The Amino Terminus of Polyomavirus Middle T ... - Journal of Virology

5 downloads 113 Views 2MB Size Report
1879-1887 ... L8S 4K1, Canada. phenylalanine for tyrosine at residue 527 ... culture dishes in Dulbecco modified Eagle medium supple-. 1879. Vol. 64, No. 5 ...
JOURNAL OF VIROLOGY, May 1990, p. 1879-1887

Vol. 64, No. 5

0022-538X/90/051879-09$02.00/0 Copyright (C 1990, American Society for Microbiology

The Amino Terminus of Polyomavirus Middle T Antigen Is Required for Transformation DONALD N. COOKt

JOHN A. HASSELLt* Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada AND

Received 22 August 1989/Accepted 3 January 1990

In polyomavirus-transformed cells, pp6Oc-src is activated by association with polyomavirus middle T antigen. These complexes have a higher tyrosine kinase activity compared with that of unassociated pp6O-src. Genetic analyses have revealed that the carboxy-terminal 15 amino acids of pp60csrc and the amino-terminal half of middle T antigen are required for this association and consequent activation of the tyrosine kinase. To define in greater detail the borders of the domain in middle T antigen required for activation of pp6Ocsrc, we constructed a set of unidirectional amino-terminal deletion mutants of middle T antigen. Analysis of these mutants revealed that the first six amino acids of middle T antigen are required for it to activate the kinase activity of pp60csrc and to transform Rat-l fibroblasts. Analysis of a series of insertion and substitution mutants confirmed these observations and further revealed that mutations affecting the first four amino acids of middle T antigen reduced or abolished its capacity to activate the kinase activity of pp60ocsrc and to transform Rat-l cells in culture. Our results suggest that the first four amino acids of middle T antigen constitute part of a domain required for activation of the pp6o0-src tyrosyl kinase activity and for consequent cellular transformation.

Polyomavirus (Py)-mediated transformation results principally from the action of one of the Py early gene products, middle T antigen (39). Middle T antigen is both necessary and sufficient to transform established (34, 51) and, in some cases, embryonic cells (24, 46) in culture and to induce tumors in hamsters (1), chickens (24), and transgenic mice (2, 52). Middle T antigen is a phosphoprotein (17, 40, 41, 44), is composed of 421 amino acids, and has a measured molecular weight of either 56,000 or 58,000, depending on its state of phosphorylation (41). Phosphorylation occurs primarily on serine and threonine residues in vivo and to a much lesser extent on tyrosine (41). By contrast, when immunoprecipitates of Py-infected or -transformed cells are incubated in vitro with [-y-32P]ATP, middle T antigen becomes phosphorylated principally on tyrosine residues (17, 40, 41). The capacity of middle T antigen to act as a substrate for phosphorylation in vitro is thought to be relevant to transformation, because there are no known transformationcompetent mutants of middle T antigen that lack this capacity. The protein kinase activity in immunoprecipitates is not intrinsic to middle T antigen (42, 43) but is due, at least in part, to its association with the cellular proto-oncogene product, pp6Oc-src (5, 14, 15). The pp6Oc-src molecules associated with middle T antigen differ from their noncomplexed counterparts by a phosphate-free tyrosine at residue 527 (9) and a higher tyrosine kinase specific activity as measured in vitro (5, 12). It has been proposed that middle T antigen may effect transformation by preventing phoshorylation of Tyr527 (12), because mutants of pp60csrc with a substitution of

phenylalanine for tyrosine at residue 527 readily transform NIH 3T3 cells, whereas wild type (wt) c-src does not or does so inefficiently (8, 23, 37). Transformation by middle T antigen may include additional effects, however, because the Tyr-527 substitution mutants of c-src do not transform as well as middle T antigen does (8), and because there are mutants of middle T antigen that activate the tyrosine kinase activity of pp6O-srs but fail to transform established rat cells (31, 36, 50). Genetic analyses have revealed that the amino-terminal half of middle T antigen is required for its capacity to transform cells (3, 6, 10, 31, 49) and, by implication, for it to associate with and activate the kinase activity of pp60csrc. Large deletions (33) and numerous point mutations (30, 38) in the carboxy-terminal half of the protein, except residues involved in anchoring the protein to the plasma membrane (7, 30, 48), do not significantly alter the transforming activity of middle T antigen or its capacity to activate the tyrosine kinase activity of pp6o0-src. By contrast, a truncated middle T antigen whose translation is initiated at the first internal methionine (Met-30) does not transform cells and does not enhance the tyrosyl kinase specific activity of pp6Oc src (49), implying that a region within the first 30 amino acids of middle T antigen is important for transformation. To define this region more precisely, we introduced deletion, insertion, and substitution mutations within sequences encoding the first 12 amino acids of middle T antigen and measured the capacity of these mutants to activate the tyrosine kinase activity of pp60-src and to transform Rat-1 cells. The results of these analyses revealed that the first four amino acids of middle T antigen are required for activation of the tyrosine kinase activity of pp60c-src and for transformation. We suggest that these amino acids form a part of the domain in middle T antigen required for the activation of pp60csrc.

