The regulation of focal adhesion kinase by the scaffolding protein RACK1

Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase with an important role in cell migration and neurite outgrowth. FAK activity is required for cell adhesion and directional cell movement. The mechanisms controlling the regulation of FAK activity are poorly characterised but are predicted to be critical as FAK activity is deregulated in developmental disorders and cancer. In non-neuronal cells, the scaffolding protein RACK1 has previously been shown to bind FAK and promote cell adhesion and polarity. The aim of this investigation was to develop a neuronal model system to investigate the role of RACK1 and its binding partners in regulating FAK activity. We refined a protocol for the dissection and homogenisation of the hippocampus from 3 week old male Sprague Dawley rats which proved useful to identify proteins recruited by RACK1 to regulate FAK activity during neurite outgrowth. We optimised a protocol to differentiate PC12 and SH-SY5Y cells into neuronal-like cells (both morphologically and biochemically). Our model system allowed us to both identify and characterise novel RACK1 interacting proteins that regulate FAK activity and neurite outgrowth. We used this model system as the basis of a mass spectrometry screen to identify 27 novel RACK1 binding partners in the rat hippocampus. We then set out to characterise some of these proteins. The first of these, Cofilin-1, is an actin severing protein that has been previously linked to the regulation of FAK activity. We determined that Cofilin-1 is scaffolded to FAK by RACK1 during differentiation. We mapped the binding site of Cofilin-1 to the WD4 domain of RACK1 and we identified that RACK1 regulates Cofilin-1 activity by directing the serine/threonine phosphatase, PP2A. In this study, we confirmed that RACK1 and FAK interact in neuronal cells and that this interaction was an absolute requirement for NGF-mediated neurite outgrowth in PC12 cells. We characterised the interaction between RACK1, AGAP2 and Arf1 and we determined that RACK1 scaffolds this multiprotein complex to FAK during differentiation to regulate FAK activity. These findings confirm RACK1 as an important adaptor protein for the regulation of FAK activity in neuronal cells through the recruitment of Cofilin-1, Arf1 and AGAP2. These findings are important for understanding how FAK activity is regulated during neuronal cell migration and will significantly contribute to our understanding of how FAK activity is regulated while providing novel therapeutic targets and approaches in neurodegenerative and developmental disorders.