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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.35 no.2 Santiago  2002

http://dx.doi.org/10.4067/S0716-97602002000200014 

Biol Res 35: 223-229, 2002

Phospholipid synthesis, diacylglycerol
compartmentation, and apoptosis

MARCIA M. WRIGHT AND CHRISTOPHER R. MCMASTER

Departments of Pediatrics and Biochemistry & Molecular Biology,
Atlantic Research Centre, IWK Health Centre, Dalhousie University,
Halifax, Nova Scotia B3H 4H7, Canada

ABSTRACT

Apoptosis is a means by which organisms dispose of unwanted cells without inducing an inflammatory response. Alterations in apoptosis is a common process by which cells become cancerous. Paradoxically, many cancer chemotherapeutics preferentially kill cancer cells by inducing apoptosis. Diacylglycerol is a lipid second messenger that regulates cell growth and apoptosis and is produced during signal transduction by hydrolysis of membrane phospholipids. Protein kinase Cs are a family of diacylglycerol responsive enzymes that are recruited to cellular membranes as a consequence of diacylglycerol production where they phosphorylate specific target proteins responsible for regulating cell growth. In this review, we will first summarize our current understanding of the role of specific proteins kinase C isoforms in the induction of cell growth/apoptosis. Subsequently, we will discuss how insights gained in lipid-mediated regulation of protein kinase Cs promotes our understanding of the role specific family members play in regulating cell growth. Finally, other diacylglycerol binding proteins involved in regulating apoptosis will be discussed.

Key terms: apoptosis, diacylglycerol, farnesol, phorbol ester, protein kinase C

INTRODUCTION

Apoptosis, or programmed cell death, is a mechanism that permits organisms to eliminate their unwanted cells without inducing an inflammatory response. Apoptosis occurs naturally during development, and is also employed for removal of damaged cells as well as normal cells that have reached the end of their lifespan. Deregulation of apoptosis is an important pathway by which cells become malignant. Paradoxically, many commonly employed chemotherapeutic drugs function by preferentially inducing apoptosis in cancer cells. Diacylglycerol (DAG) is a lipid second messenger that acts as a signaling molecule by binding directly to many different proteins. Depending on the target, DAG-induced cellular responses can be proliferative or apoptotic. DAG is produced in large quantities during lipid biosynthesis as well as by lipid turnover

during signaling. Agonist stimulated release of DAG from phospholipid stores is believed to represent the main mechanism by which DAG acts a lipid second messenger, while biosynthetic DAG does not appear to be directly involved in regulating cell growth or apoptosis. Protein kinase Cs (PKCs) are an important class of DAG binding proteins. DAG promotes membrane recruitment and activation of different PKCs family members and, depending on the isoform involved, can trigger proliferative or apoptotic signals.

DAG-dependent activation of PKCs

PKCs are a family of serine/threonine protein kinases that are differentially regulated by calcium and lipids. PKCs were first described by Nishizuka and his co-workers over 20 years ago (Takai et al., 1979) and to date at least 11 isoenzymes have been described. These are subdivided into the three main groups termed classical (cPKCs), novel (nPKCs), and atypical (aPKCs) isoforms, depending on their mode of activation and the presence of specific regulatory domain elements. The classical isoenzymes (a, ßI, ßII, and g) require DAG, calcium, and the acidic phospholipid, phosphatidylserine (PS), for maximal activity in vitro. The novel isoenzymes (d, e, h and q) are also activated by DAG and the presence of PS, but are calcium independent. The atypical isoenzymes (z, and i/l (human/mouse)) are activated by phospholipids alone in a DAG and calcium independent manner.

All PKC isoenzymes contain an amino-terminal regulatory region with different motifs for lipid and calcium binding that is connected to the carboxy- terminal ATP binding kinase domain through a hinge region (Figure 1). Activation via the regulatory region often involves translocation from the cytoplasm to cellular membranes in response to DAG production. Two tandem DAG responsive elements, refered to as cysteine rich motifs, are located within the regulatory domain C1 region. Each spans roughly 50 amino acids in length and contains 15 absolutely conserved residues (6 of them cysteines), several essential hydrophobic residues, and distinct protein folds including a loop between two ß-strands (Quest and Bell, 1994; Kazanietz et al., 1995; Quest et al., 1997). For a more detailed discussion of PKC regulation in general, the interested reader is referred to available reviews (Quest, 1996, 2000; Ron and Kazanietz, 1999).


Figure 1. Protein Kinase C Domains.

