Functional implications of mutations within polyomavirus large T antigen Rb-binding domain: effects on pRb and p107 binding in vitro and immortalization activity in vivo.

In this study, we have extensively modified the Rb-binding domain of polyomavirus large T antigen. Mutant polyomavirus large T antigens were tested for their ability to bind pRb and p107 in vitro and assayed for their capacity to immortalize primary rat embryo fibroblasts in vivo. Polyomavirus large T antigen bound pRb and p107 through a common region located between amino acids 141 to 158, containing the consensus Rb-binding sequence D/N-L-X-C-X-E. Substitution of any amino acid within the core Rb-binding sequence abolished pRb and p107 binding in vitro and immortalization activity in vivo. Substitution of amino acids outside the core Rb-binding sequence reduced pRb and p107 binding in vitro and decreased or abolished immortalization of rat embryo fibroblasts in vivo. Although duplication of the Rb-binding domain within the polyomavirus large T antigen results in a molecule that can bind at least twice as much pRb and p107 in vitro, this mutant displayed an essentially wild-type level of immortalization activity. More importantly, we found that the addition of acidic residues within the casein kinase II consensus phosphorylation region flanking the Rb-binding domain, or the deletion of amino acids 256 to 272, increased the immortalizing activity of the mutant polyomavirus large T antigen. These two mutants displayed a greater than wild-type level of pRb binding in vitro, while in contrast, a decreased affinity for p107 binding in vitro was observed. Together, these results indicate that while pRb binding appears to be an essential event for immortalization, there is no tight correlation between the frequency of immortalization and the absolute level of pRb binding in vitro, indicating that other large T antigen functions are important for cellular immortalization.     

Structural features and ligand binding properties of tandem WW domains from YAP and TAZ, nuclear effectors of the Hippo pathway

The paralogous multifunctional adaptor proteins YAP and TAZ are the nuclear effectors of the Hippo pathway, a central mechanism of organ size control and stem cell self-renewal. WW domains, mediators of protein-protein interactions, are essential for YAP and TAZ function, enabling interactions with PPxY motifs of numerous partner proteins. YAP has single and double WW domain isoforms (YAP1 and YAP2) whereas only a single WW domain isoform of TAZ has been described to date. Here we identify the first example of a double WW domain isoform of TAZ. Using NMR, we have characterized conformational features and peptide binding of YAP and TAZ tandem WW domains (WW1-WW2). The solution structure of YAP WW2 confirms that it has a canonical three-stranded antiparallel β-sheet WW domain fold. While chemical shift-based analysis indicates that the WW domains in the tandem WW pairs retain the characteristic WW domain fold, 15N relaxation data show that, within the respective WW pairs, YAP WW1 and both WW1 and WW2 of TAZ undergo conformational exchange. 15N relaxation data also indicate that the linker between the WW domains is flexible in both YAP and TAZ. Within both YAP and TAZ tandem WW pairs, WW1 and WW2 bind single PPxY-containing peptide ligand concurrently and noncooperatively with sub-mM affinity. YAP and TAZ WW1-WW2 bind a dual PPxY-containing peptide with approximately 6-fold higher affinity. Our results indicate that both WW domains in YAP and TAZ are functional and capable of enhanced affinity binding to multi-PPxY partner proteins such as LATS1, ErbB4, and AMOT.     

Immortalization of rat embryo fibroblasts by mutant polyomavirus large T antigens deficient in DNA binding.

We have identified a putative DNA-binding domain in polyomavirus large T antigen. Mutations introduced into the gene between amino acids 290 and 310 resulted in proteins that no longer bound to the high-affinity binding sites on the polyomavirus genome, showed no detectable nonspecific DNA binding, and were not able to initiate DNA replication from the viral origin. These mutant T antigen genes were introduced into rat embryo fibroblasts together with the neomycin resistance gene to allow selection for growth in the presence of G418. All the mutations tested facilitated the establishment of these cells in long-term culture at an efficiency indistinguishable from that of the wild-type protein.     

Determination of the origin-specific DNA-binding domain of polyomavirus large T antigen.

To map the DNA-binding domain of polyomavirus large T antigen, we constructed a set of plasmids coding for unidirectional carboxy- or amino-terminal deletion mutations in the large T antigen. Analysis of origin-specific DNA binding by mutant proteins expressed in Cos-1 cells revealed that the C-terminal boundary of the DNA-binding domain is at or near Glu-398. Fusion proteins of large T antigen lacking the first 200 N-terminal amino acids bound specifically to polyomavirus origin DNA; however, deletions beyond this site resulted in unstable proteins which could not be tested for DNA binding. Testing of point mutants and internal deletions by others suggested that the N-terminal boundary of the DNA-binding domain lies between amino acids 282 and 286. Taken together, these results locate the DNA-binding domain of polyomavirus large T antigen to the 116-amino-acid region between residues 282 and 398.     

