Mechanisms of Specificity and Integration in Cell Signaling

The research in my laboratory is aimed at achieving an integrated molecular and systems-level understanding of the mechanisms by which intracellular signal transduction cascades execute a diverse repertoire of responses with efficiency and fidelity, and how this impacts human disease.  We focus not just on individual pathways, but also on network-level interactions of multiple pathways.  To this end, we study conserved signaling pathways controlling growth, development and stress responses in yeast and mammalian cells, using the techniques of molecular cell biology, biochemistry and biophysics, genetics and genomics, and mathematical & computational biology.  We are particular motivated by trying to answer the following questions:

      How to protein kinases find their substrates?  Can we use this knowledge to identify new substrates and regulatory strategies, and to find new ways to treat cancer and other diseases?

      How is specificity from signal to cellular response maintained when networks are highly interconnected, and different pathways use similar or overlapping components?

      What evolutionary logic underlies the structure of signaling and gene regulatory networks?  Why are they so complicated and interconnected?  What performance objectives might these designs achieve?

      Can we combine biochemistry, genetics, math/computation and cutting-edge imaging to visualize, model and understand what really happens in space and time during intracellular signal transduction?

      How can we translate our increasingly sophisticated systems-level understanding of regulatory processes into new ideas for treating human disease?

Our present emphasis is on mitogen-activated protein kinase (MAPK) signaling pathways.  MAPK cascades participate in the regulation many biologically (and medically) important processes, including normal and pathological aspects of cell growth, division, differentiation, and death.  The ubiquity and versatility of MAPK cascades make them ideal for addressing the above questions.


Kinase-target recognition.  Clearly, one important component of understanding specificity in protein kinase signaling concerns the question of how do protein kinases recognize their protein substrates?  Our work has focused on the concept that high-affinity protein-protein interactions away from the active site are perhaps just important as the interaction of the active site with the target phosphoacceptor residues. We believe that this concept has additional implications for targeting protein kinases with therapeutic drugs, and for predicting substrates.  We have published extensively on the recognition of MAPKs by their activating kinases, the MEKs or MKKs [21, 22, 27, 29, 30, 32, 33, 38, 39, 46].  We were the first to identify a short, evolutionarily-conserved motif (the MAPK-docking site, or D-site) near the N-terminus of MEKs, that is necessary and sufficient for high-affinity MAPK binding [21, 22].  This initial finding was made in yeast, but we subsequently extended it to six different human MEKs [27, 29 , 30, 39, 46].    Contemporaneous and subsequent to our initial discovery, work in many labs showed that D-sites are found not only in MEKs, but also in other MAPK regulators such as scaffolds and phosphatases, as well as in multiple MAPK substrates; we have contributed to these developments [29, 30, 33].  Underscoring the importance of docking in MAPK signaling, others have shown that the D-sites in MEKs are the only known substrates of Anthrax lethal factor protease, and we demonstrated that the lethal-factor cleavage products of MEKs are indeed defective for MAPK binding [32]. 

      Our current interests in this area include (1) understanding how docking, and the related process of scaffolding, contribute to signal transmission and specificity in vivo [e.g. 27, 33, 38]; (2) using advanced computational algorithms and our knowledge of the D-site consensus to predict novel MAPK substrates [49]; (3) exploring how the competitive docking of multiple substrates and regulators to MAPKs influences signaling dynamics [e.g., 29, 30, 37, 46]; and (4) using modeling and simulation based approaches, backed by experiment, to gain further insight into how docking, scaffolding and other specificity-promoting mechanisms actually work [e.g. 36, 43, 47, 48].

       Signal specificity and insulation.  Another major focus of my laboratory is to dissect the mechanisms that encode signal identity in two different developmental options in yeast – mating and invasive growth.  Mutations exist that cause leaking between the signaling pathways regulating these processes, which are normally well insulated from one another.  The study of these mutants has provided insights into the mechanisms that prevent such leaking in the first place.  We first found feedback circuits that control the magnitude and duration of pathway activation are crucial for specificity [28].  We next characterized the key role in selective activation of the Ste5 scaffold promoting in signal insulation [35].  We continue to actively investigate the multiple mechanisms encoding specificity in this system, using both experiment and theory [36, 43, 47, 48].  In the future, I am also interested in applying these paradigms to issues of specificity in human embryonic stem cells.



Figures.   The first figure shows a conception of how docking is used in MEK-MAPK recognition and phosphorylation and then in MAPK-substrate recognition and phosphorylation [41].  The figure immediately above shows the problem of specificity when two pathways share a common component (basic architecture), and then several specificity-promoting insulating mechanisms known to exist [36 & 43].


       New directions.  I have had a long-standing interest in mathematical modeling and analysis as it applies to signal transduction, and recently we have begun to publish in this area [36, 43, 45, 47, 48, 50].  In 2008-09, I was on sabbatical in the Laboratory for Regenerative Medicine, run by Prof. Roger A. Pedersen at the University of Cambridge, United Kingdom, where I studied signal transduction in human embryonic stem cells.  This field has a lot of interesting questions with regard to signaling specificity, as many of the factors required to keep the cells pluripotent are also required in a slightly different context to induce their differentiation.  I am currently applying for funding and to continue some of this work in my own laboratory.


Note: The citations above refer to publication numbers listed in my online CV.