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Professional Interests - Publications - Contact Information - Lab Personnel Professional Interests of James M. Pipas
Simian virus 40 (SV40) is a polyomavirus capable of inducing tumors when injected into newborn hamsters or immuno-compromised mice. SV40 also induces neoplastic transformation in cell culture. Numerous studies have shown that expression of one particular viral component, the large TAg protein (TAg), is necessary and often sufficient to cause these transformations (Figure 1). Expression of this viral protein is sufficient to transform multiple cell types in vitro and, when expressed ectopically in transgenic mice, it causes neoplasia in numerous tissues (Figure 2). As a consequence, TAg has been used exhaustively to elucidate the mechanisms of growth regulation and control of cell proliferation.
Transformation induced by TAg is accomplished by targeting cellular components, with their subsequent inactivation or modification of function. For instance, two regions in the amino-terminal region of TAg (Figure 3), an LXCXE motif that mediates binding to the retinoblastoma (Rb) family of tumor suppressors and a J-domain that interacts with the mammalian DnaK homologue hsc70, are responsible for the inactivation of the Rb-proteins. This inactivation causes a concomitant increase in cell proliferation and tumorigenesis. The carboxy-terminal region of TAg contains a third domain important for transformation that binds, stabilizes and inactivates the tumor suppressor p53. Chaperone Model. One aim of our lab is to elucidate the biochemical mechanism by which TAg inactivates Rb proteins. Previous work has established that TAg is a DnaJ molecular chaperone. DnaJ chaperones work in concert with a chaperone partner, a member of the DnaK family. TAg binds hsc70, and like other DnaJ-DnaK interactions, this binding leads to an activation of hsc70's intrinsic ATPase activity. The TAg J-domain, and its interaction with hsc70, is essential for several TAg functions. One well defined role for the J-domain in transformation is the disruption of Rb-E2F complexes. During both productive infection and transformation TAg induces the release of E2F transcription factors from the three retinoblastoma proteins, pRb, p107, and p130, thus stimulating the expression of E2F-dependent genes. The release of E2F from Rb proteins is dependent on a functional J-domain and hsc70-mediated ATP hydrolysis. These observations led us to propose a chaperone model in which TAg first binds to Rb-E2F complexes, and then energy derived from ATP hydrolysis by hsc70, recruited by the J-domain, is used to liberate E2F from Rb.
This model is consistent with in vivo and in vitro data on the TAg-mediated disruption of p130-E2F complexes. However, in vivo the ultimate biological consequence of TAg action on each of the three Rb family members is different. Thus, in vivo, TAg expression leads to the degradation of p130, an elevation in p107 levels, and no change in the phosphorylation or steady-state level of pRb. The molecular basis for this discrimination is unknown. Furthermore, if the chaperone model is correct then the J-domain must be able to properly orient hsc70 for each target with which it interacts. Finally, genetic and biochemical studies indicate that additional cellular proteins associate with TAg via the J-domain and participate in the chaperone reaction. To explore this model, we are currently purifying all the components of the chaperone machine. We are collaborating with the laboratory of Dr. Xiaojiang Chen (USC) to use X-ray crystallography to elucidate the various structures of these TAg complexes, and the laboratory of Dr. Walter Chazin (Vanderbilt) to apply NMR spectroscopy to understanding the molecular dynamics of the chaperone machine. Novel Targets of T antigen. Several reports suggest that TAg targets additional cellular proteins other than pRb and p53, and that these interactions contribute to transformation as well. Moreover, different TAg functions seem to be required to elicit transformation depending on the cell lineage studied. To expand these studies we use different model systems expressing TAg or mutant derivatives, including cell culture in vitro and the intestinal epithelium of transgenic mice. Our results from numerous gene microarray experiments indicate that TAg induces extensive changes in cellular gene expression and that these changes are not always the same in different cell types. Our goal is to determine the molecular basis for this cell-type specificity and to map the regulation of cellular pathways to specific domains on TAg. We are actively pursuing how specific changes in these pathways are associated with transformation. In particular, we have found that the expression of SV40 TAg in intestinal epithelial cells results in a significant decrease in the levels of mRNAs encoding several drug metabolizing/detoxifying enzymes and transporters from the P450 pathway. This pathway is responsible for the metabolization, processing and final elimination of toxic compounds, including carcinogens and drugs. We have found that TAg blocks both the endogenous levels of P450 components as well as the induction of these mRNAs by xenobiotics. Furthermore, this ability requires an intact LXCXE motif, which binds to and inactivates the retinoblastoma family of tumor suppressors, suggesting that inactivation of this family plays a role in the process. We are actively investigating the connection between Rb, tumorigenesis and the P450 detoxification pathway and its possible significance in the metabolism and mechanism of action of both carcinogens and prescription drugs. Publication
Archive Recent Publications of James M. Pipas
Saenz-Robles, M.T., J.A. Markovics, J.L. Chong, R. Opavsky, R.H. Whitehead, G. Leone, and J.M. Pipas (2007) Intestinal hyperplasia induced by simian virus 40 large tumor antigen requires E2F2. J. Virol. 81:13191-13199
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