Elsevier

Molecular Brain Research

Volume 64, Issue 2, 5 February 1999, Pages 222-235
Molecular Brain Research

Research report
Isoform-specific translocation of protein kinase C following glutamate administration in primary hippocampal neurons

https://doi.org/10.1016/S0169-328X(98)00324-6Get rights and content

Abstract

High concentrations of glutamate, the major excitatory neurotransmitter in the mammalian brain, lead to intracellular calcium overload resulting in excitotoxic damage and death of neurons. Since protein kinase C (PKC) is involved in neuronal degeneration resulting from cerebral ischemia and from glutamate excitotoxicity, we investigated the effect of glutamate on changes in the cellular distribution of various PKC isoforms in cultured hippocampal neurons in comparison with the effects elicited by the PKC activator phorbol ester. Out of the expressed PKC isoforms α,γ,ε,ζ and λ only the conventional isoforms PKC α and γ responded to glutamate. Using subcellular fractionation and Western blotting with isoform-specific antibodies and immunocytochemical localization with confocal laser scanning microscopy, we observed that phorbol ester and glutamate have different effects on PKC isoform redistribution: Whereas phorbol ester resulted in translocation of PKC α and PKC γ toward a membrane fraction, the glutamate-mediated rise in intracellular calcium concentration induced a translocation mainly toward a detergent-insoluble, cytoskeletal fraction. Immunocytochemical analysis revealed an isoform-specific translocation following glutamate treatment: PKC γ was translocated mainly to cytoplasmic, organelle-like structures, whereas PKC α redistributed to the plasma membrane and into the cell nucleus. The latter result is of special interest, as it indicates that nuclear PKC may play a role in processes of excitotoxic cell damage.

Introduction

The term protein kinase C (PKC) refers to a family of serine/threonine kinases comprising 11 isoforms in mammals. On the basis of structural differences and enzymatic properties, the PKC isoforms can be divided into (a) conventional or `classical' isoforms (PKC α, βI/II and γ) which require both Ca2+ and phospholipid for activation, (b) Ca2+-independent novel isoforms (PKC δ, ε, η, θ), and (c) atypical isoforms (PKC ζ, ι (λ)) which require neither Ca2+ nor phospholipid for activation and in contrast to the conventional and novel isoforms do not respond to phorbol ester (for review, see 28, 40). The various PKC isoforms are differentially distributed within a cell and, upon stimulation, undergo translocation toward different cellular compartments. Activation of the conventional isoforms leads not only to their translocation from cytosol to plasma membrane, but also to various intracellular membranes and to the cell nucleus 4, 10. Nuclear translocation of PKC may be particularly important because it may enable PKC to exert a direct role in nuclear signaling by phosphorylating nuclear substrates including DNA-modifying enzymes or transcription factors [10].

PKC is present in high concentrations in neuronal tissue [49]and has been implicated in a broad spectrum of neuronal functions. Substantial evidence has been accumulated for a role of PKC in learning and memory (for review, see: Refs. 42, 54) and in long term potentiation, a model for activity dependent synaptic plasticity 3, 6. Besides in normal neuronal functions PKC is critically involved in processes underlying disorders like Alzheimer's disease 14, 21, 35, 56and other disorders, e.g., resulting from glutamate excitotoxicity (see below).

Glutamate is the most important excitatory neurotransmitter in the mammalian brain 24, 44. The major consequence of glutamate binding to its receptors on postsynaptic neurons is an increase in the concentration of free cytoplasmic Ca2+ ([Ca2+]i). Overstimulation of glutamate receptors with subsequent rise of [Ca2+]i leads to alterations of neuronal homeostasis and have been proposed to play, at least, a contributory role in the etiopathogenesis of ischemia/hypoxia, epilepsy, and neurodegenerative diseases 12, 13, 15, 17, 34. PKC is involved in neuronal degeneration resulting from cerebral ischemia 11, 57and from glutamate excitotoxicity 19, 36, and phorbol ester-binding-assays showed that excitotoxic doses of glutamate lead to a redistribution of PKC 52, 53. Therefore it is of great importance to understand which PKC isoforms are involved and, since compartmentalization is an important means to define the function of activated PKCs, to which intracellular sites the various isoforms are directed.

