(660.6) Lights, Camera, PAKtion: Design and Engineering of a Subcellular Targeting Optogenetic PAK1 Kinase and Sensor

Poster Board Number: A229

William Kinney (University of North Carolina at Chapel Hill), Lauren Haar (University of North Carolina at Chapel Hill ), David Lawrence (University of North Carolina at Chapel Hill)

p21-activated kinase 1 (PAK1) is a major pathogenic kinase that directs cell motility, cytoskeletal remodeling and has been shown to function as a downstream regulator for various cancer signaling cascades. Such biological roles require a tight spatial and kinetic control of its localization and activity. Unfortunately, the spatiotemporal nature of these pathways renders the application of conventional tools inadequate for studying localized PAK1 induced cell behavior. Therefore, we have designed and engineered an optogenetic analog of PAK1 to address the difficulties in assessing PAK1 intracellular activity.  

Optogenetics is a powerful light-triggered technology that, via control of protein biochemical activity, allows for the spatial and temporal control over a variety of biological processes. We developed an engineering strategy to construct a genetically expressed, light-responsive optogenetic analog of PAK1 (optoPAK1) and demonstrated that, upon illumination, optoPAK1 migrates to specified intracellular sites. OptoPAK1 was designed to function independently of endogenous biochemical regulation in a constitutively active manner with minimal activity in the dark state. We employed the improved light-induced dimer (iLid) system as a means to recruit and photoactivate the protein analog at discrete subcellular domains. Furthermore, we designed intracellular spatially segregated reporters of optoPAK1 activity and preliminary data displayed that optoPAK1 phosphorylates these reporters in a light-dependent fashion. Taken together, optoPAK1 and its corresponding biosensor offer the opportunity to appropriately study the relationship between PAK1 compartmentalized biochemical activity and cell behavior.

This work was supported by the National Institute of Neurological Disorders and Stroke 5R01NS103486-04





Fig 1. Domain configuration of an engineered optoPAK1 (top) and membrane targeting iLid-PAK1 sensor (bottom); Fig 2. Light-triggered recruitment, photoactivation and sensor phosphorylation by optoPAK1 at the (A) cytoskeleton and (B) plasma membrane in human U-373 glioblastoma astrocytomas. Left, iLid-PAK1 sensor anchored expression (green).  Middle, optoPAK1 localized expression (red). Right, immunofluorescence of phosphorylated PAK1 sensor (yellow)