designation Peptide sequence

152

163

041

Reactivity

CGYGPKKKRKVGG CGYGPKNKRKVGG CGYGPKKKRKV CGYGPKKKRK CGYGPKKKR CGYGm

(100)

(100)

(100)

193

201

043

(100)

5%

(100)

3

t2

65

92

100

(100) 100

12

15 70 75 40

100 12 -3