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Journal of Virology, August 2005, p. 9982-9990, Vol. 79, No. 15
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.15.9982-9990.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Genetic Analysis of the Polyomavirus DnaJ Domain

Kerry A. Whalen, Rowena de Jesus, Jennifer A. Kean, and Brian S. Schaffhausen^*

Department of Biochemistry, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111

Received 10 January 2005/ Accepted 5 April 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Polyomavirus T antigens share a common N-terminal sequence that comprises a DnaJ domain. DnaJ domains activate DnaK molecular chaperones. The functions of J domains have primarily been tested by mutation of their conserved HPD residues. Here, we report detailed mutagenesis of the polyomavirus J domain in both large T (63 mutants) and middle T (51 mutants) backgrounds. As expected, some J mutants were defective in binding DnaK (Hsc70); other mutants retained the ability to bind Hsc70 but were defective in stimulating its ATPase activity. Moreover, the J domain behaves differently in large T and middle T. A given mutation was twice as likely to render large T unstable as it was to affect middle T stability. This apparently arose from middle T's ability to bind stabilizing proteins such as protein phosphatase 2A (PP2A), since introduction of a second mutation preventing PP2A binding rendered some middle T J-domain mutants unstable. In large T, the HPD residues are critical for Rb-dependent effects on the host cell. Residues Q32, A33, Y34, H49, M52, and N56 within helix 2 and helix 3 of the large T J domain were also found to be required for Rb-dependent transactivation. Cyclin A promoter assays showed that J domain function also contributes to large T transactivation that is independent of Rb. Single point mutations in middle T were generally without effect. However, residue Q37 is critical for middle T's ability to form active signaling complexes. The Q37A middle T mutant was defective in association with pp60^c-src and in transformation.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Polyomavirus T antigens function both in replication of the virus and in transformation of the host cell. Large T is central to virus production as the initiator of viral DNA replication (20). Middle T and small T also play important roles in different aspects of polyomavirus infection (21, 23, 55). Defects in viral DNA replication and transcription, as well as defects in viral assembly, have been observed in different mutants of middle T and small T (1, 7, 8, 22, 38). Each of the viral early proteins also contributes to regulation of host cell function. Large T is able to immortalize primary cells (44), to block differentiation (37), and to provoke apoptosis (18, 48). These activities are mediated via association with the retinoblastoma susceptibility (Rb) family of tumor suppressors. Middle T, the major transforming protein, works through activation of cellular signaling pathways that are regulated by src-family tyrosine phosphorylation (15). Small T is able to promote cell cycle progression via association with protein phosphatase 2A (PP2A) (39).

All three T antigens are produced by differential splicing of common primary transcripts (56). As a result, they have the identical N-terminal sequence of 79 amino acids that encompasses a DnaJ domain. DnaJ domains, consisting of approximately 70 amino acids, have a helical structure in which a conserved HPD motif is found between helix 2 and helix 3 (2, 13, 32, 43, 54). DnaJ domains, found in a broad range of proteins, function to stimulate the activity of DnaKs (6, 14). The domains of polyomavirus and simian virus 40 (SV40), like other DnaJs, have been shown to activate the ATPase activity of DnaKs (45, 50, 52). Substitution experiments have shown that SV40 or BK domains can function biologically as DnaJ domains in Escherichia coli (31).

The DnaJ/K cochaperones have diverse cellular activities, including protein folding (19), protein degradation (57), vesicle sorting/uncoating (16, 36), and DNA replication (5, 33). The importance of the J domain to both polyomavirus and SV40 large T function is clear (47, 53). The ability of SV40 large T to drive DNA replication in vivo is highly dependent on an intact DnaJ domain (5). The ability of SV40 large T to transform is dependent on DnaJ function (50, 58). The J domain of SV40 large T has also been implicated in sensitizing cells to apoptosis after genotoxic damage (10). A recent report indicates that the J domain is important for the ability of SV40 large T to release VP1 from complexes with Hsc70 (9). Polyomavirus large T promotes cell cycle progression in a DnaJ-dependent manner (47). Much, but not all (48), of large T's ability to act on the Rb family is dependent on an intact J domain (47, 51, 52, 58). Mutations of the HPD loop that abolish interaction of large T with Hsc70 (5, 48) affect the ability of SV40 large T to alter the turnover of p130 (51). Work done with SV40 by the Pipas group has demonstrated an intact HPD loop is critical for dissociation of Rb/E2F complexes (52), which would account for large T promoter activation at E2F sites. Less is known about the role of the J domain in polyomavirus middle T and small T. Although some mutations in the middle T J domain have been shown to affect transforming ability (11), mutation of the HPD loop was found not to alter the ability of middle T to transform cells (4, 26). This suggests that activation of DnaK, the normal role for DnaJ, is not required for transformation.

