J domain-independent regulation of the Rb family by polyomavirus large T antigen

The ability of polyomavirus large T antigen (LT) to promote cell cycling, to immortalize primary cells, and to block differentiation has been linked to its effects on tumor suppressors of the retinoblastoma susceptibility (Rb) gene family. Our previous studies have shown that LT requires an intact N-terminal DnaJ domain, in addition to an Rb binding site, for activation of simple E2F-containing promoters and stimulation of cell cycle progression. Here we show that some LT effects dependent on interaction with the Rb family are largely DnaJ independent. In differentiating C2C12 myoblasts, overexpression of LT caused apoptosis. Although this activity of LT completely depended on Rb binding, LTs with mutations in the J domain remained able to kill. Comparisons of Rb(-) and J(-) LTs revealed additional differences. Wild-type but not Rb(-) LT activated the cyclin A promoter under serum starvation conditions. Genetic analysis of the promoter linked the Rb requirement to an E2F site in the promoter. LTs with mutations in the J domain were still able to activate the promoter. Finally, J mutant LTs caused changes in phosphorylation of both pRb and p130. In the case of p130, Thr-986 was shown to be a site that is regulated by J mutant LT. Taken together, these observations reveal that LT regulation of Rb function can be separated into both DnaJ-dependent and DnaJ-independent pathways.     

Enterocyte proliferation and intestinal hyperplasia induced by simian virus 40 T antigen require a functional J domain

Transgenic mice expressing the simian virus 40 large T antigen (TAg) in enterocytes develop intestinal hyperplasia that progresses to dysplasia with age. This induction requires TAg action on the retinoblastoma (Rb) family of tumor suppressors and is independent of the p53 pathway. In cell culture systems, the inactivation of Rb proteins requires both a J domain in TAg that interacts with hsc70 and an LXCXE motif that directs association with Rb proteins. Together these elements are sufficient to release E2Fs from their association with Rb family members. We have generated transgenic mice that express a J domain mutant (D44N) in villus enterocytes. In contrast to wild-type TAg, the D44N mutant is unable to induce enterocyte proliferation. Histological and morphological examination revealed that mice expressing the J domain mutant have normal intestines without loss of growth control. Unlike mice expressing wild-type TAg, mice expressing D44N do not reduce the protein levels of p130 and are also unable to dissociate p130-E2F DNA binding complexes. Furthermore, mice expressing D44N in a null p130 background are still unable to develop hyperplasia. These studies demonstrate that the ectopic proliferation of enterocytes by TAg requires a functional J domain and suggest that the J domain is necessary to inactivate all three pRb family members.     

Genetic analysis of the polyomavirus DnaJ domain

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.     

The J Domain of Simian Virus 40 Large T Antigen Is Required To Functionally Inactivate RB Family Proteins

Transformation by simian virus 40 large T antigen (TAg) is dependent on the inactivation of cellular tumor suppressors. Transformation minimally requires the following three domains: (i) a C-terminal domain that mediates binding to p53; (ii) the LXCXE domain (residues 103 to 107), necessary for binding to the retinoblastoma tumor suppressor protein, pRB, and the related p107 and p130; and (iii) an N-terminal domain that is homologous to the J domain of DnaJ molecular chaperone proteins. We have previously demonstrated that the N-terminal J domain of TAg affects the RB-related proteins by perturbing the phosphorylation status of p107 and p130 and promoting the degradation of p130 and that this domain is required for transformation of cells that express either p107 or p130. In this work, we demonstrate that the J domain of TAg is required to inactivate the ability of each member of the pRB family to induce a G₁ arrest in Saos-2 cells. Furthermore, the J domain is required to override the repression of E2F activity mediated by p130 and pRB and to disrupt p130-E2F DNA binding complexes. These results imply that while the LXCXE domain serves as a binding site for the RB-related proteins, the J domain plays an important role in inactivating their function.     