NMR structure of the N-terminal J domain of murine polyomavirus T antigens. Implications for DnaJ-like domains and for mutations of T antigens

The NMR structure of the N-terminal, DnaJ-like domain of murine polyomavirus tumor antigens (PyJ) has been determined to high precision, with root mean square deviations to the mean structure of 0.38 A for backbone atoms and 0.94 A for all heavy atoms of ordered residues 5-41 and 50-69. PyJ possesses a three-helix fold, in which anti-parallel helices II and III are bridged by helix I, similar to the four-helix fold of the J domains of DnaJ and human DnaJ-1. PyJ differs significantly in the lengths of N terminus, helix I, and helix III. The universally conserved HPD motif appears to form a His-Pro C-cap of helix II. Helix I features a stabilizing Schellman C-cap that is probably conserved universally among J domains. On the helix II surface where positive charges of other J domains have been implicated in binding of hsp70s, PyJ contains glutamine residues. Nonetheless, chimeras that replace the J domain of DnaJ with PyJ function like wild-type DnaJ in promoting growth of Escherichia coli. This activity can be modulated by mutations of at least one of these glutamines. T antigen mutations reported to impair cellular transformation by the virus, presumably via interactions with PP2A, cluster in the hydrophobic folding core and at the extreme N terminus, remote from the HPD loop.     

Identification of binding sites on the regulatory A subunit of protein phosphatase 2A for the catalytic C subunit and for tumor antigens of simian virus 40 and polyomavirus.

Protein phosphatase 2A is composed of three subunits: the catalytic subunit C and two regulatory subunits, A and B. The A subunit consists of 15 nonidentical repeats and has a rodlike shape. It is associated with the B and C subunits as well as with the simian virus 40 small T, polyomavirus small T, and polyomavirus medium T tumor antigens. We determined the binding sites on subunit A for subunit C and tumor antigens by site-directed mutagenesis of A. Twenty-four N- and C-terminal truncations and internal deletions of A were assayed by coimmunoprecipitation for their ability to bind C and tumor antigens. It was found that C binds to repeats 11 to 15 at the C terminus of A, whereas T antigens bind to overlapping but distinct regions of the N terminus. Simian virus 40 small T binds to repeats 3 to 6, and polyomavirus small T and medium T bind to repeats 2 to 8. The data suggest cooperativity between C and T antigens in binding to A. This is most apparent for medium T antigen, which can only bind to those A subunit molecules that provide the entire binding region for the C subunit. We infer from our results that B also binds to N-terminal repeats. A model of the small T/medium T/B-A-C complexes is presented.     

Studies of partially transforming polyomavirus mutants establish a role for phosphatidylinositol 3-kinase in activation of pp70 S6 kinase.

Infection of mouse fibroblasts by wild-type polyomavirus results in increased phosphorylation of ribosomal protein S6 (D.A. Talmage, J. Blenis, and T.L. Benjamin, Mol. Cell. Biol. 8:2309-2315, 1988). Here we identify pp70 S6 kinase (pp70S6K) as a target for signal transduction events leading from polyomavirus middle tumor antigen (mT). Two partially transforming virus mutants altered in different mT signalling pathways have been studied to elucidate the pathway leading to S6 phosphorylation. An upstream role for mT-phosphatidylinositol 3-kinase (PI3K) complexes in pp70S6K activation is implicated by the failure of 315YF, a mutant unable to promote PI3K binding, to elicit a response. This conclusion is supported by studies using wortmannin, a known inhibitor of PI3K. In contrast, stable interaction of mT with Shc, a protein thought to be involved upstream of Ras, is dispensable for pp70S6K activation. 250YS, a mutant mT which retains a binding site for PI3K but lacks one for Shc, stimulates pp70S6K to wild-type levels. Mutants 315YF and 250YS induce partial transformation of rats fibroblasts with distinct phenotypes, as judged from morphological and growth criteria. Neither mutant induces growth in soft agar, indicating that an increase in S6 phosphorylation, while necessary for cell cycle progression in normal mitogenesis, is not sufficient for anchorage-independent cell growth. In the polyomavirus systems, the latter requires integration of signals from mT involving both Shc and PI3K.     