In this study we investigated the effect of glutamate on changes in the cellular distribution of PKC isoforms in cultured hippocampal neurons in comparison with the effects elicited by phorbol ester. Utilizing biochemical approaches, we have found that glutamate leads to the translocation of classical PKC isoforms α and γ mainly towards a detergent-insoluble cellular fraction, whereas phorbol ester leads to a translocation mainly to the membrane fraction. Additionally, by investigating the subcellular localization of PKC isoforms by confocal laser scanning microscopy, we show that, following glutamate administration, PKC α and γ differ significantly in the pattern of intracellular translocation. PKCγ undergoes translocation mainly into cytoplasmic organelle-like structures, whereas PKCα translocates to the plasma membrane and also into the cell nucleus.

Section snippets

Materials

Neurobasal culture medium (NB) and B27 medium supplement (B27), were from Life Technologies (Gaithersburg, MD). Fetal calf serum (FCS) and horse serum were from HyClone Laboratories (Logan, UT). Poly-d-lysine (m.w. 30 000–70 000), cytosine β-d-arabinofuranoside (ara-c), and phorbol-12 myristate, 13 acetate (PMA) were from Sigma (St. Louis, MO). Fura-2 PE3 acetoxymethylester (fura-2 PE3/AM) was from TefLabs (Austin, TX). Pluronic F-127 was from Molecular Probes (Eugene, OR). Dizocilpine maleate

Expression of PKC isoforms

The first objective of this study was to determine which PKC isoforms were expressed in rat hippocampal neurons in culture. All determinations were performed on neurons 13–16 days in vitro (DIV). Cell homogenates were subjected to SDS-PAGE and immunoblotted with isoform specific antibodies. In a control preparation, rat brain homogenate, all isoform-specific antibodies showed a positive signal (Fig. 1). In the case of PKC θ immunoreactivity was rather low and for PKCμ several additional bands

Expression of PKC isoforms in cultured hippocampal neurons

Previous studies have shown that the expression of specific PKC isoforms in the hippocampus is under developmental regulation, both in vivo [45]and in vitro 45, 50. As expected, hippocampal neurons 13–18 days in vitro expressed the conventional isoforms PKC α and PKC γ. No immunoreactivity was, however, detected with the antibodies raised towards PKC β and PKC δ. The very low or lacking expression of PKC β is in agreement with the recent report of Roisin and Barbin [45]. The absence of PKC β is

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 515) and The National Institute on Aging (AG10916).

References (60)

  • G. Grynkiewicz et al.

    A new generation of Ca2+ indicators with greatly improved fluorescence properties

    J. Biol. Chem.

    (1985)
  • T. Jones et al.

    Cellular relocalization of protein kinase C-theta caused by staurosporine and some of its analogues

    Biochem. Pharmacol.

    (1997)
  • M.P. Mattson

    Evidence for the involvement of protein kinase C in neurodegenerative changes in cultured human cortical neurons

    Exp. Neurol.

    (1991)
  • A.C. Newton

    Protein kinase C: structure, function, and regulation

    J. Biol. Chem.

    (1995)
  • X. Nogues

    Protein kinase C, learning and memory: a circular determinism between physiology and behaviour

    Prog. Neuro-Psychopharmacol. Biol. Psychiatry

    (1997)
  • R. Raulli et al.

    N-Methyl-d-aspartate receptor-induced translocation of protein kinase C to the nucleus in rat cerebellar slices

    Neurochem. Int.

    (1994)
  • M.P. Roisin et al.

    Differential expression of PKC isoforms in hippocampal neuronal cultures: Modifications after basic FGF treatment

    Neurochem. Int.

    (1997)
  • T.B. Shea et al.