There has been little systematic genetic analysis of J domains, even for E. coli DnaJ (17, 24, 25). To map the DnaJ residues necessary for polyomavirus large T function, we carried out extensive mutagenesis of the J domain. This identified residues important for the stability of large T and for the ability of large T to act on the Rb family. These experiments also suggested that the domain is important for large T functions beyond those related to Rb. Further, we compared the effects of mutations in the J domain of large T to the same mutations in middle T. Our results indicated that the two proteins use the J domain differently.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell lines and transfections. NIH 3T3 cells were originally obtained from the American Type Culture Collection. The cells were grown in Dulbecco modified Eagle medium (DMEM; Gibco) and supplemented with 10% calf serum (HyClone). Transfections were performed by the calcium phosphate precipitation method (47). NIH 3T3 cell lines expressing different middle T antigens were obtained by cotransfection of NIH 3T3 cells with a vector for puromycin resistance and the relevant pCMV MT construct at a ratio of 1:10. At 2 days posttransfection the cells were placed in selection medium of DMEM containing 10% calf serum and puromycin at 5 µg/ml to obtain pools of puromycin-resistant colonies.

C33A cells were obtained from Vimla Band and were grown in DMEM supplemented with 10% fetal calf serum (HyClone). C33A cell transfections were performed using HEPES-buffered saline (28). For starvation experiments, cells were washed twice with phosphate-buffered saline (PBS) 18 h after transfection and placed in DMEM containing 0.2% fetal calf serum.

Plasmids and mutagenesis. pCMV large T, pCMV Rb^–LT, pCMV P43S large T, HA-Rb, HA-p130 (47), pCMV middle T, pCMV NG59 middle T (12), and INS107AL (5) have all been described previously. Point mutants were constructed on wild-type pCMV large T or pCMV middle T. In general, oligonucleotides of 18 to 20 bases with one or two mismatches to the J domain sequence were used. Standard PCR methods based on PFU Turbo polymerase (Stratagene) were used to generate the mutants. All mutations were confirmed using the dideoxy sequencing method (46). A summary of the mutants constructed is shown in Fig. 1.

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FIG. 1. Mutations in the polyomavirus DnaJ domain. The amino acid sequence encoded by the first exon (residues 1 to 79) of the polyomavirus T antigens is shown for wild type. Mutations were created in expression vectors for either middle T or large T. The amino acid changes are shown above the wild-type sequence for the large T mutants and below the wild-type sequence for middle T mutants. The dashes represent residues that were not mutated for these studies. Unstable mutants are indicated by an asterisk. Mutants defective in function are indicated by a dagger ().

The E2F-luciferase construct contains six E2F sites attached to a luciferase construct and was obtained from Amy Yee. The myc-tagged Hsc70 was obtained from Tom Roberts. The cyclin A –89-to-+11 promoter construct has been described previously (48).

Antibodies. The PN116 monoclonal and rabbit anti-T antibody used in Western blots have been described previously (30). Middle T was immunoprecipitated with rabbit polyclonal antibody 45-1 (39). HA11 was obtained from Covance. Myc antibody 9E10 was obtained from James DeCaprio. Polyclonal PP2A-A subunit antibody was obtained from Gernot Walter and Ralf Ruediger. Anti-src antibody (GD11) was obtained from Larry Feig.

Luciferase assays. NIH 3T3 cells plated on 60-mm dishes were transfected at a confluence of 20% with 2 µg of E2F-luciferase or with 1 µg cyclin A-luciferase and 0.5 µg of the relevant LT expression vectors and harvested approximately 40 h posttransfection. For the cyclin A-luciferase assays, cells were placed under serum-starved conditions at 24 h posttransfection. Cells were suspended in buffer (25 mM Tris [pH 7.5], 1 mM EDTA) and subjected to freezing-thawing three times. The lysates were cleared by Eppendorf centrifugation and assayed for luciferase activity.

Extraction and immunoprecipitations. The cells were washed in PBS and extracted in T extraction buffer (TEB; 137 mM NaCl, 10 mM Tris-Cl [pH 8.0 for HA-Rb, pH 7.0 for myc-Hsc70, and pH 9.0 for middle T], 1 mM MgCl₂, 1 mM CaCl₂ 10% [vol/vol] glycerol, and 1% [vol/vol] Nonidet P-40 supplemented with protease inhibitor cocktail, including leupeptin at 1 µg/ml, pepstatin 1 at µg/ml, phenylmethylsulfonyl fluoride at 100 µg/ml, and aprotinin at 2 µg/ml) for 20 minutes at 4°C. The cleared extracts were incubated with the appropriate antibody and protein G- or protein A-Sepharose beads (Pharmacia). The immunoprecipitates were washed with PBS. The immunoprecipitates were boiled in dissociation buffer (62.5 mM Tris-Cl [pH 6.8], 5% [wt/vol] sodium dodecyl sulfate [SDS], 25% [vol/vol] glycerol, 0.0075% [wt/vol] bromophenol blue, 5% [vol/vol] ß-mercaptoethanol).

