Phospholipase A2 products retain a neuron specific γ isoform of PKC on the plasma membrane through the C1 domain—a molecular mechanism for sustained enzyme activity

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Abstract

To clarify molecular mechanism for sustained activation of gamma protein kinase C (γPKC), a neuron-specific subtype, we investigated the involvement of phospholipase A2 (PLA2) products in the membrane association of γPKC upon activation of G protein coupled purinoceptors in CHO-K1 and NG 108-15 cells. In addition, the functional domain responsible for PLA2-product mediated retention of γPKC on the plasma membrane was determined by simultaneously monitoring two different fluorescence-tagged γPKCs and mutants in the same living CHO-K1 cells.

Purinoceptor activation by UTP induced a transient translocation of γPKC from the cytoplasm to the plasma membrane. Interestingly, PLA2 inhibitors, bromoenol lactone (BEL) and arachidonyl-trifluoromethyl ketone (AACOF3), shortened the retention time of γPKC on the plasma membrane in cells treated with UTP, while a DAG kinase inhibitor did not affect it. The C1 domain deficient mutant (ΔC1-γPKC) also showed short membrane association compared with wild type γPKC, when cells are treated with UTP or arachidonic acid (AA) plus a Ca2+ ionophore. However, deletion of C1A or C1B subdomains (ΔC1A-γPKC or ΔC1B-γPKC) did not alter the retention time on the plasma membrane, whereas PLA2 inhibitor shortened the retention times of both mutants. These results indicate that PLA2 products prolong the retention of γPKC on the plasma membrane through the C1A and/or C1B subdomain in purinoceptor-stimulated CHO-K1 cells. The importance of PLA2 product and C1 domain for the retention of γPKC on the membrane was also confirmed using neuronal cell line, suggesting that these are part of molecular machinery for sustaining enzyme activity in neurons.

Introduction

Protein kinase C (PKC) is a family of at least 10 subtypes encoded by at least nine genes. These are divided into three groups based on structural differences in the regulatory domain; conventional PKC (cPKC; α-, βI-, βII- and γ-subtypes), novel PKC (nPKC; δ-, ε-, η- and θ-subtypes) and atypical PKC (aPKC; ζ- and ι/λ-subtypes) (Nishizuka, 1988). The regulatory domain of cPKC is composed of two conserved membrane-targeting modules, the C1 and C2 domains. The C1 domain has two cysteine-rich zinc finger-like subdomains (C1A and C1B) and was first identified as a receptor for diacylglycerol (DAG) and its structural analogs, phorbol esters, while the C2 region functions as a calcium-binding site (Nishizuka, 1988, Ono et al., 1989). The roles of the C1 and C2 domains in membrane binding and activation of cPKC have been extensively studied (Cho, 2001, Newton, 1995). Recently, it has been reported that the C1A and C1B subdomains of PKCs have disparate ligand affinities and distinct roles (Slater et al., 2002, Szallasi et al., 1996, Irie et al., 2002, Medkova and Cho, 1999). For example, C1B domains of PKCs have higher affinities for PDBu than C1A domains with the exception of γPKC whose the C1A domain has almost the same affinity for PDBu as C1B domain (Irie et al., 2002).

The receptor-mediated hydrolysis of inositol phospholipids is known to produce two signaling molecules, DAG and triphosphate (IP3), which releases into the cytoplasm and mobilizes Ca2+ from its intracellular store. The DAG and Ca2+ transduce various extracellular signals into cells to regulate many cellular functions. However, both DAG and Ca2+ signals are transient and are metabolized very rapidly and disappear quickly. Signal-dependent hydrolysis of additional phospholipids such as phosphatidylcholine (PC) by phospholipase D or phospholipase A2 (PLA2) also provide DAG and two additional active molecules, free fatty acids and lysophosphatidylcholine (lyso PC) (Nishizuka, 1992). PLA2 products such as arachidonic acid (AA) and lyso PC may have a function to potentiate the PKC signal pathway, particularly by causing long-term responses (Nishizuka, 1995, Nomura et al., 2001).

