Structural & Kinetic Studies of CaMK1D Interactions

Abstract

Protein kinases are critical players in cell signaling processes and their activity leads to the phosphorylation of substrates, often regulating enzymatic activity. 518 protein kinases are encoded in the human genome, however, there are many features within the catalytic domain of these proteins that have been evolutionarily conserved. These similarities pose problems for the drug discovery of kinase targets as off-target effects are a threat with substrate mimetic inhibitors. Therefore, understanding the structure and regulation of these cell signaling proteins is key for the discovery of therapeutics that regulate enzymatic activity through novel binding space. This thesis focuses on CaMK1D, an understudied member of the calcium/calmodulin dependent protein kinase (CaMK) family. CaMK1D is a putative driver of Triple Negative Breast Cancer (TNBC), a particularly aggressive breast cancer subtype with no targeted treatment option. Overexpression of CaMK1D in TNBC tumours leads to increased cell proliferation and migration, moreover, it is a validated TNBC target as the knockdown of CaMK1D reverses tumourigenesis. The work in this thesis shows that calmodulin (CaM) binds CaMK1D in a two-part mechanism wherein CaM has a final resting place on the C-terminal lobe of CaMK1D as shown with structural data from small angle x-ray scattering. Phosphorylation plays an important role in the activation of CaMK family members, and this work shows that the phosphorylation of the CaMK1D activation loop occurs post CaM binding to sustain activity. Lastly, the development of therapeutics to inhibit CaMK1D utilizing novel binding space is the overall goal of the group working on this target and this thesis explores Protein Thermal Shift assays as an inexpensive, rapid screening method for the initial steps in drug discovery.