|Bidur Paudel, 2nd Year
Program: Veterinary and Animal Sciences
Advisor: Pablo Visconti
Education: West Virginia University, Morgantown, WV, MS, Animal and Nutritional Sciences, 2012
Northeastern University, Boston, MA, BS, Biotechnology, 2009
Structural studies of protein kinases
Protein kinases control several signaling pathways. Dysregulation of kinases result in serious health issues such as cancer, infertility and others. Thus understanding the mechanisms how kinases are regulated is the first step towards drug development, cure and treatment. In our lab, we are interested in the regulation of protein kinases family by phosphorylation. PKC-θ (PRCQ) plays an integral role in activating a range of signaling cascades that ultimately results in a transcriptional network in T cells. As part of my fellowship project, I proposed that phosphorylation of phosphorylatable residues; Serine (Ser-389), present on the consensus ATP binding kinase domain will render the kinase inactive. This hypothesis is based on the evidence we see in other kinases especially cyclin-dependent kinases (CDKs) where phosphorylation of two phosphorylatable residues rendered the kinase inactive and this event is necessary to control cell cycle. Therefore, to test our hypothesis, we aimed to gain insight into the regulation of kinase domain of protein kinase c theta (PRKCQ) that contains similar consensus ATP-binding domain. Thus as part of the project, we obtained recombinant PRKCQ plasmid and generated inactive mutants by replacing Ser for phosphomemetic aspartic acid (D) and for glutamic acid (E) using Site Directed Mutagenesis approach. We also replaced Ser (in PRKCQ sequence) for non-phosphorylatable residue Alanine (A), which we predict will maintain PRKCQ activity. Next with an aim to produce corresponding recombinant proteins, we cloned each mutant into pET E. coli T7 expression vector and subsequently transformed into BL21 (DE3) competent E. coli cells. Successful transformation of each insert was confirmed with colony Polymerase Chain Reaction (PCR). Currently, we are on the process to optimize conditions to produce wild type and mutant PRCQ recombinants proteins. Because our goal is to test the activity of PRKCQ and compare it with phosphomimetic and non-phosphorylatable mutants, we also purchased wild type PRKCQ recombinant protein and performed in vitro kinase assay using [32P]-ATP to monitor its activity in the presence and absence of lipid activator. After we figured out the optimal concentration necessary to achieve maximum PRKCQ activity we are now testing if we can inhibit the activity of PRKCQ using Rottlerin, an inhibitor known to inhibit activity of PKC isoforms. Our next step is to successfully produce recombinant proteins from these mutants and monitor their kinase activity using similar approach.