©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Association of Polyomavirus Middle Tumor Antigen with Phospholipase C-1(*)

Wen Su (1)(§), Wei Liu (1), Brian S. Schaffhausen (2), Thomas M. Roberts (1)

From the (1) Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115 and the (2) Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Middle tumor antigen (MT) is the primary transforming protein of murine Polyomavirus. MT transforms by associating with and modulating the activities of cellular proteins involved in control of cell proliferation. MT binds to and is phosphorylated by cellular tyrosine kinases. The phosphorylated tyrosines become docking sites for SH2 (Src homology 2) domain-containing molecules. Tyrosine 322 of MT is known to be phosphorylated but has no known binding protein. We have found that phospholipase C-1 (PLC-1), a SH2 domain-containing protein, coimmunoprecipitates with MT. Tyrosine phosphorylation of PLC-1 is elevated in cells expressing MT, suggesting activation of this enzyme by MT. A Tyr-322 Phe mutation in MT renders it defective in MT-PLC-1 interaction and in transformation. From the correlation between transformation and MT-PLC-1 interaction, we suggest that PLC-1 may play a role in transformation.


INTRODUCTION

Polyomavirus induces a variety of tumors in rodents and is capable of transforming primary rodent cell lines. Middle tumor antigen (MT)() is the major transforming protein of Polyomavirus. MT is a membrane protein with no known enzymatic activity (1) . It transforms by associating with and modulating the activities of cellular proteins involved in control of cell proliferation. MT appears to function much like a growth factor receptor. It binds to and activates pp60 and other members of the src family of tyrosine kinases (2, 3, 4, 5, 6) . As a result, MT is phosphorylated on tyrosines 250, 315, and 322 (7, 8, 9) . When phosphorylated, tyrosines 250 and 315 with adjacent amino acids, respectively, form the binding sites for the key signaling molecules SHC (10, 11) and phosphatidylinositol 3-kinase (PI3-kinase) (12, 13) . Mutation of tyrosine 250 or 315 to phenylalanine renders MT defective both in transformation (14, 15) and in its ability to bind SHC or PI3-kinase, respectively. Of the three phosphorylated tyrosines in MT, the least studied is tyrosine 322.

To investigate the potential role of tyrosine 322 in MT in transformation and in regulating cellular signaling proteins, we have mutated the tyrosine 322 to phenylalanine (Tyr-322 Phe MT). As judged by focus-formation assay, the Tyr-322 Phe MT is less transforming than wild type MT. We have found that phospholipase C-1 (PLC-1), a SH2 domain-containing signaling molecule, coimmunoprecipitates with MT and that tyrosine 322 is important for this interaction. The tyrosine phosphorylation level of PLC-1 is elevated in cells expressing wild type MT but not in cells expressing Tyr-322 Phe MT. From the correlation between transformation and MT-PLC-1 interaction, we suggest that PLC-1 may play a role in transformation.


EXPERIMENTAL PROCEDURES

Plasmids and Cell Lines

Site-directed mutagenesis was performed to create two unique restriction enzyme sites adjacent to Tyr-315 and Tyr-322 codons in MT cDNA, without altering the corresponding amino acids. An oligonucleotide-directed mutagenesis system from Amersham Corp. was used. A StuI site was generated immediately upstream to the Tyr-315 codon and a XbaI site downstream to the Tyr-322 codon (Polyomavirus sequence 1176 and 1202, respectively). Excision of the DNA between these two sites from MT cDNA and insertion of double-stranded oligonucleotides generate the Tyr-315 Phe and Tyr-322 Phe mutant MTs (``315F'' and ``322F''). The pLJ retrovirus vector (16) was used to express the wild type and mutant MTs. The plasmids were transfected into 2 retrovirus packaging cells by the CaCl method (17) . The transient virus supernatants were used to infect BALB/3T3 cells. BALB/3T3 cell lines expressing various MT alleles were isolated as described previously (18) . Cells were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 0.37% bicarbonate, penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% calf serum.