Protein Kinase Cs in apoptosis

Differences in regulation are but one cause of diversity between PKC isoforms. Also significant differences exist between PKCs in their expression pattern. PKCs a, ßI, ßII, d, e, and z are ubiquitously expressed, while PKCg exists predominantly in the brain. PKCh is primarily expressed in the epithelial tissues of the skin and lung, and PKCq is mainly expressed in skeletal muscle. Many PKCs have been shown to be involved, in either a positive or negative way, in the control of mitotic signaling which involves on one hand, stimulation of DNA synthesis and cell growth and on the other, inhibition of apoptosis. Therefore, to understand the role of each PKC isoform it is necessary to consider effects on both aspects of mitogenic signaling. The discussion of PKCs in apoptosis will initially centre on the two ubiquitous isoforms a and d. PKCa is primarily reported as an anti-apoptotic PKC while PKCd is mostly considered to be pro-apoptotic (see for instance, Mandil et al., 2001). Roles for other PKC isoforms in the regulation of cell growth through control of apoptosis have also recently emerged and are briefly discussed subsequently.

PKCa is chiefly thought of as an anti-apoptotic, or a proliferative PKC. Overexpression of PKCa prevents, or attenuates apoptosis in many different cell types, while down regulation of PKCa potentiates apoptosis (Le et al., 2001). In apoptotic markers (Jao et al., 2001). One mechanism by which PKCa is thought to prevent apoptosis is by regulating Bcl-2. Activated PKCa colocalizes with Bcl-2 at the mitochondrial membrane and has been shown to phosphorylate Bcl-2 on serine 70 (Ruvolo et al., 1998), an event required for Bcl-2 to prevent apoptosis (Ito et al., 1997).

PKCd, an important PKC isoform involved in pro-apoptotic signalling is cleaved to a constitutively active 40kDa catalytic fragment by caspase 3 in many cell types under a variety of conditions, including senescence, irradiation, and the presence of DNA-damaging agents such as arabinofuranosylcytosine (Emoto et al., 1996). This cleavage event involves loss of the N-terminal regulatory domain (Emoto et al., 1995). Ensuing apoptosis can be prevented by Bcl-2, Bcl-xL, and by caspase inhibitors (Mizuno et al., 1997). Also, over-expression of the catalytically active PKCd fragment causes apoptosis in a variety of cell types (Ghayur et al., 1996). PKCd has been shown to phosphorylate a number of potentially relevant substrates including myristoylated alanine-rich C kinase substrate (MARCKS), ribosomal S6 protein, and DNA-dependent protein kinase (Emoto et al., 1996; Weaver, 1995), whereby the latter appears to be important for PKCd dependent induction of apoptosis. Phosphorylation of DNA-dependent protein kinase by PKCd inactivates the enzyme thereby reducing its ability to repair DNA damage. Also, cells lacking DNA-dependent protein kinase are resistant to apoptosis caused by expression of the PKCd catalytic fragment (Weaver, 1995).

In addition to its role in the nucleus, PKCd has also been implicated in the regulation of cytochrome c release from mitochondria (Majumder et al., 2000) since PKCd translocation to mitochondria was required for apoptosis in keratinocytes. PKCd also phosphorylates a scramblase and enhances its activity, resulting in increased PS exposure on the cell surface (Frasch et al., 2000). PS binds to a receptor on the macrophage cell surface and represents one of the main signals responsible for triggering engulfment of apoptotic cells (Fadok et al., 2000). Each of these examples underscores the notion that uncontrolled activation of PKCd is pro-apoptotic. PKCd knock-out mice develop normally but contain circulating autoreactive antibodies and show evidence of B-lymphocyte expansion (Miyamoto et al., 2002). Thus, it appears that PKCd is required for negative regulation of B-cell proliferation and specially in development of B-cell tolerance. These observations correlate well with the tissue distribution, as this isoform is most abundantly present in lymphoid organs, the cerebrum and intestine.

Phorbol esters, metabolically stable pharmacological DAG analogs, induce macrophage differentiation similar to that seen in macrophages during an immune response. Once a macrophage has completed its immune response function, it is eliminated via apoptosis and replaced by new macrophages. It appears that PKCß is necessary for macrophage elimination by apoptosis. In the HL-60 human myeloid cell line, PKCß activation precedes PMA-induced apoptosis. The human myeloid cell line HL-525 is devoid of endogenous PKCß and resistant to PMA-induced apoptosis. Reexpression of PKCß in HL-525 cells restores their susceptibility to PMA-induced apoptosis (Laouar et al., 1999), implying that PKCß is pro-apoptotic and may be required to induce macrophage apoptosis at the end of their lifespan.

