The Role of Src-Homology-3 in the Activation Mechanism of MLK3

Waleed Brinjikji Ramy Goueli
Under the direction of Dr. Kathleen Gallo, Physiology

Mixed-lineage kinases (MLKs) are mammalian protein kinases that play critical roles in mitogen-activated protein kinase (MAPK) signaling pathways. MLK3 is activated by the small GTPase, Cdc42. Our lab has demonstrated that the src-homology 3 (SH3) domain of MLK3 is involved in an autoinhibitory interaction with a single proline containing sequence located between the Cdc42-binding region and leucine zipper domain of MLK3 The purpose of this study is to determine the thermodynamic parameters (?G, ?S, ?H and Ka) of the binding interaction between the SH3 domain of MLK3 and a peptide corresponding to the MLK3 autoinhibitory region using isothermal titration calorimetry (ITC). The SH3 domain of MLK3 was digested from pGEX-2T-1-SH3 plasmid and re-ligated into a bacterial expressed vector pGEX-6P-1, which contains a PreScission protease sequence, using the BamHI and EcoRI restriction sites. pGEX-6P-1-SH3 plasmids were transformed into Rosetta (DE3) pLysS cells. The GST-SH3 fusion proteins were induced by adding IPTG and then purified using glutathione affinity agarose. We are currently working to remove the GST tag from the purified fusion protein in order to produce pure SH3 of MLK3 for ITC experiments.