Focus Formation Assay

Focus formation assay was performed by infecting BALB/3T3 cells with retroviruses carrying wild type MT, Tyr-322 Phe MT, or no MT. The retroviruses were produced using the virus packaging cell line BOSC 23, which is capable of producing high titer retroviruses (19) . Ten micrograms of the plasmids pLJ-MTwt, pLJ-MT322F, or pLJ were transfected into BOSC 23 cells using the CaCl method (17) . The resulting culturing supernatants containing the retroviruses were collected, aliquoted, and stored at -80 °C. The high titer virus stocks generated in the BOSC 23 cell line allow freezing of aliquots, a process that significantly lowers virus titers. Titers of the frozen aliquots of these retroviruses were determined by infection of BALB/3T3 cells (as described below) and subsequent selection by 400 µg/ml G418. After 10 days of incubation with medium changed every 2 days, the G418-resistant colonies were counted. For focus formation assay, an equal number of each retrovirus (1.0 10 colony-forming units) in 2.5 ml of medium (DMEM supplemented with 0.37% bicarbonate, penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% calf serum) was used to infect the BALB/3T3 cells (50% confluent) in 100-mm tissue culture dishes. Polybrene were then added to a concentration of 0.8 µg/ml. After 4 h of incubation at 37 °C and 10% CO, 7.5 ml of the same medium was added. Cultures were incubated at 37 °C overnight in the presence of 10% CO. The infected cell populations were then split into two sets of 60-mm tissue culture dishes. One set was cultured in DMEM medium supplemented with 10% calf serum, and another set was cultured in DMEM medium supplemented with 3% calf serum. Media were changed accordingly every 3 days. At 10 days, cultures were fixed and stained with 0.2% crystal violet in 10% phosphate-buffered formalin (pH 7.0).

Immunoprecipitation and Immunoblotting

Cells were allowed to grow to confluency and lysed in a lysis buffer containing 1% Nonidet P-40, 20 mM TrisCl (pH 8.0), 137 mM NaCl, and 10% glycerol. For anti-p85 immunoblotting, immunoprecipitation was carried out by incubating anti-MT monoclonal antibody PAb 750 (20) (40 µl of antibody-containing tissue culture fluid) or 1 µg of anti-PLC-1 monoclonal antibody (UBI) with cell lysates (600 µg of total protein for every cell line) from BALB/3T3 cells expressing MT and from BALB/3T3 control (``vector'') cells. After 2 h of incubation at 4 °C, protein G beads (40 µl of 50% slurry, Pharmacia Biotech Inc.) were added, and the tubes were rocked at 4 °C for 1 additional hour. Immune complexes were sequentially washed with Nonidet P-40 lysis buffer (1% Nonidet P-40, 20 mM TrisCl (pH 8.0), 137 mM NaCl and 10% glycerol), 0.5 M LiCl, 20 mM TrisCl (pH 7.6), and TNE (10 mM TrisCl (pH 7.6), 0.1 M NaCl, and 1 mM EDTA). Washed immunoprecipitates were suspended in 15 µl of sample buffer (10% glycerol, 2% SDS, 100 mM dithiothreitol, 50 mM TrisCl (pH 6.8), and 0.1% bromphenol blue), heated for 3 min at 95 °C, and analyzed by 8% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes, and anti-p85 antibody R33 (rabbit serum, 1:1000 dilution) was used for immunodetection. Immunodetection was performed using the chemiluminescence reagents and the protocol from DuPont NEN.

For anti-PLC-1 immunoblotting, immunoprecipitation was carried out by incubating 2 µl of anti-MT polyclonal antibody 18-8 (rabbit serum) or 2 µl of anti-p85 antibody R33 (rabbit serum) with cell lysates (700 µg of total protein for every cell line) from BALB/3T3 cells expressing MT and from BALB/3T3 control (vector) cells. After 2 h of incubation at 4 °C, protein A beads (20 µl of 50% slurry, Pharmacia) were added, and the tubes were rocked at 4 °C for 1 additional hour. Washing and immunoblotting were performed as described above except anti-PLC-1 monoclonal antibody (0.5 µg/ml, UBI) was used for immunodetection.

For anti-phosphotyrosine immunoblotting, immunoprecipitation was carried out by incubating 1 µg of anti-PLC-1 monoclonal antibody (UBI) with the cell lysates (700 µg of total protein for each lysate). After 2 h of incubation at 4 °C, protein G beads in a volume of 40 µl (50% slurry, Pharmacia) were added, and the mixtures were further incubated with rocking at 4 °C for 1 h. Washing and immunoblotting of the PLC-1 immunoprecipitates were carried out essentially the same as described above, except that 0.7 µg/ml anti-phosphotyrosine antibody 4G10 was used for immunodetection.


RESULTS

To test the role of tyrosine 322 in MT function, we mutated tyrosine 322 to phenylalanine (Tyr-322 Phe MT) and conducted a focus formation assay. When the focus formation assay was carried out in 10% serum, Tyr-322 Phe MT is only slightly less transforming than wild type MT (Fig. 1). However, when the assay was conducted using medium containing 3% calf serum, the difference in transformation between wild type MT and Tyr 322 Phe MT was pronounced.


