Evidence that the Modulation of Membrane‐Associated Protein Kinase C Activity by an Endogenous Inhibitor Plays a Role in N1E‐115 Murine Neuroblastoma Cell Differentiation

Abstract

Murine neuroblastoma cells, N1E‐115, were induced to differentiate into neuron‐like cells by serum deprivation for 18 h. As previous studies have shown that the suppression of protein kinase C (PKC) activity by selective inhibitors or neutralizing antibodies induces neuroblastoma cells to differentiate, we tested the hypothesis that serum deprivation may cause a rapid loss in membrane PKC activity that occurs well before the morphological changes that are characteristic of cell differentiation. A significant reduction in particulate (membrane) PKC activity was indeed observed within 3 h of serum withdrawal when enzyme activity was measured in intact native membranes by the recently described in vitro “direct” assay. This rapid reduction in enzyme activity was confirmed by the decreased phosphorylation of the MARCKS protein, an endogenous PKC‐selective substrate, in intact cells. The decrease in membrane PKC activity occurred without any loss in the amount of membrane‐associated enzyme, suggesting that some factor(s) resident in neuroblastoma membranes was suppressing PKC activity. Indeed, results indicate the presence of an endogenous inhibitor of PKC tightly associated with neuroblastoma membranes. This inhibitory activity increased in the membranes of cells subjected to serum deprivation, raising the possibility that it was likely responsible for the decline in membrane PKC activity in differentiating N1E‐115 cells. Preliminary characterization indicated that the inhibitory activity is a protein and is localized mainly in the membrane fraction. Thus, these results demonstrate directly that endogenous inhibitor can regulate membrane‐associated PKC activity in cells and thereby modulate PKC‐related neuronal functions.

Evidence that the Early Loss of Membrane Protein Kinase C Is a Necessary Step in the Excitatory Amino Acid‐Induced Death of Primary Cortical Neurons

“…Current studies are addressing the possibility that reversible inactivation of PKC following glutamate receptor hyperactivation in cells 16 DIV results from the suppression of enzyme activity by a membrane-bound PKC inhibitor that may regulate PKC or perhaps specific PKC isoforms, located proximally to ionotropic glutamate receptors. We have recently demonstrated that such a membrane-bound protein inhibitor of PKC exists in NIE-l IS neuroblastoma cells and that this protein appears responsib!e for the loss of PKC activity needed for the differentiation of these cells into neurons under serum-free conditions (Chakravarthy et al, 1995).…”

Alzheimer’s Disease and Its Possible Evolutionary Origin: Hypothesis

“…They have shown that this peptide targets AβO aggregates when microinjected into the hippocampi of living double-transgenic AD mice harboring PSEN1dE9 and APP SWE transgenes [ 73 ] and also ex vivo in brain sections of both transgenic mice and postmortem human AD patients. Interestingly, ABP, which can also inhibit membrane-associated PKC activity) [ 75 , 76 ] is actually the 1276–1314 region of the human PCM1 described above.…”

“…Li et al [71] have also shown that an antibody that selectively targets the N-tails, but not one that targets the C-termini, of soluble AβOs rescues the ability of the hippocampal CA1 cells to establish LTP in AD mice. As discussed below, there may be other cellular proteins such as pericentriolar material-1 (PCM1) protein and myristoylated alanine-rich C kinase substrate (MARCKS) protein that can potentially bind AβOs through their cationic patches, 1276 KTFKTRKASAQASLASKDKTPKSKSKKRNSTQLKSRVKNI 1314 and 152 KKKKKRFSFKKSFKLSGFSFK 172 , respectively, and mediate their effects [72][73][74][75][76].…”

Characterization of Pituitary Adenylate Cyclase‐Activating Polypeptide 38 (PACAP38)‐, PACAP27‐, and Vasoactive Intestinal Peptide‐Stimulated Responses in N1E‐115 Neuroblastoma Cells

“…The relatively low potency of VIP in stimulating cAMP accumulation suggests that VIP may be interacting with the type I PACAP receptors. Because NIE-l 15 cells have frequently been used for the study of molecular mechanisms of neuronal differentiation (Richelson, 1973;de Laat and van der Saag, 1982;Chakravarthy et al, 1995: Kranenburg et al, 1995, to understand the trophic action of PACAP, it is important to establish the effect of PACAP on neurite outgrowth and the signaling mechanisms activated by PACAP/VIP in these cells. Therel'ore, the objectives of this study were (a) to characterize the signaling mechanisms activated by the three peptides, PACAP38, PACAP27, and VIP.…”

“…Furthermore, two other signaling mechanisms of importance to neuronal differentiation, elevation of [Ca2 jãnd PKC (Reboulleau, 1986;Herrick-Davis et al, 1991), interact with the PACAP-stimulated cAMP response. Because NI E-115 cells are a useful model for the study of molecular mechanisms underlying neuronal differentiation (Richelson, 1973;Chakravarthy et al, 1995;Kranenburg et al, 1995), it would be important to identify the downstream mechanisms through which PACAP induces neurite outgrowth in NIE-1l5 cells.…”