Living cells are remarkably sophisticated information-processing systems: they take in vast amounts of information from the environment, integrate this information, and use it to make complex decisions about cell response or fate. Many of these responses are mediated by complex networks of signaling proteins. How are signaling circuits built from a system of molecules? How do signaling networks maintain specificity? How has such a diversity of signaling responses evolved? Moreover, since many diseases are caused by malfunctions in signaling, how can we control signaling behaviors for therapeutic purposes?
Over the past decade, it has become increasingly clear that eukaryotic signaling systems are organized in a modular and hierarchical fashion (Figure 1). Most signaling proteins are built from a finite set of modular components, which includes catalytic domains (e.g., kinases) and a large number of protein-protein interaction domains. These domains are found in diverse proteins, in diverse combinations. Evolution appears to have used these domains to build complexity—organisms as different as worms and humans share nearly all of the same modules, but they are combined in different ways in higher organisms. Many of the modular signaling proteins are themselves sophisticated information-processing devices capable of allosteric gating functions. At a higher level, these individual signaling proteins are functionally linked into pathways and networks, which can also show conserved structures and patterns. Thus in many ways, signaling systems share the hierarchical organization of complex engineered devices, such as a computer. Our goal is to understand the hierarchical logic by which complex biological signaling systems are built.
Switches and Scaffolds: The Molecular Mechanism of Modular Signaling Devices
Many signaling proteins act as switches—information relay devices that only switch on a particular output activity when stimulated by the proper upstream inputs. Others act as specificity elements. We are particularly interested in scaffold and adapter proteins—molecules that have multiple interaction sites that bind and appear to "wire" multiple signaling proteins into specific pathways. We are using biochemical and cell biological approaches to understand the structure and function of these types of signaling devices. We have been focusing on dissecting the mechanism of MAP kinase cascades involved in transmitting stress responses, and how scaffold proteins (such as Ste5) interact with and modulate kinase specificity and function. Another area of focus is on molecular switches that are involved in regulating the actin cytoskeleton, and understanding how these proteins work as a network to yield complex spatial responses, such as cell movement.
Synthetic Cell Signaling Networks: Learning How to Reprogram Cellular Responses
Biology is traditionally a field of observation and analysis. More recently, however, we have begun to apply synthetic or forward engineering approaches to understand the design principles governing cell signaling systems. In such approaches, we take a toolkit of biological modules and ask how one can use them to build systems with new or precisely modified behaviors. Why take this synthetic approach? First, by trying to link biological components together into functional systems, one can begin to understand the basic rules of biological hierarchy and modularity and gain insight into the process by which novel behaviors evolve. Second, by building minimal or alternative systems that carry out a particular target behavior, one can not only understand how one example system works but also begin outlining the general design principles underlying this class of behaviors. Synthetic networks can be analyzed through well-controlled structure-function analysis, in which network connectivity and parameters are systematically perturbed to learn what is both necessary and sufficient to carry out a biological task. Such systematic studies are difficult to perform on purely natural systems that are encumbered by idiosyncratic evolutionary histories. Synthetic approaches are commonly used as research approaches in chemistry and physics, and their application to biology is likely to be extremely valuable. Finally, by tinkering with biological systems, we will move toward learning how to precisely and predictably engineer cells to carry out useful therapeutic tasks, much in the way that today's electrical engineers can predictably build diverse microchips containing many millions of transistors.
Over the past several years, we have been exploring how modular signaling domains can be assembled in novel combinations to build new molecular switches and scaffolds. Reconnecting signaling modules to generate new functions has proved to be unexpectedly straightforward, highlighting the remarkable modularity of these components and how they facilitate system evolvability. We have built synthetic Rho GEF proteins that, because of novel input control, can generate new cytoskeletal responses. We have used synthetic scaffolds to redirect the flow of kinase pathway information in a cell, yielding novel signaling pathways in yeast. We have also used scaffolds to create synthetic feedback circuits that result in complex behaviors, such as adaptation. We are currently testing whether such approaches could be used to modulate immune cell behavior. One of our goals is to develop the ability to build combinatorial libraries of synthetic circuits that could then be screened for particular complex signaling functions. To achieve this goal, we are developing complementary experimental and computational methods.
We are also interested in using synthetic biology for applications. For example, we are developing novel switch proteins that can be used as tools in cell biology research. The ability to reliably engineer cells is likely to become more important in medicine as we move toward increasing use of cells as therapeutic agents—it will be critical to be able to tune and reprogram their behavior precisely to suit their target action. In the long term it may be possible to engineer cells that can target specific lesions and deliver a therapeutic payload, carry out regenerative/repair functions at precisely the right place and time, or search for and destroy microscopic tumors. Toward this goal, we are attempting to rewire neutrophils, a highly motile cell, to chemotax toward novel inputs.
Tracing the Evolution of Signaling Systems
A complementary way to understand how signaling modules are used to generate new functions is to trace the evolution of signaling systems. One way we are doing this is by studying intracellular pathogens that have evolved fascinating ways to interface with and hijack host signaling systems, often taking advantage of their modularity. An alternative approach is to track the evolution and functional divergence of MAP kinase circuits in fungal species. We are also interested in how novel types of signaling currencies evolved. For example, phospho-tyrosine signaling is critical for mammalian development and growth, but this relatively new mode of communication only arose ~1 billion years ago—closely linked to the emergence of multicellular organisms. To understand the origins of this new system and how it rose in functional importance in the metazoan lineage, we are studying the use of phospho-tyrosine signaling components (kinase, phosphatases, and SH2 domains) in premetazoan lineages, including the choanoflagellate Monosiga brevicollis and the slime mold Dictyostelium discoideum.
