The transfer of the terminal phosphate group from adenosine triphosphate (ATP) to specific tyrosine residues on defined protein targets has profound regulatory effects on all the processes that sustain life in higher eukaryotes. Indeed, there are nearly a hundred distinct human enzymes, known as tyrosine kinases, that facilitate this transfer. Dysregulation of tyrosine kinase activity has been implicated in a variety of human diseases including several cancers making these enzymes ideal targets for therapeutic intervention. Tyrosine phosphorylation driven signal transduction was considered to be restricted to the eukaryotic domain of life and indeed the presence of many distinct tyrosine kinases was believed to be the hallmark of multicellularity. However, recent discoveries have provided clear evidence that bacterial cells also encode numerous unique tyrosine kinases that play a similarly important role in bacterial physiology. The largest family of these bacterial tyrosine kinases, the BY-kinases, are conserved across the bacterial kingdom, contain none of the sequence signatures characteristic of eukaryotic tyrosine kinases, are structurally unique, and are activated and regulated through distinct, yet incompletely understood, mechanisms. The research proposed, using an integrated application of experimental and computational tools will decipher in terms of structure and mechanism, the activation and regulation of BY-kinase function. This research will provide multi-disciplinary training to a broad range of scholars at various career stages ranging from high-school students to postdoctoral trainees. The work will be facilitated by the outstanding intellectual environment at the City College of New York enabled by a student body that reflects the unique and diverse demographics of New York City.
The long-term goal of the research is to understand the activation, activity and regulation of BY-kinases in atomic detail. Towards that goal, the mechanism/s employed by the catalytic subunit of BY-kinases in achieving efficient auto-phosphorylation and the nature of their interaction with counteracting PTPs to reverse this covalent modification, will be determined. The proposed research will utilize solution-state nuclear magnetic resonance and high-resolution mass-spectrometric techniques informed by all-atom computational studies and validated by biochemical experiments in vitro and in cell. The proposed studies will provide an expanded view of protein phosphorylation in life’s kingdoms through detailed insight into a divergent platform utilized by nature to perform this simple chemistry that has profound biological consequences. The archetypal BY kinase, Escherichia coli Wzc, and its cognate protein tyrosine phosphatase, Wzb will be utilized in these studies. Deploying proteins from a non-pathogenic, genetically tractable organism will allow the robust validation of the in vitro biophysical/biochemical insights within the cellular context. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
