Selective Roles of Protein Kinase C Isoforms on Cell Motility of GT1 Immortalized Hypothalamic Neurones

Abstract

Recently, we demonstrated that activation of the protein kinase C (PKC) signalling pathway promoted morphological differentiation of GT1 hypothalamic neurones via an increase in beta-catenin, a cell-cell adhesion molecule, indicating a possible involvement of PKC in cellular motility. In this study, we explored the differential roles of PKC isoforms in GT1 cell migration. First, we transiently transfected GT1 cells with enhanced green fluorescence protein (EGFP)-tagged actin to monitor the dynamic rearrangement of filamentous-actin (F-actin) in living cells. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, markedly promoted lamellipodia formation, while safingol (a PKC alpha-selective inhibitor) blocked the TPA-induced lamellipodial actin structure. Both wound-healing and Boyden migration assays showed that TPA treatment promoted neuronal migration of GT1 cells; however, cotreatment of TPA with safingol or rottlerin (a PKC delta-selective inhibitor) clearly blocked this TPA effect, indicating that both PKC alpha and PKC delta may be positive regulators of neuronal migration. By contrast, PKC gamma-EGFP-expressing GT1 cells exhibited decreased cellular motility and weak staining for actin stress fibres, suggesting that PKC gamma may act as a negative mediator of cell migration in these neurones. Among the PKC downstream signal molecules, p130Cas, a mediator of cell migration, and its kinase, focal adhesion kinase (FAK), increased following TPA treatment; phosphorylation of p130Cas was induced in a PKC alpha-dependent manner. Together, these results demonstrate that PKC alpha promotes GT1 neuronal migration by activating focal adhesion complex proteins such as p130Cas and FAK.