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 molecular chaperone activity of simian virus 40 large T antigen is required to disrupt Rb-E2F family complexes by an ATP-dependent mechanism

The simian virus 40 large T antigen (T antigen) inactivates tumor suppressor proteins and therefore has been used in numerous studies to probe the mechanisms that control cellular growth and to generate immortalized cell lines. Binding of T antigen to the Rb family of growth-regulatory proteins is necessary but not sufficient to cause transformation. The molecular mechanism underlying T-antigen inactivation of Rb function is poorly understood. In this study we show that T antigen associates with pRb and p130-E2F complexes in a stable manner. T antigen dissociates from a p130-E2F-4-DP-1 complex, coincident with the release of p130 from E2F-4-DP-1. The dissociation of this complex requires Hsc70, ATP, and a functional T-antigen J domain. We also report that the "released" E2F-DP-1 complex is competent to bind DNA containing an E2F consensus binding site. We propose that T antigen disrupts Rb-E2F family complexes through the action of its J domain and Hsc70. These findings indicate how Hsc70 supports T-antigen action and help to explain the cis requirement for a J domain and Rb binding motif in T-antigen-induced transformation. Furthermore, this is the first demonstration linking Hsc70 ATP hydrolysis to the release of E2F bound by Rb family members.     

The role of the J domain of SV40 large T in cellular transformation.

SV40 large T antigen (TAg)-mediated transformation is dependent on binding to p53 and the retinoblastoma tumor suppressor protein (pRB) and inactivating their growth suppressive functions. Transformation minimally requires three regions of TAg: a C-terminal domain that mediates binding to p53; the LXCXE motif (residues 103-107), necessary for binding to pRB and the related proteins p107 and p130; and an N-terminal domain (residues 1-82) that contains homology to the J domain found in cellular DnaJ/Hsp40 molecular chaperone proteins. We have found that the N-terminal J domain of T Ag cooperates with the LXCXE motif to inactivate the growth suppressive functions of the pRB-related proteins.     

Inhibition of lens fiber cell morphogenesis by expression of a mutant SV40 large T antigen that binds CREB-binding protein/p300 but not pRb

Simian virus (SV) 40 large T antigen can both induce tumors and inhibit cellular differentiation. It is not clear whether these cellular changes are synonymous, sequential, or distinct responses to the protein. T antigen is known to bind to p53, to the retinoblastoma (Rb) family of tumor suppressor proteins, and to other cellular proteins such as p300 family members. To test whether SV40 large T antigen inhibits cellular differentiation in vivo in the absence of cell cycle induction, we generated transgenic mice that express in the lens a mutant version of the early region of SV40. This mutant, which we term E107KDelta, has a deletion that eliminates synthesis of small t antigen and a point mutation (E107K) that results in loss of the ability to bind to Rb family members. At embryonic day 15.5 (E15.5), the transgenic lenses show dramatic defects in lens fiber cell differentiation. The fiber cells become post-mitotic, but do not elongate properly. The cells show a dramatic reduction in expression of their beta- and gamma-crystallins. Because CBP and p300 are co-activators for crystallin gene expression, we assayed for interactions between E107KDelta and CBP/p300. Our studies demonstrate that cellular differentiation can be inhibited by SV40 large T antigen in the absence of pRb inactivation, and that interaction of large T antigen with CBP/p300 may be enhanced by a mutation that eliminates the binding to pRb.     

Edwardsiella tarda DnaJ is a virulence-associated molecular chaperone with immunoprotective potential

Members of the DnaJ/Hsp40 family play an important role in protein homeostasis by regulating the activity of DnaK/Hsp70. In this study, we examined the activity and function of the DnaJ from Edwardsiella tarda, a serious fish pathogen that can also infect humans and birds. In silico analysis indicated that E. tarda DnaJ contains structural features, i.e. the J domain, the glycine/phenylalanine-rich region, and the zinc-finger domain, that are conserved among Type I Hsp40. Purified recombinant DnaJ was able to stimulate the ATPase activity of DnaK. Pull-down assay indicated that DnaJ could interact specifically with DnaK. Mutation of the conserved HPD site in the J domain completely abolished the DnaK-stimulating effect of DnaJ. To examine the functional importance of DnaJ, a dnaJ-defective mutant was constructed. Compared to the wild type, the dnaJ mutant (i) was retarded in growth and more sensitive to H?O?-induced oxidative damage, (ii) dramatically reduced in general bacterial virulence and in blood dissemination capacity, and (iii) significantly weakened in the ability to block macrophage activation and to survive within macrophages. Furthermore, when used as a subunit vaccine, purified recombinant DnaJ induced protective immunity in Japanese flounder (Paralichthys olivaceus). Taken together, these results indicate that DnaJ plays an important role in the pathogenesis of E. tarda probably by functioning as a DnaK partner and that DnaJ, with its immunoprotective property, may be useful in the control of E. tarda infection in aquaculture.     

