Non-Canonical Modular Domain Interactions Dictate PKCα Function

Abstract

The use of modular protein domains has emerged as a prominent feature of increasing phylogenetic complexity. Linking modular domains within a single protein allows complex regulation while conserving the sequence and structure of the individual domains. For instance, spatio-temporal control of signaling proteins is often achieved by stringing together a conserved catalytic domain with one or more regulatory modules. These modules can play multiple roles including masking the catalytic site to inhibit basal activity (auto-inhibition), releasing auto-inhibition through conformational changes triggered by second messenger stimuli, and facilitating translocation to subcellular compartments through binding secondary messengers or scaffolding proteins. Each additional module in a signaling protein provides a combinatorial enhancement to its regulation and cellular function. The protein-context independent structure and cellular function of individual modules have been extensively researched using biophysical approaches such as x-ray crystallography and NMR. Most modular domains have an evolutionarily conserved canonical function. However, coordination of interactions between these domains remains largely unexplored primarily due to the reliance on reductionist structural and biochemical approaches. As a corollary, our current structural understanding of modular signaling proteins does not adequately address the versatility of their cellular function. Using the uniquely persistent ER/K α-helix derived from the lever arm of myosin VI combined with genetically encoded fluorophores we have previously developed a methodology termed SPASM to both observe and modulate intra- molecular interactions between domains in multi-domain proteins. Using human protein kinase C α (PKCα) as a model multi-domain signaling protein, we have uncovered intra- and inter- molecular interactions involving each of its modular domains. These interactions contribute to context-dependent spatio-temporal regulation of PKC function in cells. Our findings highlight the importance of intra-molecular interactions in biologically critical multi-domain proteins.