Klarissa Hollander
Kern Laboratory
Department of Biochemistry
Brandeis University
In Vitro Myristoylation: the Path to a Clearer Picture of Abl Kinase Regulation
Poster Abstract
Abelson tyrosine kinase (Abl) plays a critical role in cell cycle regulation. Misregulation of Abl has been identified in various cancers. When functioning normally, the kinase populates an inactive, closed conformation, and active open conformation. For closing, the SH2 and SH3 domains pack against the kinase domain and an N-terminal myristoyl group inserts into the kinase domain. While the regulatory role of the above mechanisms is generally described, there is much more debate about the role of the 80 residues connecting the myristoyl moiety to the SH3 domain which are not conserved and have no defined structure.
Abl can be purified in its myristoylated state only from eukaryotic cells, and in vitro myristoylation experiments have never been performed, which are necessary for NMR and other biophysical experiments. Previous in vitro research has utilized a myristoylated peptide which binds to Abl in trans to simulate the inhibitory effect of a myristoylated tail. By myristoylating the tail directly in vitro, we have obtained pure states of both myristoylated and non-mysristoylated Abl, allowing for a direct comparison of the levels of inhibition provided by the myristoyl group how it happens in the cell.
Using UV-Vis coupled assays, we determined that myristoylation in trans provides less inhibition than myristoylation in cis, and removal of the unstructured tail leads to even less inhibition. However, the addition of peptides which bind to the SH2 and SH3 domains, preventing them from packing against the kinase domain, activates each of the three constructs by 5.5 fold.
Personal Statement
I am extremely grateful for the support of the M.R. Bauer Foundation this summer, which has allowed me to continue my work in the Kern lab and experience full-time academic lab work. These ten weeks have allowed me to focus on my research without the stress of classes and spend the time necessary to hone my skills, enabling me to confidently perform each of the tasks necessary for my experiments. This intensive period has been crucial for the completion of my senior thesis not only because I collected data, but also because I have learned how to design, plan, and efficiently execute experiments, skills which will allow me to accomplish more in the upcoming year and for the rest of my career.
Additionally, this experience has cemented my resolve to pursue my PhD after graduation. This opportunity to work full time, doing what I will be doing for most of the time that I spend in grad school, showed me that it really was a future that I could readily see myself in and one that I think I would enjoy. This time has also greatly informed the choices that I will make when it comes to picking interesting programs. I have a much better idea of what I want to get out of a graduate school experience and having interacted much more closely with my PI, other PhD students and fellow undergrads I have a much better idea of what kind of environment will give that to me. I am very thankful for the opportunity to learn, grow, and become a better scientist.