Inactivation of the PKR protein kinase and stimulation of mRNA translation by the cellular co-chaperone P58(IPK) does not require J domain function

P58(IPK) was discovered as an inhibitor of the interferon-induced, protein kinase, PKR. Upon virus infection, PKR can, as part of the host defense system, inhibit mRNA translation by phosphorylating the alpha subunit of protein synthesis eukaryotic initiation factor 2 (eIF-2alpha). We previously found that influenza virus recruits the cellular P58(IPK) co-chaperone to inhibit PKR activity and thus facilitate viral protein synthesis. P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs in addition to the highly conserved J domain found in all DnaJ chaperone family members. To define the role of molecular chaperones in regulating cell growth in addition to PKR regulation, we performed a detailed analysis of the P58(IPK) J domain. Using growth rescue assays, we found that the P58(IPK) J domain substituted for the J domains of other DnaJ proteins, including DnaJ in Escherichia coli and Ydj1 in Saccharomyces cerevisiae. This is the first time a cellular J domain from a mammalian DnaJ family member was shown to be functional in both prokaryotic DnaJ and eukaryotic Ydj1 constructs. Furthermore, point mutations within the conserved HPD residue cluster of the P58(IPK) J domain disrupted P58(IPK) J function including stimulation of ATPase activity of Hsp70. However, the P58(IPK) HPD mutants still inhibited PKR activity and thus supported cell growth in a yeast rescue assay. Overexpression of the HPD mutants of P58(IPK), similar to their wild-type counterpart, also stimulated mRNA translation in a mammalian cell system. Taken together, our data necessitate a model of P58(IPK) inhibition of PKR kinase activity and stimulation of mRNA translation, which does not require classical J domain function found in the DnaJ molecular chaperone family.     

Simian Virus 40 Large T Antigen J Domain and Rb-Binding Motif Are Sufficient To Block Apoptosis Induced by Growth Factor Withdrawal in a Neural Stem Cell Line

Serum-free mouse embryo (SFME) cells are a neural stem cell line that is dependent upon epidermal growth factor (EGF) for survival. Removal of EGF results in the G1 arrest and apoptosis of SFME cells. We have shown that the expression of simian virus 40 large T antigen in SFME cells blocks apoptosis and allows cell survival and division in the absence of EGF. Therefore the presence of T antigen abrogates the EGF requirement. The steady-state levels of p53, p21, and mdm-2 do not increase as SFME cells undergo apoptosis upon EGF withdrawal. Furthermore, the amino-terminal 136 amino acids (N136) of T antigen are sufficient to block death and to promote proliferation in the absence of EGF, while the carboxy-terminal fragment (C251-708), which contains the p53 binding site, is unable to block death. Taken together, these data suggest that SFME cells deprived of EGF undergo p53-independent apoptosis. Mutations that disrupt either the J domain or Rb family binding abolish the ability of T antigen to block SFME cell apoptosis and to promote cell growth. We conclude that T antigen must act on one or more members of the Rb family to inhibit SFME cell apoptosis.     

Structural basis for the inactivation of retinoblastoma tumor suppressor by SV40 large T antigen

Inactivation of the retinoblastoma (Rb) tumor suppressor by Simian virus 40 (SV40) large T antigen is one of the central features of tumorigenesis induced by SV40. Both the N-terminal J domain and the LxCxE motif of large T antigen are required for inactivation of Rb. The crystal structure of the N-terminal region (residues 7-117) of SV40 large T antigen bound to the pocket domain of Rb reveals that large T antigen contains a four-helix bundle, and residues from helices alpha2 and alpha4 and from a loop containing the LxCxE motif participate in the interactions with Rb. The two central helices and a connecting loop in large T antigen have structural similarities with the J domains of the molecular chaperones DnaJ and HDJ-1, suggesting that large T antigen may use a chaperone mechanism for its biological function. However, there are significant differences between large T antigen and the molecular chaperones in other regions and these differences are likely to provide the specificity needed for large T antigen to inactivate Rb.     