Electrophoresis. Samples were analyzed on discontinuous buffer SDS gels (34) of 7.5% acrylamide. Radiolabeled gels were exposed and quantified by PhosphorImager (Molecular Dynamics) using ImageQuant software. All of the figures were prepared in Adobe Photoshop.

Large T NT purification. The N-terminal domains (NT; residues 1 to 259) of large T expressed as glutathione S-transferase fusions in pGEX3X were produced in E. coli BL21. Expression was induced with 50 µM IPTG (isopropyl-ß-D-thiogalactopyranoside) overnight at 30°C. Cell extracts were incubated with glutathione-beads (Sigma) to harvest the fusion proteins. Washed beads were treated overnight at 4°C with factor Xa (Haematologic Technologies, Inc.) in buffer (20 mM Tris-Cl [pH 8.0], 1 mM calcium chloride, 100 mM NaCl) to release NT. The soluble NT was concentrated by Amicon ultrafiltration.

ATPase assays. The assays were performed in a 20-µl total volume containing 75 mM NaCl, 1.5 mM CaCl₂, 40 mM HEPES (pH 7.2), and 2 µCi of [-³²P]ATP with 0.4 µg of mouse Hsc70 (Stressgen). In some cases the reactions were supplemented with 0.8 µg of wild-type or mutant purified NT protein. The reaction mixtures were assembled on ice and then incubated at 37°C. The reactions were stopped by plating 2-µl aliquots in triplicate onto polyethyleneimine thin-layer chromatography plates. ATP and ADP were separated by chromatography in 1 M LiCl-1 M formic acid solution for 3 h. The plates were dried and analyzed by using a Molecular Dynamics PhosphorImager.

Middle T kinase assays. NIH 3T3 cells plated onto 100-mm dishes were transfected at a confluence of 20% with 5 µg of different middle T expression vectors and harvested approximately 40 h posttransfection. After extraction in TEB in the presence of protease inhibitors, the cleared extracts were incubated with rabbit polyclonal antibody to middle T (45-1) and protein A-Sepharose beads for 1 h. Washed immunoprecipitates were incubated in 200 µl of kinase buffer (20 mM Tris [pH 7.5], 5 mM MnCl₂) containing 5 to 10 µCi of [-³²P]ATP (2,000 Ci/mmol; NEN) for 15 min at room temperature. The reactions were washed with PBS, boiled in dissociation buffer, and subjected to SDS-polyacrylamide gel electrophoresis (PAGE). After electrophoresis, the gel was alkaline treated (0.5 M potassium hydroxide) for 1 h at 55°C to remove background serine/threonine phosphorylation. The gel was then fixed (10% acetic acid, 30% methanol), dried, and analyzed with a PhosphorImager.

Transformation assays. NIH 3T3 cells were grown in 10% calf serum and transfected with 7 µg of carrier DNA and 2 µg of the middle T cDNAs by using the calcium phosphate method. When the confluency reached 100%, the cells were maintained in 5% calf serum-DMEM and allowed to grow for 10 to 14 days. The plates were then washed twice with PBS and fixed for 30 min with 3.7% formaldehyde. The fixed plates were stained with 0.2% crystal violet in 3.7% formaldehyde. Foci were then counted.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mutant analysis of the polyomavirus J domain demonstrates differences between the middle T and large T proteins. Site-directed mutagenesis was used to screen the J domains of middle and large T antigens. Point mutants were made in vectors expressing either middle T or large T using conservative substitutions such as lysine to arginine or leucine to valine. The mutants are listed in Fig. 1. In all, 63 mutants of large T and 51 mutants of middle T were examined.

The stabilities of mutant large T antigens were compared to wild type after transient expression in NIH 3T3 cells. Cell extracts prepared approximately 40 h after transfection were subjected to SDS-PAGE. Immunoblotting was then used to assess the level of large T protein. Figure 2A shows an example of the results. A majority of the mutants, including P43S, Q36A, and F62Y, retained wild-type levels of expression, whereas W59F large T showed a very low level of expression. In all, nine of the large T mutants showed a substantial decrease in protein levels (Fig. 1). The simplest interpretation of this observation is that the mutations cause unfolding of the domain leading to degradation of the protein. Figure 3A shows the positions in the J structure of mutations that caused loss of large T stability. Four DnaJ mutations also rendered middle T unstable (Fig. 1 and 3C). Two examples, R12K and L13V, are shown in Fig. 2B. Seven mutations (K10R, L17V, M30V, G25A, F27Y, L55V, and W59F) that rendered large T unstable did not appreciably affect the stability of middle T. On the other hand, mutation R12K (Fig. 2B) was unstable in the middle T background but not in large T.

