Persistent activation of protein kinase Cα is not necessary for expression of cerebellar long-term depression
Introduction
Simultaneous activation of parallel fiber (PF) and climbing fiber (CF) inputs to a cerebellar Purkinje cell (PC) induces persistent and input-specific attenuation of PF-PC synaptic transmission (Sakurai, 1987, Hirano, 1990, Crepel and Jaillard, 1991, Ito, 2001). This phenomenon, called cerebellar long-term depression (LTD), has been regarded as a cellular basis for some types of motor learning, such as adaptation of vestibulo-ocular reflex and eye blink conditioning (Li et al., 1995, De Zeeuw et al., 1998, Ito, 2001). Induction and expression of LTD are postsynaptic. Activation of mGluR1 at the PF-PC synapse and the depolarization-induced increase in intracellular Ca++ concentration by CF input cooperatively evoke LTD (Linden, 1994, Ito, 2002). As both inputs are required for LTD induction, signals from PF and CF inputs should converge at some points in the molecular signaling cascade. It is important to identify the molecule whose amount or activity changes more when a PC receives two inputs within a certain time window, because such a molecule may play a critical role in the coincidence detection. The candidates are Ca++, IP3 receptor and protein kinase Cα (Ito, 2002). In this study, we focused on PKCα and examined its regulation during induction of LTD.
PKC family comprises of 11 isoforms, which are divided into three subfamilies (cPKC, nPKC and aPKC) based on differences in regulatory domains (Dekker et al., 1995). PKCα, βI, βII and γ belong to the cPKC subfamily, which are activated by binding to both Ca++ and diacylglycerol (DAG) (Kishimoto et al., 1980, Newton, 2001, Ohno and Nishizuka, 2002, Corbalán-García and Gómez-Fernández, 2006). According to the sequential activation model, cPKC is activated in two steps (Oancea and Meyer, 1998). First, cPKC binds Ca++ through the C2 domain and translocates from the cytosol to the plasma membrane. Then it binds to DAG in the plasma membrane through the C1 domain and is fully activated. The following scheme has been proposed for the induction of LTD. The intracellular Ca++ increased by CF input and DAG produced by PF inputs through mGluR1/phospholipase Cβ (PLCβ) cascade cooperatively activate cPKC (Ito, 2002). Of the four cPKC subtypes, PKCα specifically phosphorylates GluR2 subunit of AMPA type glutamate receptors for LTD induction (Leitges et al., 2004). AMPA receptors containing phosphorylated GluR2 are internalized from the plasma membrane and the postsynaptic response at PF-PC synapse is depressed (Matsuda et al., 1999, Xia et al., 2000, Chung et al., 2003). Some researchers have postulated that near simultaneous activation of CF and PF input pathways induce supralinear and persistent activation of cPKC (Hirono et al., 2001, Kuroda et al., 2001). However, precise regulation of PKCα activity during induction of LTD in a PC has not been demonstrated.
We wished to know whether simultaneous activation of the two input pathways evokes persistent activation of PKCα or not. To this end, we used PKCα and its mutants fused to a fluorescent protein GFP. The GFP-fused proteins were expressed in a cultured PC and their translocation in response to various stimuli was monitored. Our results indicate that translocation is primarily controlled by Ca++, enhanced by binding DAG, and that the expression of LTD does not require prolonged activation of PKCα, although its activity is necessary for the induction of LTD.
Section snippets
Monitoring translocation of PKCα
To examine the intracellular translocation of PKCα, PKCα or its mutants fused to GFP were constructed (Fig. 1A). The fusion proteins expressed in HEK 293T cells were estimated to be about 110 kDa by Western blotting as expected (data not shown), and uniformly distributed in the cytosol of HEK 293T cells and in cultured PCs as endogenous PKCα (Figs. 1B, C). We first examined the translocation of GFP-PKCα using a phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA, 5 μM) that induces strong
Regulation of PKCα in PCs
PKCα is involved in the induction of cerebellar PF-PC LTD and is proposed to integrate signals downstream of CF and PF inputs (Kuroda et al., 2001, Ito, 2002, Leitges et al., 2004). Although intensive studies on the activation and translocation mechanisms of PKC have been conducted in non-neuronal cells (Sakai et al., 1997, Oancea and Meyer, 1998, Bartlett et al., 2005), precise regulatory mechanism of PKCα in a neuron has not been uncovered. To investigate the regulation mechanism of PKCα with
Culture
Methods of preparing primary culture of cerebellar neurons were similar to previous studies (Kawaguchi and Hirano, 2006). Briefly, cerebella were dissected out from E20 Wistar rats and were incubated in Ca++ and Mg++-free Hank's balanced salt solution containing 0.1% trypsin and 0.05% DNase for 15 min at 37 °C. Neurons were dissociated by trituration and seeded on poly-d-lysine-coated coverslips in DMEM/F12-based medium containing 2% fetal bovine serum (Furuya et al., 1998). On the next day,
Acknowledgments
We thank S. Kawaguchi and Y. Tagawa for comments on the manuscript. We thank N. Saito for providing us PKCα and PKCγ cDNA. This work was supported by the grants-in-aid for scientific research in Japan.
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