Figure 1: Focus formation assay of wild type and Tyr-322 Phe MTs. BALB/3T3 cells were infected with an equal number (1.0 10 colony-forming units) of control retrovirus (Vector), wild type MT (WildType), and Tyr-322 Phe MT (Tyr322Phe) as described under ``Experimental Procedures.'' The lower three cultures were incubated in DMEM plus 10% calf serum (CS) while the upper three cultures were incubated in the same medium plus 3% calf serum.



We next explored the function of tyrosine 322 in transformation. When phosphorylated, a tyrosine often forms the core of the binding sites for SH2 domains of intracellular signaling proteins. Previous work indicated that tyrosine 322 in MT might be involved in the activation of PI3-kinase (21) . Therefore, we analyzed the interaction between MT and p85 (the SH2 domain-containing subunit of PI3-kinase) in wild type and Tyr-322 Phe MTs using immunoprecipitation in conjunction with immunoblotting. BALB/3T3 cells and BALB/3T3 cells expressing wild type, Tyr-315 Phe, and Tyr-322 Phe MTs were used for the study. These MT cell lines expressed comparable levels of MT as judged by anti-MT immunoblotting. Anti-MT immunoprecipitates were immunoblotted using an anti-p85 antibody to determine the level of p85 associated with each MT allele. As expected, Tyr-315 Phe MT is defective in its p85 signal (Fig. 2). However, the p85 signal of Tyr-322 Phe MT is comparable with that of wild type MT. Therefore, tyrosine 322 appears unimportant for p85 binding by MT.


Figure 2: Effect of Tyr-322 Phe mutation in MT on its association with p85. Immunoprecipitation with anti-MT antibody and subsequent immunoblotting of the immunoprecipitates with anti-p85 antibody were performed as described under ``Experimental Procedures.'' BALB/3T3 cells expressing wild type, Tyr-315 Phe and Tyr-322 Phe MTs were constructed and were used in the experiments. vector represents the pLJ vector for the expression of MT cDNAs (16). wt, 315F, and 322F represent wild type, Tyr-315 Phe, and Tyr-322 Phe mutant MTs, respectively. The arrow indicates the position of p85. The numbers at right represent the positions of the molecular mass standards (kDa).



While the core of an SH2 binding site is formed by the phosphotyrosine, each SH2 domain achieves binding specificity by interacting with the amino acids just carboxyl to the phosphotyrosine. Amino acids 323, 324, and 325 in MT are Leu, Asp, and Ile. Of the SH2 domains studied to date, only the amino-terminal SH2 domain of PLC-1 has a preference for Tyr(P)-Leu-Asp-Ile (22) . Hence, we investigated the possibility of interaction between PLC-1 and MT. Immunoblotting using anti-PLC-1 antibody was carried out on anti-MT immunoprecipitates. A distinct band at 145 kDa, the mobility of which corresponds to PLC-1, is easily observed in the MT immunoprecipitate from cells expressing wild type MT (Fig. 3). The PLC-1 signal in the Tyr-322 Phe mutant MT (322F) immunoprecipitate is reduced to the control (vector) level. The Tyr-315 Phe mutant MT (315F), in which the p85 binding site is abolished, however, gives a comparable PLC-1 signal to that of wild type MT. Thus, the data of Fig. 3provide a strong indication that MT associates with PLC-1 and that tyrosine 322 in MT is important for the association.


Figure 3: Association of MT and PLC-1. Anti-PLC-1 immunoblotting was performed on anti-MT immunoprecipitates. lysate represents the cell lysate (2 µg of total protein from BALB/3T3) that was loaded in the lane to visualize the exact position of PLC-1. The arrow indicates the position of PLC-1. The rest of the lettering is explained in the legend to Fig. 2.



Tyrosine phosphorylation of PLC-1 is required for its activation in vivo and can increase its catalytic activity in vitro(23, 24) . MT complexes contain activated members of the src family tyrosine kinases (2, 3, 4, 5, 6) , and thus the level of tyrosine phosphorylation of PLC-1 in MT transformed cells might be expected to be elevated. As shown in Fig. 4 , the tyrosine phosphorylation level of PLC-1 is indeed increased in cells expressing MTs that bind PLC-1, i.e. wild type MT and Tyr-315 Phe MT. Cells expressing Tyr-322 Phe MT, which is defective in PLC-1 binding, showed a level of PLC-1 tyrosine phosphorylation comparable with the background level found in control cells (Fig. 4). The same membrane was striped and immunoblotted by anti-PLC-1 antibody in order to determine the position and the relative quantity of PLC-1 in these immunoprecipitates. The PLC-1 signals appear exactly at the same position of the signals in the anti-phosphotyrosine blot, and equal intensities of PLC-1 signals were observed in each lane.