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FIG. 2. Effects of DnaJ mutants on large T and middle T stability. (A) Examples of expression of large T DnaJ mutants. NIH 3T3 cells in 100-mm dishes at 20% confluence were transfected with 3 µg of empty vector (lane 1) or vectors expressing wild-type (lane 2), P43S (lane 3), Q36A (lane 4), W59F (lane 5), F62Y (lane 6), and T64S (lane 7) large T antigens. At ca. 40 h posttransfection, cells were extracted. After SDS-PAGE of the cell extracts, large T was blotted with anti-T antibody. (B) Comparison of large T and middle T DnaJ mutant stability. Extracts from K10R (lane 1), E11D (lane 2), R12K (lane 3), or L13V (lane 4) mutants large T or middle T, as well as control (lane 5) and wild-type (lane 6), transfected cells were blotted with anti-T antibody as in panel A. (C) Cells transfected with wild-type middle T (lane l), NG59 middle T (lane 2), G25A (lane 3), G25A/NG59 double mutant (lane 4), L55V (lane 5), and L55V/NG59 double mutant (lane 6) were extracted at ca. 40 h posttransfection. After SDS-PAGE, blotting was done with anti-T antibody.

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FIG. 3. Locations in the DnaJ structure of large T and middle T mutations that affect stability or function. (A) Mutated residues that result in protein instability in the large T background are highlighted in yellow: K10R, L13V, L14V, L17V, P20A, G25A, F27Y, M30V, L55V, and W59F. The structure was originally reported by Berjanskii et al. (2) and is displayed using Visual Molecular Dynamics. (B) The large T mutations that cause the protein to be inactive in E2F or cyclin A promoter assays are shown in blue: Q32A, A33G, Y34F, H49R, M52V, and N56T. The unstable mutants are shown in yellow. (C) Mutated residues that cause middle T instability are highlighted in yellow: R12K, L13V, L14V, and M30V. (D) The defective mutant at position Q37A is highlighted in blue. In all panels the HPD residues are red.

The results just described suggest that mutations of the J domains of large T and middle T have different effects on each protein even though the N-terminal sequences are identical. Possible explanations for the divergence lie within the differences in the C-terminal residues of the two proteins, as well as their association with different cellular proteins. A key difference between middle T and large T is middle T association with PP2A. Furthermore, it is also known that PP2A and Hsc70 compete for binding to middle T (4) suggesting contact may exist between PP2A and Hsc70. If PP2A were to contact the J domain, this interaction might provide additional stability. To test this hypothesis, L55V and G25A mutants of middle T were combined with the NG59 mutation that prevents PP2A binding (49). Figure 2C shows that the two double mutants are significantly less stable than a middle T mutant only in the J domain. This result is also true in the case of the M30V middle T mutant (data not shown). This result suggests that PP2A binding renders middle T more resistant to mutations in the J domain.

Effects of J mutations on large T Rb function. Large T activation of promoters containing E2F sites was shown to be dependent upon an intact Rb binding site, as well as an intact HPD loop in the J domain (52). Earlier experiments suggested a role for some residues outside of the HPD loop in large T for the activation of E2F promoters (47). Each of the mutants in large T was tested for the ability to activate the E2F promoter. Mutants Q32A, A33G, Y34F, H49R, M52V, and N56T were all defective in E2F promoter activity as assayed by cotransfection of the relevant large T constructs and an E2F-luciferase reporter construct (Fig. 4A, upper panel). As expected, the HPD mutant, P43S, was defective in E2F promoter activity (Fig. 4A). The non-HPD mutants defective in E2F activation are found in helix 2 and helix 3 (shown in blue in Fig. 3B). Previously, it has been shown that mutations in the HPD loop that abolish E2F activation have no effect on binding of Rb (47). Coimmunoprecipitation experiments showed that this was also true for the non-HPD mutants (not shown). In our E2F analysis, we also tested C-terminal mutants, including M71V and T76S. Structural analysis (2) suggested that these sequences, while part of the first exon of the T antigens, may not be part of the J domain but rather serve as a linker. These mutants were all wild type for E2F activation, so our data are consistent with this hypothesis.

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FIG. 4. Effects of non-HPD large T J mutants on the Rb-family members. (A) The upper panel depicts E2F-containing promoter activation. NIH 3T3 cells were cotransfected with the 6XE2F-luciferase construct, and the indicated cytomegalovirus (CMV) expression plasmids (wild type, P43S, Q32A, A33G, Y34F, H49R, M52V, and N56T). Error bars indicate the standard error of the mean. The P value of wild type compared to each of the mutants was <0.05 as determined by one-way analysis of variance. Cells were extracted ca. 40 h posttransfection and assayed for luciferase activity. The lower panel shows results for extracts from the promoter assays separated by SDS-PAGE and blotted for large T antigens with anti-T antibody: control (lane 1), wild-type large T (lane 2), P43S (lane 3), Q32A (lane 4), A33G (lane 5), Y34F (lane 6), H49R (lane 7), M52V (lane 8), and N56 T (lane 9). (B) DnaJ mutant large T-induced p130 mobility shift in C33A cells. C33A cells on 60-mm dishes at 80% confluence were transfected with 3 µg of HA-p130 and 1 µg of the CMV expression vectors wild-type large T (lane 1), wild type (1.5 µg) (lane 2), Q32A (lane 3), A33G (lane 4), Y34F (lane 5), H49R(lane 6), M52V(lane 7), N56T (lane 8), and P43S (lane 9). Cell extracts made approximately 40 h posttransfection were subjected to SDS-PAGE and blotted for HA-p130 using HA11. The arrows indicate differentially phosphorylated species of p130.

