Our laboratory is using a combination of molecular, genetic, and biochemical approaches to investigate biological functions regulated by the highly conserved and ubiquitous Ras G-proteins.
Ras proteins act as switches for signal transduction pathways governing numerous essential biological activities, such as cell division, development, cell death, and organization of the cytoskeleton. One of the most daunting and perplexing activities involving Ras is tumorigenesis. Constitutively active forms of Ras proteins are commonly found in many types of tumors, and can efficiently transform cells in culture.
In humans, there are three RAS genes, which encode four Ras proteins with more than 90 percent identity in amino acid sequence. The biochemical properties of these proteins are nearly the same and very straightforward (Fig. 1). Ras can bind either GTP or GDP. In the resting state, Ras is primarily GDP-bound and inactive. In response to signals, Ras switches to the active GTP-bound state, a process catalyzed by guanine nucleotide exchange factors (GEFs). Activated Ras stimulates effector proteins to turn on downstream pathways. How a given Ras protein functions in the cell, however, is anything but straightforward. Despite the fact that several proteins have been identified to act downstream of Ras, how they affect cancer formation remains largely unknown. The best documented Ras effector in animal cells is the Raf protein kinase. Ras also interacts with the conserved Rho G-proteins, such as Cdc42, for oncogenesis. The link between the Ras-Raf pathway and cancer is quite obvious as Ras and Raf mediate the signal for cell division generated by growth factors. By contrast, how Ras and Cdc42 interact to affect tumor formation is unclear. In addition, by one count, there are at least three Ras effectors and five GEFs. Under experimental conditions, most known Ras effectors and GEFs can interact with nearly all the Ras proteins, but how they actually match up with one another in the cell is poorly understood.
The Chang lab uses the fission yeast, Schizosaccharomyces pombe, as a model system, which is amenable to an array of genetic manipulations, including gene deletion, that are not readily available to more complex systems (Learn more about the S. pombe genome sequencing project).
The power of this system was first exploited by Paul Nurse, whose lab discovered Cdk (cyclin-dependent kinase) as a universal machine governing eukaryotic cell division. Because of this pioneering contribution, Paul Nurse was awarded the Nobel prize in 2001 (Listen to his acceptance speech). S. pombe contains a single Ras homolog, Ras1, which shares a striking 67 percent identity in the amino acid sequence with the human Ras. This suggests that yeast Ras1 can interact with its GEFs and effectors in a fashion very similar to those in humans.
Indeed, we and others have shown that Ras1 controls two conserved pathways with distinct outputs (Fig. 2 and see PDF). Ras1 interacts with the Byr2 protein kinase, which mediates signals generated by the mating pheromone. The interaction between Ras1 and Byr2 is analogous to that between human Ras and Raf. We also discovered a second Ras1 effector, Scd1, which is a presumptive GEF for Cdc42. Activated Cdc42 in turn stimulates a protein kinase, Shk1/PAK1.
The inactivation of the Ras1-Cdc42 pathway readily leads to alterations of many cellular functions (see below), including the change of cell morphology (from being rod-shape to round).
Our current research program is broadly divided into two areas:
To reveal biological functions controlled by the yeast Ras1-Cdc42 pathway that are important to tumorigenesis in humans:
To this end, we are focusing on a novel component in this pathway, Yin6 (PDF). Intriguingly, Yin6 is structurally and functionally homologous to the mammalian Int6, which was first discovered as an integration site by the mouse mammary tumor virus (Fig 3). What role Int6 plays in breast cancer formation is not clear. Our yeast data support a model in which Int6 cooperates with Ras to regulate the function of the 26S proteasome (Fig. 4). Inactivation of this process leads to accumulation of polyubiquitinated proteins that block proper cell cycle control and genetic stability (PDF, Fig 5). We plan to delineate the mechanism by which Yin6 and Ras1 regulates the proteasome functioning and to test our ideas in the mammalian cell.
To delineate conserved mechanisms by which a single Ras can coordinate interactions with multiple factors:
We have shown that the specificity of the Ras pathways can be regulated by the GEF (PDF). In S. pombe, Ras1 has two GEFs, Ste6 and Efc25 (Fig. 6). The former controls the Ras1-Byr2 pathway, while the latter Ras1-Scd1 pathway. We are currently testing a hypothesis that GEF can influence the binding between Ras and downstream effectors and we plan to test this to see if it also operates in other eukaryotes.