Abstract
Protein kinase C-ε (εPKC) induces neurite outgrowth in neuroblastoma cells but molecular mechanism of the εPKC-induced neurite outgrowth is not fully understood. Therefore, we investigated the ability of phosphatidylinositol 4,5-bisphosphate (PIP2) binding of εPKC and its correlation with the neurite extension. We found that full length εPKC bound to PIP2 in a 12-ο-tetradecanoylphorbol-13-acetate dependent manner, while the regulatory domain of εPKC (εRD) bound to PIP2 without any stimulation. To identify the PIP2 binding region, we made mutants lacking several regions from εRD, and examined their PIP2 binding activity. The mutants lacking variable region 1 (V1) bound to PIP2 stronger than intact εRD, while the mutants lacking pseudo-substrate or common region 1 (C1) lost the binding. The PIP2 binding ability of the V3-deleted mutant was weakened. Those PIP2 bindings of εPKC, εRD and the mutants well correlated to their neurite induction ability. In addition, a chimera of pleckstrin homology domain of phospholipase Cδ and the V3 region of εPKC revealed that PIP2 binding domain and the V3 region are sufficient for the neurite induction, and a first 16 amino acids in the V3 region was important for neurite extension. In conclusion, εPKC directly binds to PIP2 mainly through pseudo-substrate and common region 1, contributing to the neurite induction activity.
Highlights
Protein kinase C-e induces neurite outgrowth in neuroblastoma cells but molecular mechanism of the ePKCinduced neurite outgrowth is not fully understood
To investigate the importance of actin-binding site between first half of C1 domain (C1A) and second half of C1 domain (C1B) in the neurite induction by regulatory domain of ePKC (eRD), we made a K/A mutant, in which lysine in the consensus sequence of the actin-binding site was substituted by alanine
regulatory domain (RD) of the K/A mutant showed both strong neurite induction activity and membrane localization as shown in Fig. 1b and c; about 35% of the cells expressing RD of the K/A mutant possessed long neurites. These results suggested that, except for the actin-binding site, unknown important factor is involved in the eRD-induced neurite induction
Summary
Protein kinase C-e (ePKC) induces neurite outgrowth in neuroblastoma cells but molecular mechanism of the ePKCinduced neurite outgrowth is not fully understood. It has been shown that the neurite-induction ability of ePKC is independent of its catalytic activity; regulatory domain of ePKC (eRD) induces neurite outgrowth (Zeidman et al 1999). In the case of the eRD-mediated neurite induction, deletion of ABS is not effective; RD of an ABS-deleted mutant still had strong neurite induction ability, while full length of the ABS-deleted mutant had about 50% less activity (Zeidman et al 2002). These results suggest that there is an additional factor, in addition to actin binding, important for the neurite induction by ePKC, especially by eRD
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