ATP-binding cassette transporter A1 contains a novel C-terminal VFVNFA motif that is required for its cholesterol efflux and ApoA-I binding activities

The stimulation of cellular cholesterol and phospholipid efflux by apolipoprotein A-I is mediated by the activity of the ATP-binding cassette transporter A1 (ABCA1). Individuals with Tangier disease harbor loss-of-function mutations in this transporter that have proven useful in illuminating its activity. Here, we analyze a mutation that deletes the last 46 residues of the 2261 amino acid transporter (Delta46) and eliminates its lipid efflux. As the final four amino acids of the C terminus represent a putative PDZ-binding motif, we initially characterized deletion mutants lacking only these residues. Although a moderate decline in lipid efflux was detected, this decline was not as profound as that seen in the Delta46 mutant. Subsequent systematic analysis of the ABCA1 C terminus revealed a novel, highly conserved motif (VFVNFA) that was required for lipid efflux. Alteration of this motif, which is present in some but not all members of the ABCA family, did not prevent trafficking of the transporter to the plasma membrane but did eliminate its binding of apoA-I. Chimeric transporters, generated by substituting the C termini of either ABCA4 or ABCA7 for the endogenous terminus, demonstrated that ABCA1 could stimulate cholesterol efflux without its PDZ-binding motif but not without the VFVNFA motif. When a peptide containing the VFVNFA sequence was introduced into ABCA1-expressing cells, ABCA1-mediated lipid efflux was also markedly inhibited. These results indicate that the C-terminal VFVNFA motif of ABCA1 is essential for its lipid efflux activity. The data also suggest that this motif participates in novel protein-protein interactions that may be shared among members of the ABCA family.     

Mechanisms of interference with simian virus 40 (SV40) DNA replication by trans-dominant mutants of SV40 large T antigen.

Mutations at multiple sites within the simian virus 40 (SV40) early region yield large T antigens which interfere trans dominantly with the replicative activities of wild-type T antigen. A series of experiments were conducted to study possible mechanisms of interference with SV40 DNA replication caused by these mutant T antigens. First, the levels of wild-type T antigen expression in cells cotransfected with wild-type and mutant SV40 DNAs were examined; approximately equal levels of wild-type T antigen were seen, regardless of whether the cotransfected mutant was trans dominant or not. Second, double mutants that contained the mutation of inA2827, a strong trans-dominant mutation with a 12-bp linker inserted at the position encoding amino acid 520, and various mutations in other parts of the large-T-antigen coding region were constructed. The trans-dominant interference of inA2827 was not affected by second mutations within the p105Rb binding site or the amino or carboxy terminus of large T antigen. Mutation of the nuclear localization signal partially reduced the trans dominance of inA2827. The large T antigen of mutant inA2815 contains an insertion of 4 amino acids at position 168 of large T; this T antigen fails to bind SV40 DNA but is not trans dominant for DNA replication. The double mutant containing the mutations of both inA2815 and in A2827 was not trans dominant. The large T antigen of dlA2433 lacks amino acids 587 to 589, was unstable, and failed to bind p53. Combining the dlA2433 mutation with the inA2827 mutation also reversed the trans dominance completely, but the effect of the dlA2433 mutation on trans dominance can be explained by the instability of this double mutant protein. In addition, we examined several mutants with conservative point mutations in the DNA binding domain and found that most of them were not trans dominant. The implications of the results of these experiments on possible mechanisms of trans dominance are discussed.     

Hsc70 contacts helix III of the J domain from polyomavirus T antigens: addressing a dilemma in the chaperone hypothesis of how they release E2F from pRb

Hsc70's expected binding site on helix II of the J domain of T antigens appears to be blocked in its structure bound to tumor suppressor pRb. We used NMR to map where mammalian Hsc70 binds the J domain of murine polyomavirus T antigens (PyJ). The ATPase domain of Hsc70 unexpectedly has its biggest effects on the NMR peak positions of the C-terminal end of helix III of PyJ. The Hsc70 ATPase domain protects the C-terminal end of helix III of PyJ from an uncharged paramagnetic probe of chelated Gd(III), clearly suggesting the interface. Effects on the conserved HPD loop and helix II of PyJ are smaller. The NMR results are supported by a novel assay of Hsc70's ATP hydrolysis showing that mutations of surface residues in PyJ helix III impair PyJ-dependent stimulation of Hsc70 activity. Evolutionary trace analysis of J domains suggests that helix III usually may join helix II in contributing specificities for cognate hsp70s. Our novel evidence implicating helix III differs from evidence that Escherichia coli DnaK primarily affects helix II and the HPD loop of DnaJ. We find the pRb-binding fragment of E2F1 to be intrinsically unfolded and a good substrate for Hsc70 in vitro. This suggests that E2F1 could be a substrate for Hsc70 recruited by T antigen to an Rb family member. Importantly, our results strengthen the chaperone hypothesis for E2F release from an Rb family member by Hsc70 recruited by large T antigen. That is, it now appears that Hsc70 can freely access helix III and the HPD motif of large T antigen bound to an Rb family member.     