PKCe is specifically activated by several mitotic signaling molecules including platelet- derived growth factor (Moriya et al., 1996), colony stimulating factor (Cai et al., 1997), and erythropoietin (Li et al., 1996). PKCe overexpression suppresses apoptosis by increasing expression of Bcl-2 (Gubiana et al., 1998) and this is inhibited in various forms of apoptosis (Sawai et al., 1997). Based on these observations activation of PKCe is generally considered to be anti-apoptotic.

Another DAG responsive regulator of apoptosis is PKCq. PKCq is cleaved by caspase-3 (Datta et al., 1997) in response to several inducers of apoptosis. This causes proteosome-mediated degradation of the enzyme and these events precede apoptosis (Villalba et al., 2001). PKCq cleavage and subsequent apoptosis can be prevented by overexpression of Bcl-xL. PKCq phosphorylates and inhibits the pro-apoptotic BAD at serine 112. Thus, degradation of PKCq would be pro-apoptotic while activation of PKCq is predicted to be anti-apoptotic.

Another anti-apoptotic PKC is PKCz. PKCz has been reported to activate the MAP kinase pathway resulting in activation of nuclear factor kB while simultaneously inactivating the inhibitor of kB, thereby promoting mitogenesis (Musashi et al., 2000). PKCz basal activity is increased by Bcl-2 and is unaffected by caspase inhibitors (Berra et al., 1997). However, PKCz has subsequently been shown to undergo caspase dependent cleavage in the hinge region which leads to its inactivation (Frutos et al., 1999).

Farnesol Induced Apoptosis and its Reversal by Diacylglycerol

Farnesol is a naturally occurring metabolite that is produced from farnesol pyrophosphate through a shunt off the isoprenoid/cholesterol biosynthetic pathway (Edwards and Ericsson, 1999). The production of farnesol through this shunt is one of many routes used to control cellular cholesterol levels. As cholesterol synthesis is upregulated, intracellular farnesol production increases for eventual secretion from cells and elimination from the body. It has been observed in cell culture that farnesol preferentially caused apoptosis in cancer cell lines versus non-malignant cells (Adany et al., 1994). In addition, farnesol also caused apoptosis in primary blasts obtained from patients with acute myeloid leukaemia but did not cause apoptosis in primary monocytes, or quiescent or proliferating T lymphocytes (Rioja et al., 2000). The mechanism by which farnesol preferentially causes apoptosis in cancer cells is not known. Identifying the mechanism of action of farnesol should pinpoint a target molecule to which small molecule drugs could be directed for use as potential chemotherapeutic agents for treatment of cancer.

A mechanism by which farnesol induces apoptosis was proposed in the late 1990's. Accordingly, farnesol was thought to directly compete with DAG for binding to the enzyme cholinephosphotransferase (CPT) and thereby inhibit its function (Anthony et al., 1999; Miquel et al., 1998). CPT transfers phosphocholine from CDP-choline onto DAG to synthesize phosphatidylcholine the major phospholipid present in eukaryotic cells. Genetic inactivation of phosphatidylcholine biosynthesis results in apoptotic cell death. Interestingly, the addition of exogenous DAG or phosphatidylcholine (but not other lipids) prevented farnesol-induced apoptosis, consistent with the notion that inhibition of phosphatidylcholine synthesis by farnesol represents the mechanism underlying farnesol-induced apoptosis.

As we were the first laboratory to isolate the human (or any mammalian) CPT gene/cDNA this enabled us to test whether farnesol directly affects the CPT-dependent step in phosphatidylcholine synthesis and thereby promote apoptosis (Henneberry and McMaster, 1999; Henneberry et al., 2000; Henneberry et al., 2001). To that end, we established a Chinese hamster ovary cell line in which CPT over-expression was inducible and found that this prevented the block in phosphatidylcholine biosynthesis associated with exposure to farnesol. However, over-expression of CPT did not prevent farnesol induced apoptosis. This implied that the inhibition of phosphatidylcholine biosynthesis by farnesol was not the primary mechanism by which farnesol induced apoptosis (Wright et al., 2001). In addition, exogenous administration of DAG, which prevented farnesol induced apoptosis, did not relieve the farnesol-induced block in phosphatidylcholine synthesis (Wright et al., 2001). Thus, we have shown through two different sets of experiments that farnesol does not cause apoptosis through the inhibition of phosphatidylcholine synthesis. However, as farnesol induced apoptosis can be prevented by exogenous addition of DAG or phosphatidylcholine, these two lipids appear to be intimately involved in the mechanism by which farnesol causes apoptosis. As phosphatidylcholine can be readily metabolized to DAG through phospholipase D followed by phosphatidic acid phosphatase, we believe that farnesol either activates a DAG-mediated process that results in the induction of apoptosis, or inhibits a DAG dependent process that is required for cell proliferation.