Figure 4: Tyrosine phosphorylation of PLC-1. Anti-phosphotyrosine immunoblotting was carried out on anti-PLC-1 immunoprecipitates. Immunoprecipitation and immunoblotting were performed as described under ``Experimental Procedures.'' The lettering is explained in the legend to Fig. 2.



Since PLC-1 and p85 both bind MT, we analyzed the interaction of the two enzymes in cells expressing MT. Immunoblotting with anti-PLC-1 antibody was conducted on anti-p85 immunoprecipitates from cells expressing the various alleles of MT. High levels of PLC-1 were detected only in the anti-p85 immunoprecipitates from cells expressing wild type MT and not from control cells or cells containing MTs, which were mutant in either the PLC-1 or p85 binding site (Fig. 5). The reverse of the above experiment was also carried out. Anti-p85 immunoblotting was performed on anti-PLC-1 immunoprecipitates. Similar results were obtained (Fig. 6). Since previous studies have shown that each MTsrc complex contains only one molecule of MT (25) , the data of Fig. 5 and Fig. 6raise the possibility that PLC-1 and p85 might bind to the same MT molecule. However, the data in Fig. 2 and Fig. 3suggest that no positive or negative cooperativity occurs between PLC-1 and p85 in their binding to MT. This follows from the fact that when MT loses the binding site for one protein, its binding to the other one is similar to wild type MT (Fig. 2, lanes3 and 4; Fig. 3, lanes2 and 3).


Figure 5: Anti-PLC-1 immunoblotting of anti-p85 immunoprecipitates. Immunoprecipitation and immunoblotting were conducted as described under ``Experimental Procedures.'' The lettering is explained in the legend to Fig. 2.




Figure 6: Anti-p85 immunoblotting of anti-PLC-1 immunoprecipitates. Immunoprecipitation and immunoblotting were performed as described under ``Experimental Procedures.'' The lettering is described in the legend to Fig. 2. Cell lysate (2 µg of total protein from BALB/3T3) was loaded in the last lane (labeled as lysate) to visualize the exact position of p85.




DISCUSSION

Previous work by other laboratories can be reexamined in light of the data reported here. The reaction catalyzed by PLC-1 produces two second messenger molecules: inositol 1,4,5-trisphosphate and diacylglycerol. It has been observed that the expression of wild type MT caused an increase in the inositol 1,4,5-trisphosphate level (26) . Diacylglycerol activates protein kinase C (27) . It has been shown that protein kinase C is activated in cells by the expression of MT (28, 29) . Both of these published results are consistent with our conclusion that PLC-1 may be involved in MT-induced transformation.

In experimental systems for the study of growth factor receptors, it has been difficult to measure the functional consequences of deleting the binding site for PLC-1 on the receptors. Tyr Phe mutations in the PLC-1 binding sites of the platelet-derived growth factor, fibroblast growth factor, and nerve growth factor receptors had no detectable biological effects (30, 31, 32) . However, restoring the PLC-1 binding site also restores a mitogenic response in a platelet-derived growth factor receptor with multiple Tyr Phe mutations (33) . We observed a pronounced difference in transformation ability of wild type MT and Tyr-322 Phe MT in 3% calf serum (Fig. 1). In 10% serum, the effects of the Tyr-322 Phe mutation are less obvious. Presumably, when certain factors in serum are less available, the PLC-1 pathway becomes limiting in MT-induced transformation. Wild type Polyomavirus raises, as its name would suggest, a wide variety of tumors in mice. Thus installation of a PLC-1 binding site mutation into the polyoma genome will create an excellenttest system to evaluate the role of PLC-1 activation in a broad spectrum of tumor types in vivo.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA30002 (to T. M. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Fellow of the Leukemia Society of America. To whom correspondence should be addressed: Dana-Farber Cancer Inst., M857, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3246; Fax: 617-632-4770; Internet: wen_su@dfci.harvard.edu.

The abbreviations used are: MT, Polyomavirus middle tumor antigen; PLC-1, phospholipase C-1; DMEM, Dulbecco's modified Eagle's medium; SH2, Src homology 2; PI3-kinase, phosphatidylinositol 3-kinase.


ACKNOWLEDGEMENTS

We thank Dr. Steve Dilworth for the generous gift of the monoclonal antibody PAb 750, and Dr. David Pallas and Dr. Charles Stiles for critical review of the manuscript.


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