Polyomavirus large T antigens affect the phosphorylation status of the pRB family member, p130 (52, 58). Mutations within the HPD loop of the J domain of polyomavirus cause a supershift in the p130 molecule seen on SDS-PAGE. The supershift is caused by phosphorylation of the p130 molecule in the C-terminal portion of the molecule. It is has been demonstrated that SV40 large T causes a dephosphorylation of the p130 molecule (57, 58). This result suggests that the large T protein is important for proper cycling of the p130 molecule and that the J mutant is unable to cycle the molecule. Instead, the J mutant leaves the p130 "stuck" in a hyperphosphorylated state. To test whether the non-HPD mutants behaved similarly, experiments were carried out in C33A cells with the various non-HPD mutants and HA-tagged p130 protein. SDS-PAGE analysis of the samples and subsequent immunoblotting with anti-HA was done. Fig. 4B shows that the non-HPD mutants also caused a similar shift in the p130 protein. Therefore, all J mutants defective in the ability to activate E2F sites also affected p130 phosphorylation.

Neither the defect in E2F activation nor the effect on p130 could be explained by differences in protein expression. As shown in Fig. 4A these mutants were expressed at levels close to wild type, suggesting that their defect was caused by loss of J domain activity rather than protein instability.

Interactions of mutant large T's with Hsc70. The HPD loop of large T is known to be critical for Hsc70 binding (5, 47). This binding is necessary for large T transactivation, since reducing large T-Hsc70 interactions by overexpression of exogenous DnaJ domains squelches transactivation of E2F promoters (47). To examine the interaction of the non-HPD mutants with Hsc70, wild-type or mutant large T was cotransfected with myc-tagged Hsc70. Coimmunoprecipitation and Western blotting were used to determine binding of Hsc70 to large T. The experiment in Fig. 5A demonstrates that the P43S HPD and two of six non-HPD (A33G and M52V) mutants were unable to bind the cellular heat shock protein. Figure 5B shows that lack of binding did not result from poor protein expression. A33G retains a low level of activity in the E2F assay despite the lack of hsc70 binding. It is likely that A33G has a lowered affinity for hsc70 that permits modest in vivo activity but does not result in stable complexes that can be immunoprecipitated. However, Q32A, Y34F, H49R, and N56T mutants that were defective in activation of E2F sites were still able to bind the Hsc70 protein in a manner similar to that of the wild type.

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FIG. 5. Interactions of large T J domain mutants with Hsc70. (A) Binding to Hsc70: NIH 3T3 cells at 20% confluence in 100-mm dishes cotransfected with 2 µg of vector expressing wild type (lanes 1 and 2), P43S (lanes 3 and 4), CMV empty vector (lanes 5 and 6), Q32A (lanes 7 and 8), A33G (lanes 9 and 10), Y34F (lanes 11 and 12), H49R (lanes 13 and 14), and M52V (lanes 15 and 16), N56T (lanes 17 and 18), and 5 µg of myc-tagged Hsc70 were extracted ca. 40 h posttransfection. After extraction, immunoprecipitation was carried out with anti-myc 9E10 antibody (even lanes) or control monoclonal (odd lanes). After SDS-PAGE, Hsc70 was blotted with 9E10 (anti-myc Hsc70) or large T was blotted with PN116 (anti-T). (B) Western blots of cell extracts used for the immunoprecipitations in panel A. (C) H49R and Y34F are defective in the stimulation of Hsc70 ATPase activity. ATP hydrolysis measurements were carried out as described in Materials and Methods. ATP hydrolysis catalyzed by Hsc70, Hsc70 plus 0.8 µg of wild-type NT, Hsc70 plus 0.8 µg of Y34F NT, and Hsc70 of 0.8 µg of H49R NT was measured. The data represent the means from experiments done in triplicate, and error bars indicate the standard error.

Since binding Hsc70 might not be sufficient for stimulation of its ATPase function, activity measurements were also performed. To do this, wild-type, Y34F, and H49R N-terminal domains (residues 1 to 259) were constructed and purified from E. coli using an approach similar to that of Riley et al. (45). The N-terminal domain was used because it can be easily expressed in E. coli as a soluble protein. A time course of ATP hydrolysis was determined for Hsc70 using [-³²P]ATP in the presence of wild-type, Y34F, or H49R proteins. In these assays, wild-type NT was able to stimulate Hsc70 ATPase activity, whereas the J domain mutants were defective (Fig. 5C). It seems likely that the defect in E2F activation by Y34F, as well as H49R and probably other mutants that retain binding, is caused by an inability to stimulate DnaK function.