Corresponding author. t Present address: Department of Pathology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7525. t Present address: Institute for Molecular Biology and Biotechnology, McMaster University, 1280 Main St. W., Hamilton, Ontario L8S 4K1, Canada. *

MATERIALS AND METHODS Cells. All cell lines were propogated on plastic tissue culture dishes in Dulbecco modified Eagle medium supple1879

1880

COOK AND HASSELL

J. VIROL.

EcoRI (1560) cc I (368) ATO (173) Xho I (154)

Xho I

digest

Bal 31 mutagenesis Hind III

Nco I

Kienow

I

AAGCTTACCATGG T1'COAATGETACC

Ugatlon of

ollgonuclotide Hind iII digest Ligation & scren

for ollgonucieotide

Eco RI

ATO

Hind

Hind

Hind

III

2V40 *erly

III

HI digest

Hind III promoter Ugation and screen

Eco

RI

ATG Hind III

Hind

III

FIG. 1. Procedure to generate amino-terminal deletion mutants of Py middle T antigen. pPymT(X) carries a cDNA encoding middle T antigen. This DNA was linearized with XhoI, and various lengths of DNA encoding the amino terminus of middle T antigen were removed from the ends of the linear DNA with Bal 31 exonuclease. Sequences required for transcription and translation were restored to these DNAs as described in the text.

mented with gentamicin (50 ,ug/ml), fungizone (2.5 ,ug/ml), and 10% fetal bovine serum and were maintained at 37°C in a humidified CO2 incubator. Construction of recombinant plasmids. All cloning procedures were performed as described by Maniatis et al. (29). Recombinant plasmids were isolated from Escherichia coli DH1 or DH5. Restriction enzymes and DNA-modifying enzymes were used according to the specifications of the manufacturers. A series of deletion mutants was generated using pPymT (X) as a substrate (Fig. 1). The plasmid has an insert of Py middle T cDNA bounded by a unique Xhol site at nucleotide (nt) 154 (a former HphI site), and an EcoRI site at nt 1560 (18). The plasmid was linearized by cleavage with XhoI and subjected to deletion mutagenesis by incubation for various periods with Bal 31 exonuclease. The recessed 3' ends were made blunt by incubation of the DNA with the Klenow fragment of E. coli DNA polymerase I (Klenow) in the

presence of all four deoxynucleotide triphosphates. The double-stranded oligodeoxynucleotide shown in Fig. 1 was then ligated to the plasmid DNA. The oligodeoxynucleotide contains an NcoI site embedded within a eucaryotic translation initiation consensus sequence (25-28) and has a HindIII recognition site. After ligation, the DNA was cleaved with Hindlll to ensure that only one oligodeoxynucleotide was retained on the plasmid DNA. The plasmid DNA was then purified from an agarose gel, circularized by ligation, and used to transform E. coli DH1. Cleavage with HindIII would also remove the NcoI site from plasmids in which the oligonucleotide had been inserted in the wrong orientation with respect to the middle T antigen coding sequences. Ampicillin-resistant colonies were therefore screened for plasmids that had acquired a new NcoI site as well as the HindlIl site, thereby confirming that the oligonucleotide was situated in the correct orientation, juxtaposing the initiator ATG to the deletion endpoint. The approximate size of the deletions was determined by polyacrylamide gel electrophoresis after cleavage of the DNA with HindIII and AccI (nt 368). The sequence of plasmids bearing small deletions in middle T antigen coding sequences was determined by the method of Maxam and Gilbert (32). Those plasmids that were predicted to encode an in-frame, truncated middle T antigen, whose terminal methionine would be encoded by the ATG within the oligonucleotide, were processed further. These DNAs were cleaved with Hindlll, dephosphorylated by incubation with calf intestinal alkaline phosphatase, ligated to the simian virus 40 (SV40) HindIII C fragment containing the SV40 early and late promoters, and used to transform E. coli. The orientation of the SV40 sequences in the recombinant plasmids was determined by cleavage of the DNA with BglI and analysis of the fragments by electrophoresis through agarose gels. Plasmids carrying the SV40 early promoter next to the middle T antigen-coding sequences were analyzed further. Three mutants (d1l92, d1201, and d1207 [Table 1]) bearing short deletions in middle T antigen-coding sequences were isolated in this way. pmT165 was constructed in a similar manner, except that the oligonucleotide was inserted in the opposite orientation with respect to the middle T antigen-coding sequences. Consequently, pmT165 possesses a Hindlll site but not an NcoI site. Two deletion-substitution mutants were generated by enzymatically correcting the reading frame of two out-of-frame deletion mutants. inl79 was constructed by altering the reading frame of an out-of-frame deletion mutant (dI179) at its novel NcoI site. Because the Py early region contains another NcoI site at nt 1211, d1179 DNA was cleaved partially with NcoI to linearize the plasmid. The cohesive ends of the linear DNA were rendered blunt by the Klenow fragment, and the oligonucleotide was ligated to them. The DNA was then cleaved with HindIlI and EcoRI, and the middle T antigen-coding sequences were isolated and inserted between the EcoRI and HindlIl sites of pML-2. The predicted sequence of this mutant was confirmed by DNA sequencing (Table 1). The SV40 early promoter-enhancer region was inserted in front of the in179-coding sequences as

described above. Another mutant of this type, in197, was constructed by fusing sequences encoding the first five amino acids of wt middle T antigen to another deletion mutant (d1197) lacking the first eight amino acids of middle T antigen as follows. DNA sequences encoding amino acid residues 8 through 421 were isolated by partial cleavage of d1197 DNA with NcoI, treatment of the recessed 3' ends with the Klenow fragment,

FUNCTIONAL DOMAINS OF POLYOMAVIRUS MIDDLE T ANTIGEN

VOL. 64, 1990

1881

TABLE 1. Sequence and transforming capacity of middle T antigens Plasmid

Nucleotide and amino acid

Transformation frequency (% of wt)

sequence"

pmT165(H) d1166

AAGCTT CCACCATC ATG GAT AGA... AAGCTTACCATGGCACCATC ATG GAT AGA...

100 10

wt

ATG GAT AGA GTT CTG AGC AGA GCT GAC AAA GAA AGG CTG CTA... Met Asp Arg Val Leu Ser Arg Ala Asp Lys Glu Arg Leu Leu...

100

d1207

ATG

GGG CTG CTA...