An Inhibitory Fragment Derived from Protein Kinase Cε Prevents Enhancement of Nerve Growth Factor Responses by Ethanol and Phorbol Esters

Abstract

We have studied nerve growth factor (NGF)-induced differentiation of PC12 cells to identify PKC isozymes important for neuronal differentiation. Previous work showed that tumor-promoting phorbol esters and ethanol enhance NGF-induced mitogen-activated protein (MAP) kinase activation and neurite outgrowth by a PKC-dependent mechanism. Ethanol also increases expression of PKC␦ and PKC⑀, suggesting that one these isozymes regulates responses to NGF. To examine this possibility, we established PC12 cell lines that express a fragment encoding the first variable domain of PKC⑀ (amino acids 2-144), which acts as an isozyme-specific inhibitor of PKC⑀ in cardiac myocytes. Phorbol esterstimulated translocation of PKC⑀ was markedly reduced in these PC12 cell lines. In addition, phorbol ester and ethanol did not enhance NGF-induced MAP kinase activation or neurite outgrowth in these cells. In contrast, phorbol ester and ethanol increased neurite outgrowth and MAP kinase phosphorylation in cells expressing a fragment derived from the first variable domain of PKC␦. These results demonstrate that PKC⑀ mediates enhancement of NGF-induced signaling and neurite outgrowth by phorbol esters and ethanol in PC12 cells. Protein kinase C (PKC)1 is a multigene family of phospholipiddependent, serine-threonine kinases that plays a central role in cell growth and differentiation. Molecular cloning studies have identified 10 isozymes encoded by 9 different mRNAs (1, 2). Based on sequence homology and biochemical properties, the PKC gene family has been divided into three groups: "conventional" PKCs (␣, ␤I, ␤II, and ␥) regulated by calcium and diacylglycerols or phorbol esters; "novel" PKCs (⑀, ␦, , and ), which are calcium-independent but diacylglycerol-and phorbol ester-sensitive; and "atypical" PKCs (, and /), which are insensitive to calcium, diacylglycerol, and PMA. In addition, two related phospholipid-dependent kinases, PKC and protein kinase D, share sequence homology in their regulatory domains to novel PKCs and may constitute a new subgroup (3, 4).Several studies with tumor-promoting phorbol esters suggest that PKC modulates neural differentiation. Phorbol esters induce neural tissue from ectoderm in Xenopus embryos (5) and elicit neurite outgrowth from chick sensory ganglia (6, 7), chick ciliary ganglion neurons (8), several human neuroblastoma cell lines (9, 10), and rat PC12 cells (11, 12). Studies using purified isozymes, kinase-defective mutants, and transgenic or mutant cell lines have implicated PKC␣, -␤, -␦, -⑀, and -in the differentiation of nonneural cells (13-17). Overexpression of PKC␣ or -␤ in Xenopus embryos enhances neural induction (18), but little else is known about the identity of specific PKC isozymes that regulate neural differentiation.Recent evidence suggests that PKC⑀ plays a role in neural differentiation and plasticity. PKC⑀ is expressed predominantly in the nervous system and is particularly abundant in the hippocampus, olfactory tubercle, and layers I and II of cerebral cortex (19). Within immunoreactiv...

The mitogen-activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic

“…In contrast to NGF treatment, epidermal growth factor (EGF) treatment of PC12 cells induced an acute phasic activation of p42 MAPkinase which returned to baseline within 30 min ( Figure 1B). These data are in agreement with data of several other laboratories [16,21,22,34,35]. Bailie et al reported that primary cultures of hepatocytes possess binding sites for NGF, and we investigated whether NGF had a similar capability to modulate the MAP kinase cascade in these cells [26].…”

“…Several studies have demonstrated that chronic elevation of plasma ethanol levels into the " 50 mM range inhibits hepatocyte proliferation\liver regeneration in i o [23,24]. Furthermore, ethanol treatment of PC12 cells has been shown to inhibit DNA synthesis and to potentiate the ability of NGF to increase neurite outgrowth, which were shown to be dependent upon signalling via the MAP kinase cascade [21,22]. Other workers examining NGF function have shown that neurite outgrowth in PC12 cells caused by treatment with this factor is dependent upon signalling via the MAP kinase cascade, which we have confirmed (data not shown) [36].…”

“…In PC12 cells, NGF has been demonstrated to chronically activate the MAP kinase cascade via the B-Raf protein kinase and to induce morphological neuronal differen-tiation and cell cycle exit [17][18][19][20]. Ethanol treatment of PC12 cells has been shown capable of inducing morphological neuronal differentiation and cell cycle exit, and data by Roivainen et al demonstrated that ethanol treatment of PC12 cells enhanced neuronal differentiation via activation of the MAP kinase cascade [21,22]. Treatment of hepatocytes either in itro or in i o with low concentrations of ethanol also inhibits DNA synthesis and liver regeneration, although the mechanism(s) by which this occurs are unknown [23,24].…”

Protein kinase Cε makes the life and death decision

“…It has been reported that overexpression of PKCε enhanced nerve growth factor (NGF)-induced phosphorylation of ERK in PC12 pheochromocytoma cells and increased NGF-induced neurite outgrowth [80]. Ceramide inhibited growth of human embryonic kidney (HEK) 293 cells by attenuating ERK activity in a PKCε-dependent manner [81].…”

Protein Kinase Cε Actin-binding Site Is Important for Neurite Outgrowth during Neuronal Differentiation

“…Based on experiments with cell lines of various origin, both PKC␦ (O'Driscoll et al, 1995;Corbit et al, 1999) and PKC⑀ (Hundle et al, 1995;Fagerström et al, 1996;Hundle et al, 1997;Brodie et al, 1999;Zeidman et al, 1999) have been proposed to be the PKC isoform that positively regulates neurite outgrowth. This could suggest that these isoforms have redundant functions, but in several other cell systems PKC␦ and ⑀ have unique and sometimes opposite effects (Mischak et al, 1993;Lehel et al, 1994;Fleming et al, 1998).…”