J domain effects on the cyclin A promoter. Whether the J domain participates in large T functions not related to Rb is an open question. Previous work has shown that large T can transactivate the cyclin A promoter (18, 48). In serum-starved cells this activation is completely dependent on Rb binding. However, when the E2F site in the promoter is mutated (–37/–33), large T still transactivates, but that activation is now completely independent of Rb binding (48). Our earlier work showed that HPD mutants in the J domain were also active, although at levels reduced from wild type (48). To determine whether the reduced activity indicated a J domain contribution or whether it simply represented a general loss of protein structure, the new set of J domain mutants were analyzed in cyclin A promoter assays. To avoid E2F effects, a mutant cyclin A (–37/–33) reporter lacking the E2F site was used. Every mutant that was wild type in the E2F assay was also fully competent in activation of the mutant cyclin A promoter (data not shown). The mutants impaired in E2F activation (Q32A, A33G, Y34F, H49R, M52V, and N56T) were similarly impaired in cyclin A activation (Fig. 6). The cyclin A data followed a pattern similar to the E2F data. For example, the mutant that had the most activity on the two promoters was Q32A, whereas Y34F had the least. Since large T activation of the mutant cyclin A promoter is independent of Rb binding, the results suggest a role for the residues within the J domain in large T activities independent of Rb.

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FIG. 6. Decreased activity of J domain mutants on the cyclin A promoter parallels that seen on E2F-containing promoters. The upper panel shows results for NIH 3T3 cells cotransfected with cyclin A –37/–33 luciferase construct along with the indicated CMV expression vectors (wild type, P43S, Q32A, A33G, Y34F, H49R, M52V, and N56T). The cells were placed in 0.2% calf serum at 24 h posttransfection. Cells were extracted approximately 40 h posttransfection and assayed for luciferase activity. Error bars represent the standard error of the mean. The P value of wild type compared to each of the mutants was <0.05 as determined by one-way analysis of variance. The lower panel shows results extracts from the promoter assays separated by SDS-PAGE and blotted for large T antigens with anti-T antibody: control (lane 1), wild-type large T (lane 2), P43S (lane 3), Q32A (lane 4), A33G (lane 5), Y34F (lane 6), H49R (lane 7), M52V (lane 8), and N56T (lane 9).

J domain mutations and middle T. As for large T, conservative mutations in the J domain were tested for their effect on middle T. The results of Fig. 1 to 3 showed that relatively few conservative mutations affect the stability of middle T. As a screen for middle T activity, immunoprecipitates made from transfected NIH 3T3 cells were tested in an in vitro tyrosine kinase reaction. Almost without exception, single-mutant middle Ts were phosphorylated in complexes with src-family members to an extent similar to that seen with the wild type (Fig. 7A). The middle T versions of non-HPD J-defective large T mutants (A33G, M52V, and N56T) were all active in the in vitro tyrosine kinase reaction and, as expected, these mutants all transformed Rat-1 cells normally (data not shown). All of these results argue against a role for the binding of heat shock proteins in fibroblast transformation assays.

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FIG. 7. Q37A middle T is defective in association with c-src. (A) NIH cells were transfected with the indicated CMV expression vectors control (lane 1), wild-type middle T (lane 2), K10R (lane 3), A33G (lane 4), G46A (lane 5), M52V (lane 6), N56T (lane 7), F62Y (lane 8), and G74A (lane 9). The cells were extracted ca. 40 h posttransfection, and extracts were immunoprecipitated with anti-T antibody bound to protein A-Sepharose beads. Immunoprecipitated middle T was labeled with []ATP in an in vitro kinase reaction. After SDS-PAGE, 32P-labeled middle T was analyzed by using a PhosphorImager (results shown in the upper panel). A fraction of the immunoprecipitates loaded in the same order as described above were separated by SDS-PAGE and blotted with anti-middle T antibody (results shown in the lower panel). (B) Pools of NIH 3T3 cells were made that express wild type, INS107A, or Q37A middle T antigens. Middle T immunoprecipitates from those or control cells were labeled in an in vitro kinase reaction and analyzed as described above (upper panel). A fraction of the immunoprecipitates were separated by SDS-PAGE and blotted with anti-middle T antibody (lower panel). (C) Middle T was immunoprecipitated from extracts of the cell pools. After separation on SDS-PAGE, Western blots were probed with GD11 anti-src antibody (upper panel). A fraction of the immunoprecipitates were separated by SDS-PAGE and blotted with anti-middle T antibody (lower panel). (D) Aliquots of the same immunoprecipitate shown in panel C analyzing src were used to test for association with PP2A by probing with antibody specific for PP2A subunit. Lanes (B to D): 1, wild type; 2, INS107A; 3, Q37A.

