A novel PIP₂ binding of εPKC and its contribution to the neurite induction ability¹

Abstract

Protein kinase C-e (ePKC) induces neurite outgrowth in neuroblastoma cells but molecular mechanism of the ePKCinduced neurite outgrowth is not fully understood. Therefore, we investigated the ability of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) binding of ePKC and its correlation with the neurite extension. We found that full length ePKC bound to PIP 2 in a 12-o-tetradecanoylphorbol-13-acetate dependent manner, while the regulatory domain of ePKC (eRD) bound to PIP 2 without any stimulation. To identify the PIP 2 binding region, we made mutants lacking several regions from eRD, and examined their PIP 2 binding activity. The mutants lacking variable region 1 (V1) bound to PIP 2 stronger than intact eRD, 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 ePKC, eRD and the mutants well correlated to their neurite induction ability. In addition, a chimera of pleckstrin homology domain of phospholipase Cd and the V3 region of ePKC 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, ePKC directly binds to PIP 2 mainly through pseudo-substrate and common region 1, contributing to the neurite induction activity. Keywords: actin, neurite outgrowth, neuroblastoma, phosphatidylinositol 4,5-bisphosphate, protein kinase C. Protein kinase C (PKC) plays pivotal roles in proliferation, differentiation, and apoptosis etc. The PKC family consists of at least 10 subtypes that are classified into three groups based on the structure of their regulatory domain (RD) (Nishizuka 1992;Shirai and Saito 2002;Newton 2006). Conventional PKCs (a, b1, b2, and c) have two common regions, C1 domain and C2 domain, in the RD. The former is responsible for diacylglycerol (DAG) and phorbol ester binding, the latter binds to calcium. Thus, calcium and DAG are required for the activation of conventional PKCs. On the other hand, novel PKCs (e, d, g, and h) are activated by DAG, but not by Ca 2+ because novel PKCs lack the C2 domain. Atypical PKCs (f and k/i) are insensitive to both Ca 2+ and DAG because of lack of the C2 domain and one of the C1 domains. Each subtype shows different enzymatic properties and distinct tissue and cellular distribution, suggesting specific functions of each PKC subtype (Ohno 1997), but the individual functions have not been fully understood.Among them, ePKC is abundant in the central nervous system and is thought to play important roles in nervous system (Tanaka and Nishizuka 1994;Akita 2002). Specifically, ePKC is localized at nerve terminus and seems to mediate synaptic function (Saito et al. 1993; Prekeris et al. Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.Abbreviations used: ABS, actin-binding site; C1, common region 1; C1A, first half of C1 domain; ...

The role of protein kinase C epsilon in neural signal transduction and neurogenic diseases

“…Shirai et al investigated the possibility of the direct binding of PKC ε to phosphatidylinositol 4,5-bis phosphate (PIP 2 ) and its correlation with the neurite outgrowth. They found that the direct binding of PKCε to PIP 2 can induce neurite and may influence the function of actin binding proteins [52]. They believed that the open conformation of PKC ε could interact with RhoGAP and/or PIP2, and this could be regulated by the binding of PKC ε to actin [8].…”

The substrates and binding partners of protein kinase Cε

“…PKC activation by DAG can be mimicked by the addition of other compounds that bind the C1 domain, such as tumor promoting phorbol esters. PKCε may also be activated by other lipids, such as arachidonic acid, or phosphoinositide bisphosphate (PIP2) [15, 16]. …”

Protein kinase Cε: function in neurons

“…How PKCε regulates RhoA and Cdc42 is still unknown. We have recently shown that PKCε binds to phosphatidylinositol 4,5‐bis phosphate (PIP 2 ) and that the PIP 2 binding is necessary for PKCε‐induced neurite induction and its membrane localization [11]. The PIP 2 binding of PKCε may influence the function of actin binding proteins, leading to actin rearrangement and neurite induction.…”

“…Alternatively, PKCε may attenuate RhoA via RhoGAP, which is reported to bind to PKCε and induce neurite outgrowth [13]. Interestingly, the binding of full length PKCε to RhoGAP and PIP 2 is dependent on 12‐ O ‐tetradecanoylphorbol 13‐acetate, but εRD can bind to PIP 2 without 12‐ O ‐tetradecanoylphorbol‐13‐acetate [11,13]. These results suggest that the open conformation is important for the binding of PKCε to RhoGAP and PIP 2 .…”