We have observed that phorbol esters prevented farnesol-induced apoptosis, while broad spectrum PKC inhibitors potentiated farnesol induced apoptosis. This implies a role for PKCs and suggests that the mechanism by which farnesol causes apoptosis is likely to involve inhibition of a proliferative PKC isoform (Figure 2). We are in the process of determining which PKC isoform is affected by farnesol and whether PKC inhibition by farnesol is direct or indirect. Several lines of experimentation are underway. These include examination of PKC translocation to membrane fractions subsequent to farnesol and/or DAG treatment to address whether alterations in subcellular location occur due to farnesol treatment. Also, PKC activity following treatment with farnesol and/or DAG will be evaluated. In addition, the construction of cell lines expressing dominant negative and constitutively active forms of PKC will help to determine their ability to modulate farnesol induced apoptosis. Finally, in vitro experiments using purified PKCs will be employed to determine whether farnesol alters the ability of PKCs to associate with membranes and be activated in this manner.


Figure 2. Current Model for Farnesol Induced Apoptosis.

Other Diacylglycerol Receptors and Apoptosis

As previously discussed, DAG interacts with cysteine-rich motifs present in the regulatory domain of PKCs. While PKCs are the best characterized DAG binding proteins this motif has been found more recently in several novel "non-kinase" proteins, collectively termed phorbol ester receptors (see Quest, 1996, 2000; Ron and Kazanietz, 1999). Phorbol esters bind to cysteine-rich domains and activate proteins by promoting translocation to specific cellular membranes. Translocation of most phorbol ester receptors has been shown to require functional cysteine-rich motifs as determined by inactivating point mutations or deletion mutagenesis. The first non-PKC, phorbol ester binding protein described was n-chimaerin (Hall et al., 1990), a GTPase activating protein (GAP) for the small G protein Rac which regulates the Erk signalling pathway. The Ras guanine exchange factors (RasGEF) involved in activation of Ras and ERK signalling cascade also contains a cysteine rich motif. RasGRP binds phorbol esters with high affinity but with lipid cofactor requirements distinct from PKCs. The transforming potential of RasGRP is dependent on the presence of a functional cysteine-rich domain (Tognon et al., 1998) and RasGRP knock-out mice generally display defective proliferative responses (Dower et al., 2000).

Munc13 is a scaffolding protein, primarily involved in the exocytic events required for neurotransmitter release, that preferentially translocates to the Golgi in response to phorbol esters. Munc13 contains a cysteine-rich motif and promotes apoptosis in response to phorbol esters (Song et al., 1999).

This burgeoning field of non-kinase phorbol ester receptors opens up a wealth of possible DAG signalling cascades involved in controlling a wide variety of responses ranging from proliferation (RasGEF) to apoptosis (Munc13).

SUMMARY

The roles of specific PKC isoforms in the regulation of cell growth and apoptosis are currently being delineated concomitantly with those of other DAG binding proteins. As the endogenous lipids themselves cannot be visualized, we need to characterize the recruitment to specific subcellular locations and activation of DAG producing phospholipases. Activation of PKCs at specific membranes needs to be temporally correlated with an increase in DAG at the same site. Understanding the molecular mechanisms by which specific PKCs regulate apoptosis will require more precise insights to the effects alterations in lipid-dependent recruitment and regulation have on the downstream targets of PKCs. Once this information is available, specific PKCs may be targeted for the development of chemotherapeutic anticancer drugs.

ACKNOWLEDGEMENTS

This research is supported by operating grants from the Canadian Institutes of Health Research, the National Sciences and Engineering Research Council, and GlaxoSmithKline to CRM. CRM is a senior clinical research scholar of the IWK Health Centre. MMW holds a PhD studentship from CancerCare Nova Scotia.

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Corresponding author: Christopher R. McMaster, 5849 University Avenue, Room C-302, Atlantic Research Centre, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada. Telephone: (902) 494-7066; Fax: (902) 494-1394; e-mail: cmcmaste@is.dal.ca.

Received: May 22, 2002. In revised form: June 26, 2002. Accepted: July 15, 2002

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