    Degradation of protein kinase Cα and its free catalytic subunit, protein kinase M, in intact human neuroblastoma cells and under cell-free conditions: evidence that PKM is degraded by calpain-mediated proteolysis at a faster rate than PKC

    FEBS Lett.

    (1994)
  • P. Tejero-Diez et al.

    Expression of protein kinase C isozymes in hippocampal neurons in culture

    FEBS Lett.

    (1995)
  • E.A. Van der Zee et al.

    Historical review of research on protein kinase C in learning and memory

    Prog. Neuro-Psychopharmacol. Biol. Psychiatry

    (1997)
  • C. Vorndran et al.

    New fluorescent calcium indicators designed for cytosolic retention or measuring calcium near membranes

    Biophys. J.

    (1995)
  • M. Wolf et al.

    The protein kinase inhibitor staurosporine, like phorbol esters, induces the association of protein kinase C with membranes

    Biochem. Biophys. Res. Commun.

    (1988)
  • R. Beckmann et al.

    Differential nuclear localization of protein kinase C isoforms in neuroblastoma×glioma hybrid cells

    Eur. J. Biochem.

    (1994)
  • T.V.P. Bliss et al.

    A synaptic model of memory: long-term potentiation in the hippocampus

    Nature

    (1993)
  • G.J. Brewer et al.

    Optimized survival of hippocampal neurons in B27-supplemented NeurobasalTM, a new serum-free medium combination

    J. Neurosci. Res.

    (1993)
  • K. Buchner

    Protein kinase C in the transduction of signals toward and within the cell nucleus

    Eur. J. Biochem.

    (1995)
  • M. Cardell et al.

    Time course of the translocation and inhibition of protein kinase C during complete cerebral ischemia in the rat

    J. Neurochem.

    (1993)
  • D.W. Choi

    Cerebral hypoxia: some new approaches and unanswered questions

    J. Neurosci.

    (1990)
  • J.T. Coyle et al.

    Oxidative stress, glutamate, and neurodegenerative disorders

    Science

    (1993)
  • M. Didier et al.

    DNA strand breaks induced by sustained glutamate excitotoxicity in primary neuronal cultures

    J. Neurosci.

    (1996)
  • Cited by (26)

    • Protein kinase C-β mediates neuronal activation of Na<sup>+</sup>/H<sup>+</sup> exchanger-1 during glutamate excitotoxicity

      2014, Cellular Signalling
      Citation Excerpt :

      Furthermore, it has been suggested that various protein kinases can directly phosphorylate NHE-1 at its amino acid serine residues in the distal C terminus phosphorylation domain and activate NHE-1 in ischemic neurons and myocardium [6,15]. Members of the PKC family are important regulators of neuronal cellular mechanism, and have been implicated in cerebral ischemia and glutamate excitotoxicity [16,17]. Neuronal cell stimulation by glutamate receptor activation leads to an increase in intracellular Ca2 +, and consequently could activate Ca2 +-dependent conventional PKC isoforms (α, βI, βII, and γ) [18].

    • Ethanol regulation of γ-aminobutyric acid<inf>A</inf> receptors: Genomic and nongenomic mechanisms

      2004, Pharmacology and Therapeutics
      Citation Excerpt :

      The effect on GABAA receptors following NMDA receptor activation is mediated by intracellular Ca2+, since the Ca2+ chelator (BAPTA) blocks the down-regulation of GABAA receptors following NMDA receptor activation (Robello et al., 1997). In addition, the application of glutamate in hippocampal cultures leads to translocation of PKCα and γ to the plasma membrane and cytoplasmic organelles, respectively, while PKCε localization remains unaltered (Etoh et al., 1991; Buchner et al., 1999). Therefore, interactions of NMDA and GABAA receptors via intracellular signaling pathways may play a vital role in GABAA receptor adaptation following chronic ethanol administration.

    View all citing articles on Scopus
    1

    Present address: Day Neuromuscular Laboratory, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129; USA.

    2

    Present address: Nathan Kline Institute for Psychiatric Research, New York University Medical Center, 140 Old Orangeburg Rd., Orangeburg, NY 10962; USA.

    View full text