Research reportIsoform-specific translocation of protein kinase C following glutamate administration in primary hippocampal neurons
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).
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- 1
Present address: Day Neuromuscular Laboratory, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129; USA.
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Present address: Nathan Kline Institute for Psychiatric Research, New York University Medical Center, 140 Old Orangeburg Rd., Orangeburg, NY 10962; USA.