Q37A was an interesting exception. This mutation lies just outside of the HPD loop on the second helix of the J domain. In vitro kinase reactions of transfected Q37A consistently showed very little middle T phosphorylation. To confirm the transfection result, cell pools of NIH 3T3 cells expressing wild type, Q37A, and INS107AL, a mutant defective in both PP2A and src binding, were made by using coselection with puromycin. INS107AL (Fig. 7B, upper panel) was completely inactive in the tyrosine kinase reaction as expected. Q37A also showed little activity in the in vitro kinase reaction even though the protein was present at a level comparable to that seen with the wild type (Fig. 7B, lower panel). To examine the basis for the defect in Q37A-associated kinase activity, middle T immunoprecipitates were made from each of the cell pools. Blotting of these immunoprecipitates for c-src showed that wild type, but not INS107A, was able to associate with src (Fig. 7C). Q37A appeared to have relatively little binding compared to the wild type, although somewhat more than INS107AL. The binding of src to middle T is thought to depend on the association of PP2A (27, 40). To test association with PP2A, cells were harvested, extracted, and immunoprecipitated with anti-middle T antibody. The resulting immunoprecipitates were blotted for the PP2A A subunit as well as the middle T protein. As shown previously, INS107A fails to bind the A subunit of PP2A (Fig. 7D). Q37A bound the PP2A protein at levels close to that of the wild type.

The inability of Q37A to bind src leads to a defect in binding of important adaptor proteins including those mentioned above, phosphatidylinositol 3-kinase and Shc. Since both molecules are critical signaling molecules involved in middle T transformation, the activity of Q37A in transformation assays was determined. The middle T Q37A mutant, along with wild-type middle T and INS107AL(PP2A-) middle T cDNA, was transiently transfected in triplicate into NIH 3T3 cells. One plate was harvested and lysed 48 h posttransfection and assayed for middle T expression. The mutant proteins were expressed at levels close to that of the wild type (data not shown). The remaining plates were placed under reduced serum conditions and grown for 14 days. The plates were fixed and stained with crystal violet, and foci were counted. Figure 8 shows an example of each middle T transfected plate. The wild-type middle T protein was capable inducing focus formation (211 and 227 foci). Q37A, defective in src binding, was highly defective compared to the wild-type protein (34 and 28 foci). Ins107AL, defective in binding PP2A as well as src, was somewhat more defective compared to Q37A (14 and 17 foci). These results are consistent with those seen for the middle T kinase and src binding assays discussed above.

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FIG. 8. Q37A middle T is defective in transformation assays. NIH 3T3 cells were transfected with vector expressing wild-type middle T (A), INS107AL (B), or Q37A middle T (C) and grown for 14 days under reduced serum conditions. The cells were fixed and stained, and foci were counted.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our primary goal was a thorough genetic analysis of the J domain of polyomavirus T antigens. Previously, the study of the polyomavirus J domain has focused primarily on the importance of the conserved HPD loop (48). Some DnaJ mutants outside the HPD loop have been examined, especially for SV40 (24, 42), but little systematic analysis on DnaJ has been done (25). This work, combined with recent work on SV40 (9, 24), begins to close this gap in our knowledge.

Site-directed mutagenesis revealed several J domain residues necessary for large T and middle T stability. Comparison to the nuclear magnetic resonance (NMR) structure of the polyomavirus J domain (2) shows that, in general, the residues necessary for stability of the protein are located within the hydrophobic core of the domain. Presumably, the mutations cause unfolding that results in rapid degradation of the protein. Short pulse-labeling of the large T mutant L17V, for example, showed significant expression compared to wild type, even though the steady-state concentration measured by Western blotting was extremely low (not shown).

A number of mutations have different effects on the stability of middle T and large T. One possible explanation could be the cellular environment. Degradation of a nuclear large T mutant by cellular proteases might be easier than degradation of a membrane-associated middle T mutant. A second possibility is that J domain contacts with the rest of the protein determine how a given mutation affects stability. Such an interaction could occur within the middle T molecule itself, for example, between the J domain and C-terminal residues. Third, any protein-protein interaction providing additional stability to the domain could counteract the destabilizing effect of any given mutation. We favor the idea that there is increased stability because of an interaction between the J domain and the middle T binding partner PP2A. Unlike other associated proteins, PP2A is stoichiometrically associated with middle T. Further, it is known that PP2A and Hsc70 compete for binding to middle T (5), and our data has demonstrated that a middle T deletion mutant lacking the J domain does not bind PP2A (not shown). Here we have shown that a second mutation that blocked PP2A binding in L55V and G25A made those previously stable mutants unstable.

