Signal Transduction and Growth Regulation Using Polyomavirus
We are interested in the cellular signal transduction regulating cell growth and survival. Most of our work starts from questions raised by murine polyomavirus, a small DNA tumor virus. Polyomaviruses subvert host cell mechanisms for their replication and transcription. Murine polyoma causes neoplastic transformation and a broad range of tumors in its host. Studies on polyoma have identified such key general mechanisms of cell regulation as tyrosine phosphorylation and phosphoinositide 3-kinase. We work both to identify new pathways that contribute to tumorigenesis and to understand the mechanisms for those we have already identified. Murine polyomavirus transformation is based on actions of three viral oncogenes: large T (LT), middle T (MT) and small T (ST) antigens. As depicted below, these three proteins work in different cell compartments. However, each T antigen is capable by itself of regulating both cell cycle progression and cell survival.
Figure 1. The drawing illustrates cellular pathways affected by polyomavirus T antigens.
Large T Antigen
Large T (LT) is a protein with multiple functions. These are related on the one hand to its role as an initiator of viral DNA replication and on the other to its ability to regulate the host cell. LT is capable of blocking differentiation of cells, of making embryonic cells immortal, and of inducing programmed cell death.
Figure 2. The diagram illustrates functions associated with different regions of large T antigen (green). More details are presented in the text below.
LT is a nuclear phosphoprotein. Its architecture is shown in Figure 2. LT is a DNA binding protein that oligomerizes using a zinc element. Acting as a helicase, LT binds ATP. It can be divided into two functional domains. The N-terminal domain is involved in regulating cell cycle progression. It interacts with tumor suppressors of the Rb family. The C-terminal domain is sufficient for initiation of viral DNA replication if the cellular environment is right, that is if the cell cycle is progressing. Phosphorylation of threonine 278 by cyclin/cdks acts as a biosensor to determine whether this is the case.
Much of LT function associated with the cell cycle depend on its interaction with members of the Rb tumor suppressor family. The ability of the N-terminal domain to promote cell cycle progression, to immortalize, to induce apoptosis and to block differentiation depends on the association of LT with Rb family members. Rb family members regulate transcription factors of the E2F family, which are important for cell cycle progression. LT disrupts E2F/Rb interactions. It turns out that much of the ability of LT to affect Rb family members depends on the LT DnaJ domain. This J domain allows interaction with hsc70, a member of the DnaK family. This connects LT to molecular chaperone systems.
There are also effects of LT on the host cell that do not involve the Rb family. In addition to transcriptional effects, these include regulation of DNA damage responses. LT sensitizes cells to the effects of DNA damage.
Figure 3. The images illustrate the effects of LT on cells following treatment with DNA damaging agents (etoposide, UV). The panel on the right illustrates DNA fragmentation, one hallmark of DNA damage.
Middle T Antigen
Middle T (MT) is the major transforming protein of polyomavirus. Mutant viruses that express LT and ST, but have altered MT fail to transform. As a transgene, MT is a potent inducer of tumors in a wide range of tissues.
Figure 4. The illustration depicts important regions of MT antigen.
MT is associated with membranes through a hydrophobic sequence at the C-terminus. It has no known enzymatic function. Rather it serves to associate with a collection of cellular signal transducers. The binding of protein phosphatase 2A (PP2A) allows MT to recruit tyrosine kinase of the src family. As a result of this association, MT is phosphorylated on a series of tyrosines. MT then associates with PLC gamma, the adaptor SHC and phosphoinositide 3-kinase (PI3K).
Figure 5. Phosphorylation sites on MT antigen and cellular pathways that are influenced by phosphorylation at these sites.
The association of MT with Shc, PI3K or PLCgamma1 sets off signaling cascades that affect the host cell. Recruitment of Shc leads to activation of the Ras pathway. PI3K causes activation of Akt kinase and the Rac GTPase. PLCgamma1 activates protein kinase C. Genetic experiments in animals indicates that the importance of particular signaling pathways depends on the tissue. It is also important to note that genetics shows there are additional functions important for transformation that remain to be elucidated.
Association of MT with PI3K is critical for transformation in vitro and tumor induction in vivo. Analysis of the interaction of MT with PI3K has also led us into structural studies. The association of MT with PI3K is mediated by the SH2 domains of the p85 subunit of PI3K. SH2 domains, modules of about 100 amino acids, function in tyrosine kinase signal transduction. Titrations of SH2 domains with MT tyrosine phosphorylated sequences can be carried out by NMR.
Figure 6. SH2 domain titrations with tyrosine phosphorylated MT analyzed by NMR.
This kind of NMR analysis can be used to map specific interactions of ligands with the SH2. It can also be used to examine the dynamics of the protein-ligand interactions. In this case, it reveals that the ligand induces conformational changes in the SH2 on the path to high affinity bound structure.
Small T Antigen
Small T (ST) antigens function in the cytoplasm or nuclear cell compartments.
Figure 7. Interaction of ST with PP2A.
ST replaces the B subunit of PP2A. Many, but not all, ST functions depend on the ability to bind PP2A. One example is shown below in Figure 8. Wild ST induces apoptosis in mouse embryo fibroblasts, as seen by DNA fragmentation, while a PP2A minus mutant does not.
Figure 8. PP2A binding and ST functions. (Left) Cell cycle profiles illustrate the effects on apoptosis. (Right) Effects on DNA fragmentation are shown.
A lesson taught by polyomavirus in a number of contexts is the importance of context to signaling. ST, which causes cell death in cells growing in serum, promotes cell survival when serum is removed. The difference appears to arise from the ways in which ST manipulate the Akt kinase.
Figure 9. Effects of ST on signaling via Akt.