Selective roles of protein kinase C isoforms on cell motility of GT1 immortalized hypothalamic neurones

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 PKCalpha-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 PKCdelta-selective inhibitor) clearly blocked this TPA effect, indicating that both PKCalpha and PKCdelta may be positive regulators of neuronal migration. By contrast, PKCgamma-EGFP-expressing GT1 cells exhibited decreased cellular motility and weak staining for actin stress fibres, suggesting that PKCgamma may act as a negative mediator of cell migration in these neurones. Among the PKC downstream signal molecules, p130(Cas), a mediator of cell migration, and its kinase, focal adhesion kinase (FAK), increased following TPA treatment; phosphorylation of p130(Cas) was induced in a PKCalpha-dependent manner. Together, these results demonstrate that PKCalpha promotes GT1 neuronal migration by activating focal adhesion complex proteins such as p130(Cas) and FAK.