It has been well known that cPKC and nPKC are translocated from the soluble fraction to the particulate fraction when activated by several stimuli (Kraft et al., 1982, Zalewski et al., 1988, Kiley and Jaken, 1990, Mochly-Rosen et al., 1990). Recently, the movement of PKC in living cells has been visualized using PKC fused with green fluorescent protein (GFP) (Sakai et al., 1997, Oancea and Meyer, 1998). This technique revealed that physiological stimuli induce a rapid and reversible translocation of PKC, and that the translocation varies depending on PKC subtypes (Shirai et al., 1998, Kajimoto et al., 2001)and the stimulation (Sakai et al., 1997, Ohmori et al., 1998, Shirai et al., 2000), suggesting that the translocation is an important process for activation of the enzyme leading to the phosphorylation of the specific substrates.

Using the imaging technique, we previously reported that in CHO-K1 cells which endogenously express the P2Y purinoceptors coupled to hetero-trimeric G-proteins (Iredale and Hill, 1993). UTP, a purinergic receptor agonist, induced a transient translocation of γPKC from the cytoplasm to the membrane (Shirai et al., 2000). The P2Y causing the translocation of γPKC appears to be P2Y1, P2Y2 and/or P2Y6 (our unpublished data). Interestingly, the translocation of γPKC induced by purinoceptor stimulation was hastened in the presence of PLA2 inhibitor, suggesting the involvement of PLA2 products in the membrane association of in CHO-K1 cells (Shirai et al., 2000). It is known that PLA2 products such as arachidonic acid from postsynapse enhance the release of neurotransmitter and act on PKC synergistically with DAG (Herrero et al., 1992, Coffey et al., 1994), suggesting the effects of PLA2 on PKC translocation may be one of the molecular basis regulating the synaptic efficacy in the nervous system. However, it has not been fully ascertained how PLA2 products alter the membrane association of γPKC in neuronal cells upon receptor-stimulation. Therefore, in a series of study on the sustained activation of PKC in neuron, we precisely investigated the possible involvement of PLA2 products in the membrane association of γPKC neuron-specific subtype, upon activation of G protein-coupled receptors, and determined the functional domain responsible for the PLA2-mediated retention on the plasma membrane by simultaneously monitoring two different fluorescence-tagged γPKC and its deletion mutants lacking the C1domain or the C1 subdomains in the same living CHO-K1 and NG 108-15 cells.

Section snippets

Materials

A23187 was purchased from Calbiochem (La Jolla, CA). Dibutyryl adenosin 3′,5′-cyclic monophosphate (Bt2cAMP), Uridine triphosphate (UTP), and 1,2-dioctanoylglycerol (DiC8) were obtained from Sigma (St. Louis, MO). Diacylglycerol kinase (DGK) inhibitor (R59022) was purchased from Research Biochemicals International. Bromoenol lactone (BEL) and arachidonyl-trifluoromethyl ketone (AACOF3) were obtained from Caymen Chemical (Ann Arbor, MI). All other chemicals used were of analytical grade.

Cell culture

The

Phospholipase A2 inhibitors, but not diacylglycerol kinase inhibitor, affect the retention of γPKC on the plasma membrane after purinoceptor stimuli

First, we examined whether PLA2 products or DAG affect the retention of γPKC on the plasma membrane using γPKC-DsRed in CHO-K1 cells.

γPKC-DsRed was abundantly expressed in the perikarya (Fig. 1A upper) and faintly in the nucleus except for the nucleolus in CHO-K1 cells as reported previously (Shirai et al., 2000). When cells were stimulated by UTP, γPKC-DsRed rapidly translocated, within 10 s, from the cytoplasm to the plasma membrane, and γPKC-DsRed fluorescence was still present on the

Discussion

To investigate the molecular machinery underlying sustained activation of enzyme activity, we focused on γPKC, which is a neuron specific subtype and has been implicated in many neuronal functions including neurotransmission, neuronal plasticity and nerve growth (Tanaka and Nishizuka, 1994). The present study demonstrated that PLA2 inhibitors, but not a DAG kinase inhibitor shorten the association of γPKC with the plasma membrane in response to stimulation of G-protein coupled receptors. The

Acknowledgements

This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology in Japan, a Grant-in-Aid for Scientific Research on Priority Areas (C)—Advanced Brain Science Project—from the Ministry of Education, Culture, Sports, Science and Technology in Japan, the Uehara Memorial Foundation and the Sankyo Foundation of Life Science, and the Hyogo Science and Technology Association.

References (43)

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