A novel PIP2 binding of epsilonPKC and its contribution to the neurite induction ability.
Protein kinase C-epsilon (epsilonPKC) induces neurite outgrowth in neuroblastoma cells but molecular mechanism of the epsilonPKC-induced neurite outgrowth is not fully understood. Therefore, we investigated the ability of phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding of epsilonPKC and its correlation with the neurite extension. We found that full length epsilonPKC bound to PIP(2) in a 12-omicron-tetradecanoylphorbol-13-acetate dependent manner, while the regulatory domain of epsilonPKC (epsilonRD) bound to PIP(2) without any stimulation. To identify the PIP(2) binding region, we made mutants lacking several regions from epsilonRD, and examined their PIP(2) binding activity. The mutants lacking variable region 1 (V1) bound to PIP(2) stronger than intact epsilonRD, 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 epsilonPKC, epsilonRD and the mutants well correlated to their neurite induction ability. In addition, a chimera of pleckstrin homology domain of phospholipase Cdelta and the V3 region of epsilonPKC 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, epsilonPKC directly binds to PIP(2) mainly through pseudo-substrate and common region 1, contributing to the neurite induction activity.
Shirai
Yasuhito
Y
Laboratory of Molecular Pharmacology, Biosignal Research center, Kobe, Japan.
Murakami
Takuya
T
Laboratory of Molecular Pharmacology, Biosignal Research center, Kobe, Japan.
Kuramasu
Maho
M
Laboratory of Molecular Pharmacology, Biosignal Research center, Kobe, Japan.
Iijima
Leo
L
Laboratory of Molecular Pharmacology, Biosignal Research center, Kobe, Japan.
Saito
Naoaki
N
Laboratory of Molecular Pharmacology, Biosignal Research center, Kobe, Japan.
D045744:Cell Line, Tumor; D005786:Gene Expression Regulation / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009154:Mutation / Q000502:physiology; D016501:Neurites / Q000187:drug effects / Q000502:physiology*; D009447:Neuroblastoma / Q000473:pathology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D051744:Protein Kinase C-epsilon / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D019269:Phosphatidylinositol 4,5-Diphosphate; D049452:Green Fluorescent Proteins; D051744:Protein Kinase C-epsilon
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