Genetic analysis of large T revealed residues besides those of the HPD loop that were important for large T effects on the Rb family. Large T proteins mutant at amino acids 32, 33, 34, 49, 52, and 56 were all defective in activating the E2F promoter. Large T HPD mutants cause hyperphosphorylation of the Rb family member p130 (47). The mutants outside the HPD loop also cause p130 hyperphosphorylation. The residues important for affecting Rb function are found in helix 2 (32-34) and helix 3 (49, 52, 56). As can be seen in Fig. 3B, these residues appear to be located on the surface of the domain and are supported by residues important for stability. The importance of sequences in helix 2 and helix 3 can also be seen in SV40 functional assays. Mutations made in residues Y34 (helix 2) and K53 (helix 3) result in an SV40 T antigen defective in J function (17). Interestingly, K53, although conserved in many polyomaviruses, is not present in murine polyomavirus and a Q53A mutant of polyomavirus retained significant activity (not shown). The importance of helix 2 residues 32 to 34 is also highlighted by NMR data on E. coli DnaJ. That study demonstrated that the outer surface of helix 2 is the binding site for DnaK (29). Mutagenesis results have shown that helix 3 residues can be important for E. coli DnaJ function, just as has been shown here for the polyomavirus J domain (25). NMR work suggested conformational changes in helix 3 upon DnaJ/DnaK interactions (35) that might be affected by mutation. The J mutants defective in Rb function can be separated into classes with respect to association with Hsc70. As measured by coimmunoprecipitation, mutants at residues 33 and 52 did not interact with Hsc70. However, other large T mutants at residues 32, 34, 49, and 56 retained near-wild-type levels of binding, even though they are defective in activating the ATPase activity of Hsc70.

Polyomavirus J domains participate in other activities of large T besides regulating the Rb family. In SV40, for example, the J domain is important for replication and can participate in VP1 assembly by promoting its dissociation from hsc70 (5, 9). The results here suggest that the polyomavirus large T J domain can participate in transcriptional activation that does not depend on Rb. Previous examination of the cyclin A promoter showed that, while the H42Q mutant retained activity, it was somewhat (4-fold) reduced in the ability to transactivate (48). Because of the uncertainties in protein stability and because the effect was independent of the E2F site, no conclusion was drawn from that result. Examination of all of the J mutants here showed that a defect in E2F activation was always matched by a decrease in activation of a mutant cyclin A promoter that lacked the E2F site. In particular, the activation of the mutant cyclin A promoter was dependent not only on the HPD residues but also upon residues 32 to 34 and 49, 52, and 56. Detailed work to be presented elsewhere will show that the actual target in the promoter is a Creb/ATF site (36a). Creb/ATF transcription factors function in complexes with other proteins such as CBP, Act, and Oct-1. It seems plausible that, as in the case for Rb/E2F complexes, the chaperone activity of large T targets such complexes.

Polyomavirus middle T does not rely on productive association between DnaK and the J domain for the ability to transform fibroblasts (4, 26). However, it is known that mutations near the N terminus can affect middle T binding to src and PP2A (11, 26), suggesting the possibility that other non-HPD residues could be important for middle T function. Of the 53 mutants examined here, Q37 was the only residue uncovered in the mutant screen that affected middle T transformation without simply destabilizing the protein. Interestingly, the Q37A mutation did not affect large T function in E2F or cyclin A assays (data not shown). This suggests that Q37 is not required for DnaJ activation of DnaK and further supports the idea that the J domains in large T and middle T have different roles. The effect of Q37A mutation on transformation suggests a role for the J domain separate from its action as an effector of DnaK. Since mutations in the N terminus of middle T have been reported to affect transformation (11), our observation that only mutation of Q37 affected middle T function was somewhat surprising. Since we used rather conservative substitutions, this suggests that the structure may tolerate modest changes in amino acids.

Q37A middle T was highly defective in association with c-src (Fig. 7B and C). It is not surprising that the mutant does not transform given the severe defect in tyrosine kinase activity. One possible explanation for the defect in binding is that src is positioned very close to, and is possibly even in direct contact with, the J domain. Q37A could be defective either because loss of direct contact reduces affinity for src or that a conformational change resulting from the mutation causes steric hindrance. This interpretation might seem unexpected since previous mapping of the src binding site has suggested that it is near residue 200 (3). However, there is some suggestion that the src protein may be close to the J domain. Some monoclonal antibodies directed against small T antigen will immunoprecipitate the large T and middle T proteins, so the epitopes must be within the conserved J domain sequence (41). Interestingly, two monoclonal antibodies did not immunoprecipitate middle T-associated kinase activity (41). The simplest interpretation is that the antibodies recognizing the N-terminal J domain sequences are directed against epitopes that are unavailable in the src-middle T complex. In turn, this suggests that src and the J domain are in proximity to one another. There is an alternative explanation for the failure of Q37A to bind src properly that is based on PP2A-middle T interactions. The association of src with middle T is known to be dependent upon recruitment of PP2A (27) in a manner independent of PP2A catalytic activity (40). As noted above, a truncated middle T lacking the J domain fails to bind PP2A. Although Q37A binds PP2A, the mutation could change the orientation so that src could not interact with the middle T-PP2A complex.

In conclusion, the genetics suggest that the J domain in middle T might be important for the formation of the src-middle T complex. For large T, the observation that the J domain participates in non-Rb transactivation suggests that chaperone function may affect other transcription factor protein complexes in the way in which it alters Rb-E2F complexes.

 
This study was supported by NIH grant CA34722 to B.S.S.

 
* Corresponding author. Mailing address: Department of Biochemistry, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-6868. Fax: (617) 636-2409. E-mail: brian.schaffhausen{at}tufts.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, August 2005, p. 9982-9990, Vol. 79, No. 15
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