The role of PKC in neuronal differentiation and in regulation of the actin cytoskeleton during neurite outgrowth

Abstract: Protein kinase C (PKC) is a family of serine/threonine kinases subgrouped into classical (?, ?I, ?II, ?), novel (?, ?, ?, ?) and atypical (?, ?/?) isoforms. PKC can be activated by phorbol esters, and prolonged treatment of neuroblastoma cells has been shown to induce neuronal differentiation with neurite outgrowth and increased expression of neuronal genes. Here we show that a classical PKC? isoform is important for the phorbol ester-induced gene expression via the MAP kinases Erk 1/2. A previous study has shown that novel PKC?, via its regulatory domain, induces neurites in neuroblastoma cells. In this thesis we show that membrane localization and an actin-binding site in the regulatory domain is important for this effect. Besides neuroblastoma cells, the regulatory domain of PKC? is sufficient to induce cellular processes in both immortalized neural cells and fibroblasts, indicating that it targets commonly used cytoskeletal proteins. Furthermore, the catalytic activity of PKC is not only dispensable, it counteracts the neurite-inducing capacity of the regulatory domain. Overexpression of PKC? does not induce the formation of long cellular extensions by itself. However, treatment with phorbol ester triggers the formation of processes in PKC?-overexpressing cells. This provides a model system to study initial cytoskeletal changes, which revealed that stress fiber loss accompanies outgrowth of processes. This indicates that the small GTPase RhoA, which is the main regulator of stress fiber formation, is suppressed. Furthermore, overexpression of a constitutively active RhoA blocked both PKC-mediated stress fiber loss and neurite outgrowth. A possible mechanism for the RhoA suppression is via p190RhoGAP, with which PKC? forms a complex. Cdc42 was further shown to be crucial for the PKC-mediated stress fiber loss, and both Rac1 and Cdc42 are important for PKC-mediated outgrowth of cellular processes.