A novel PIP(2) binding of εPKC and its contribution to the neurite induction ability1

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 (PIP(2)) binding of εPKC and its correlation with the neurite extension. We found that full length εPKC bound to PIP(2) in a 12-ο-tetradecanoylphorbol-13-acetate dependent manner, while the regulatory domain of εPKC (εRD) bound to PIP(2) without any stimulation. To identify the PIP(2) binding region, we made mutants lacking several regions from εRD, and examined their PIP(2) binding activity. The mutants lacking variable region 1 (V1) bound to PIP(2) stronger than intact εRD, while the mutants lacking pseudo-substrate or common region 1 (C1) lost the binding. The PIP(2) binding ability of the V3-deleted mutant was weakened. Those PIP(2) 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 PIP(2) 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 PIP(2) mainly through pseudo-substrate and common region 1, contributing to the neurite induction activity.