Human T cell leukemia virus type 1 Tax associates with a molecular chaperone complex containing hTid-1 and Hsp70.

Tax, an oncogenic viral protein encoded by human T cell leukemia virus type 1 (HTLV-1), induces cellular transformation of T lymphocytes by modulating a variety of cellular gene expressions [1]. Identifying cellular partners that interact with Tax constitutes the first step toward elucidating the molecular basis of Tax-induced transformation. Here, we report a novel Tax-interacting protein, hTid-1. hTid-1, a human homolog of the Drosophila tumor suppressor protein Tid56, was initially characterized based on its interaction with the HPV-16 E7 oncoprotein [2]. hTid-1 and Tid56 are members of the DnaJ family [2,3], which contains a highly conserved signature J domain that regulates the activities of heat shock protein 70 (Hsp70) by serving as cochaperone [4-6]. In this context, the molecular chaperone complex is involved in cellular signaling pathways linked to apoptosis, protein folding, and membrane translocation and in modulation of the activities of tumor suppressor proteins, including retinoblastoma, p53, and WT1[7-12]. We find that expression of hTid-1 inhibits the transformation phenotype of two human lung adenocarcinoma cell lines. We show that Tax interacts with hTid-1 via a central cysteine-rich domain of hTid-1 while a signature J domain of hTid-1 mediates its binding to Hsp70 in HEK cells. Importantly, Tax associates with the molecular chaperone complex containing both hTid-1 and Hsp70 and alters the cellular localization of hTid-1 and Hsp70. In the absence of Tax, expression of the hTid-1/Hsp70 molecular complex is targeted to perinuclear mitochondrial clusters. In the presence of Tax, hTid-1 and its associated Hsp70 are sequestered within a cytoplasmic "hot spot" structure, a subcellular distribution that is characteristic of Tax in HEK cells.     

Structural features required for the interaction of the Hsp70 molecular chaperone DnaK with its cochaperone DnaJ.

Hsp70 family members together with their Hsp40 cochaperones function as molecular chaperones, using an ATP-controlled cycle of polypeptide binding and release to mediate protein folding. Hsp40 plays a key role in the chaperone reaction by stimulating the ATPase activity and activating the substrate binding of Hsp70. We have explored the interaction between the Escherichia coli Hsp70 family member, DnaK, and its cochaperone partner DnaJ. Our data show that the binding of ATP, subsequent conformational changes in DnaK, and DnaJ-stimulated ATP hydrolysis are all required for the formation of a DnaK-DnaJ complex as monitored by Biacore analysis. In addition, our data imply that the interaction of the J-domain with DnaK depends on the substrate binding state of DnaK.     

Simian virus 40 large T antigen's association with the CUL7 SCF complex contributes to cellular transformation

Simian virus 40 large T antigen (T Ag) is capable of immortalizing and transforming rodent cells. The transforming activity of T Ag is due in large part to perturbation of the tumor suppressor proteins p53 and the retinoblastoma (pRB) family members. Inactivation of these tumor suppressors may not be sufficient for T Ag-mediated cellular transformation. It has been shown that T Ag associates with an SCF-like complex that contains a member of the cullin family of E3 ubiquitin ligases, CUL7, as well as SKP1, RBX1, and an F-box protein, FBXW8. We identified T Ag residues 69 to 83 as required for T Ag binding to the CUL7 complex. We demonstrate that delta69-83 T Ag, while it lost its ability to associate with CUL7, retained binding to p53 and pRB family members. In the presence of CUL7, wild-type (WT) T Ag but not delta69-83 T Ag was able to induce proliferation of mouse embryo fibroblasts, an indication of cellular transformation. In contrast, WT and delta69-83 T Ag enabled mouse embryo fibroblasts to proliferate to similarly high densities in the absence of CUL7. Our data suggest that, in addition to p53 and the pRB family members, T Ag serves to bind to and inactivate the growth-suppressing properties of CUL7. In addition, these results imply that, at least in the presence of T Ag, CUL7 may function as a tumor suppressor.