In other cell cycle work, David Beach and Konstantin Galaktionov made the important discovery of the regulatory target of the myc onco-gene. That myc is an oncogene has long been known, as has its role in cell cycle: the Myc protein is a driving force in cell division, and in partnership with a protein called Max, can promote either oncogenic transformation (cancer) or apoptosis (programed cell death). Myc is known to be a transcription factor for controlling gene expression, but the critical target genes whose expression it regulates remained obscure. David and Konstantin have found that the cdc25A protein is the target of Myc's function. They were studying Cdc25A because their earlier studies in yeast showed that it was an important CDK regulator, a so-called protein phosphatase. Interestingly, elevated levels of cdc25A have been found in a significant number of breast tumors and other cancers, making these cell cycle components a prime target for anti-cancer drug discovery efforts.

Nicholas Tonks' lab has continued to move forward in their work on the role of protein tyrosine phosphatases (PTPases). These enzymes catalyze dephosphorylation--the removal of phosphate groups from molecules which have been phosphorylated by protein kinases. In 1994, Nick and then-Cold Spring Harbor Fellow and X-ray crystallographer David Barford determined the first three-dimensional structure of a PTPase: the human PTPase 1B (PTP1B). The crystal structure of a molecule frequently sheds a great deal of light on how the molecule might function--scientists can then see which domains are exposed on the surface, where potential binding sites are located, etc. This information can then be used to develop molecules that effectively block specific actions or interactions. In 1995, Nick and David (by then at Oxford University) built on their success with another "first:" they solved the structure of PTP1B bound to a phosphorylated substrate (the molecule on which an enzyme acts).

In order to accomplish this, Nick and Postdoctoral Fellow Andrew Flint developed a mutant that would bind to its substrate but not dephos-phorylate it, and David crystalized and solved the structure of the bound pair. These structures of PTP1B demonstrated that the unique signature motif that is the defining feature of the PTP enzyme family acts as a rigid cradle structure that binds to the phosphate group on a target substrate. The motif lies at the base of a cleft on the surface of the protein and when the substrate binds, the cleft closes, locking the substrate within the enzyme.

These studies are illuminating how a PTPase works; the next question was with what does it work? It has long been thought that there were a limited number of PTPases each of which dephosphorylated a great many substrates. Nick and his colleagues suspected, and have now shown, that PTPases are actually very selective. Together they have developed "substrate-trapping" mutants for a variety of PTPases, like the one used to solve the structure of the PTP1B substrate complex. By expressing substrate-trapping mutants in cells, they can see which substrates the phosphatases recognize. They have made the surprising discovery that many PTPs examined in a physiological context are exquisitely selective, binding consistently to only a small number of substrates. This work has implications for the development of finely targeted therapeutics, as phosphorylation and dephosphorylation are critical to many cellular processes including cell growth, proliferation, and differentiation.