The tumor antigens and the early functions of polyoma virus

Summary Polyoma virus (Py) tumor (T) antigens are the proteins specified by the early region of the viral genome. They are responsible for most biological effects caused by this oncogenic virus, i.e. induction of tumors, cell transformation and most of the virus-induced events observed in productive and transforming infection. By immunoprecipitation with antitumor serum followed by gel electrophoresis three major Py T-antigens have been characterized: large Tantigen (IT) with an apparent M_T of about 100 000, middle T-antigen (mT) of about 55 000 Mr and small T-antigen (sT) of about 23 000 Mr. In addition, there may exist one or more minor species by Py T-antigens. Analysis of the tryptic peptides showed that IT, mT and sT have a common N-terminal amino acid sequence, but differ from each other in the size and the sequence of the C-terminal part of the molecule as a consequence of different splicing of their mRNAs. With the nucleotide sequence of the Py genome being known, the coding regions for each of the Py T-antigens have been identified and consequently the amino acid sequence of IT, mT and sT was deduced. Cell fractionation experiments showed that the major part of 1T is located in the nucleus, mT was found in plasma membranes and sT is mainly present in the cytoplasm. Large T is a phosphoprotein and undergoes posttranslational modification. Two-dimensional gel electrophoresis of Py T-antigens revealed considerable charge heterogeneity particularly for mT and sT.All Py transformed cell lines analyzed contained mT and sT. Large T was not detected in virtually all Py transformed mouse cell lines and in about one third of Py transformed rat and hamster cell lines. Instead of 1T often new immunoreactive proteins were found which are probably truncated forms of 1T. These and other recent results suggest that IT is required neither for initiation nor for maintenance of cell transformation. For tumor induction in hamsters, similar conclusions were reached from analysis of Py T-antigens and viral DNA sequences in cell lines derived from tumors that had been induced either by virus or by viral DNA digested with various restriction enzymes. Experiments done with several deletion mutants indicated that mT is required for cell transformation by Py. In a protein kinase assay done in vitro with Py T-antigen immunoprecipitates, a kinase activity associated with Py mT was found which phosphorylates tyrosine residues mainly of mT and less frequently of 1T and of rat immunoglobulins. In all transformation defective mutants, kinase activity measured by this assay was absent or strongly reduced.In a concluding chapter I discuss the events occurring in wild-type virus and mutant infected cells trying to attribute specific functions to each of the three Py T-antigens. At least two functions are known for 1T, one is initiation of viral DNA replication, the other induces a mitotic response of the host cell, i.e. the events leading to and including host chromatin duplication. Middle T-antigen is certainly involved in cell transformation, possibly by its presence in the membrane. No function has been defined yet for sT. Since there are more virus-induced events observed in infected cells than Py T-antigens at least one of them must be a multifunctional protein.     

Nonspecific DNA binding activity of simian virus 40 large T antigen: evidence for the cooperation of two regions for full activity.

We generated a series of COOH-terminal truncated simian virus 40 large tumor (T) antigens by using oligonucleotide-directed site-specific mutagenesis. The mutant proteins [T(1-650) to T(1-516)] were expressed in insect cells infected with recombinant baculoviruses. T(1-623) and shorter proteins [T(1-621) to T(1-516)] appeared to be structurally changed in a region between residues 269 and 522, as determined by increased sensitivities to trypsin digestion and by altered reactivities to several monoclonal antibodies. These same mutant proteins bound significantly less nonorigin plasmid DNA (15%) and calf thymus DNA (25%) than longer proteins [T(1-625) to T(1-708)]. However, all mutant T antigens exhibited a nearly wild-type level of viral origin-specific DNA binding and binding to a helicase substrate DNA. This indicated that binding to origin and helicase substrate DNAs is separable from about 85% of nonspecific binding to double-stranded DNA. As an independent confirmation that a region distinct from the origin-binding domain (amino acids 147 to 247) is involved in nonspecific DNA binding, we found that up to 96% of this latter activity was specifically inhibited in wild-type T antigen by several monoclonal antibodies which collectively bind to the region between residues 269 and 522. In order to investigate the relationship between the origin-binding domain and the second region, we performed origin-specific DNA binding assays with increasing amounts of calf thymus DNA as competitor. The results suggest that this second region is not an independent nonspecific DNA binding domain. Rather, it most likely cooperates with the origin-binding domain to give rise to wild-type levels of nonspecific DNA binding. Our results further suggest that most of the nonspecific binding to double-stranded DNA is involved in a function other than direct recognition and binding to the pentanucleotides at the replication origin on simian virus 40 DNA.