Growth of melanocytic cells is associated with down-regulation of protein kinase C α,δ and ε isoforms: Possible role of diacylglycerol

Protein kinase C (PKC) down-regulation has been shown to correlate with the growth of murine melanocytic cells in culture (Brooks, G., Wilson, R. E., Dooley, T. P., Goss, M. W., and Hart, I. R. (1991) Cancer Res. 51, 3281-3288). We now show that PKC alpha, delta, epsilon, and zeta isoforms are present at the protein level in quiescent, non-transformed Mel-ab melanocytes, maintained in the absence of phorbol ester. Proliferation of Mel-ab cells, achieved by incubation in the continual presence of phorbol 12,13-dibutyrate, was associated with a down-regulation of the PKC alpha, delta, and epsilon isozymes. Examination of two transformed syngeneic lines (the B16 murine melanoma and the long terminal repeat Ras.2 line), that grew in the absence of exogenous phorbol esters, showed that PKC alpha protein levels were either partially down-regulated or unaffected, the PKC delta and epsilon isoforms were down-regulated completely, and the levels of PKC zeta protein remained unaltered relative to quiescent Mel-ab cells. Basal levels of total diacylglycerol were elevated 5-fold in B16 melanoma cells compared with levels found in quiescent or proliferating Mel-ab melanocytes and appear to arise largely from the breakdown of phosphatidylinositol phospholipids accompanied by a significant rise in phospholipase C activity. Hourly treatments of quiescent Mel-ab melanocytes with the synthetic diacylglycerol analogue, 1,2-dioctanoyl-sn-glycerol, for 24 h, resulted in an induction of DNA synthesis which was associated with a significant down-regulation of PKC levels mediated largely via post-translational rather than transcriptional mechanisms. These results show for the first time that specific isoforms of PKC are down-regulated at the protein level during proliferation of murine melanocytic cells and suggest that the constitutive down-regulation of PKC in transformed melanoma cells may arise as a consequence of elevated endogenous phosphatidylinositol-derived diacylglycerol levels.

Protein kinase C down-regulation, and not transient activation, correlates with melanocyte growth

The nontumorigenic, immortal line of murine melanocytes, Mel-ab, requires the continual presence of biologically active phorbol esters for growth (R. E. Wilson et al., Cancer Res., 49: 711–716, 1989). Comparable treatments of B16 murine melanoma cells result in partial inhibition of cell proliferation. The role of protein kinase C (PKC) in the modulation of growth of cells from these two melanocytic cell lines has been investigated. Significant levels of PKC were present in quiescent Mel-ab cells as determined by Western blotting, whereas no immunoreactive protein was detected in cell extracts from either proliferating Mel-ab or B16.F1 cells. Phosphorylation of a Mr 80,000 protein, which by one- and two-dimensional gel analysis comigrated with the known Mr 80,000 protein substrate of PKC in fibroblasts, was induced in 12-O-tetradecanoylphorbol-13-acetate-stimulated quiescent Mel-ab cells but not in proliferating Mel-ab cells or B16.F1 melanoma cells. Direct measurement of PKC activity in these cells demonstrated a 10-fold greater level of activity in quiescent Mel-ab cells (262 ± 50 pmol/min/mg SD) compared with growing cells (22.8 ± 11.8 pmol/min/mg SD). An intermediate level of activity was detected in proliferating B16.F1 melanoma cells (148.5 ± 20.4 pmol/min/mg SD). The subcellular distribution of PKC was dependent upon the growth state of the cells such that quiescent Mel-ab cells displayed a higher level of activity in the cytosol, whereas growing Melab cells displayed greater activity in the particulate fraction. Like many other transformed lines, B16.F1 melanoma cells constitutively expressed the majority of enzyme activity in the particulate fraction. Measurement of [3H]phorbol ester binding in intact cells paralleled the PKC activation data such that quiescent Mel-ab cells displayed binding of 1612 ± 147 cpm/106 cells, whereas proliferating Mel-ab and B16.F1 melanoma cells displayed binding of 652 ± 28 and 947 ± 81 cpm/106 cells, respectively. Membrane-permeant diacylglycerol analogues, which activated but did not down-regulate PKC, were devoid of growth-stimulating effects on melanocytes, even in the presence of the specific diacylglycerol kinase inhibitor, R59022. Together, these data show that PKC down-regulation, and not activation, correlates with the growth of melanocytes in culture.

Both tumour-promoting and non-promoting phorbol esters inhibit 125I-EGF binding and stimulate the phosphorylation of a 80kd protein kinase C substrate protein in intact Swiss 3T3 cells

Sapintoxin A (SAP A) and 12-deoxyphorbol 13-phenylacetate (DOPP), are two biologically active but non-turnour-promoting phorbol esters that potently bind to and activate the phorbol ester receptor, protein kinase C (PKC). SAP A and DOPP cause a dose-dependent increase in the phosphorylation of an 80 kd (80K) substrate protein for PKC in Swiss 3T3 cells. A similar dose—response effect was seen with sapintoxin D (SAP D), the stage 2 promoting analogue of 12-O-tetradecanoylphorbol-13-acetate and the complete promoter phorbol 12,13-dibutyrate (PDB). The doses resulting in a half maximal phosphorylation of this protein (Ka were 20 nM (SAP A), 45 nM (DOPP), 23 nM (SAP D) and 37 nM (PDB). Both non-promoting and phorbol esters induced a dose-dependent inhibition of [125I]epidermal growth factor (EGF) binding to its receptor in Swiss 3T3 cells. The doses required for 50% inhibition of binding (Ki) were: 8 nM (SAP A), 16 nM (DOPP), 14 nM (SAP D) and 17 nM (PDB). The results clearly demonstrate that induction of phosphorylation of the Pu 80K phosphoprotein and inhibition of [125I]EGF binding in Swiss 3T3 cells following exposure to phorbol esters is independent of the tumour-promoting activity of these compounds. The fact that SAP A, DOPP, SAP D and PDB are mitogenic for a variety of cell types and that exposure to these compounds leads to 80K phosphorylation and inhibition of [125I]EGF binding, suggests that these early biological events may play a role in the mitogenic response induced by these compounds.

The effects of phorbol esters with different activities on protein kinase C

The sapintoxins are a series of naturally occurring fluorescent phorbol esters with a range of selective biological activities (e.g. pro-inflammatory but non-tumour promoting). Their ability to activate protein kinase C (PKC) in vitro has been studied. Both tumour promoting and non-promoting phorbol derivatives activate the enzyme in vitro at low concentrations. 12-deoxyphorbol-13-phenylacetate-20 acetate (DOPPA) acts as a partial agonist in the activation of protein kinase C. Structurally distinct phorbol esters may therefore preferentially activate different forms of protein kinase C. -sapinine, a biologically inactive compound, binds to protein kinase C without stimulating the enzyme and prevents subsequent activation by phorbol esters such as 12-O-tetradecanoyl phorbol-13-acetate (TPA).

Arresting developments in the cardiac myocyte cell cycle: Role of cyclin-dependent kinase inhibitors

Like most other cells in the body, foetal and neonatal cardiac myocytes are able to divide and proliferate. However, the ability of these cells to undergo cell division decreases progressively during development such that adult myocytes are unable to divide. A major problem arising from this inability of adult cardiac myocytes to proliferate is that the mature heart is unable to regenerate new myocardial tissue following severe injury, e.g. infarction, which can lead to compromised cardiac pump function and even death. Studies in proliferating cells have identified a group of genes and proteins that controls cell division. These proteins include cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors (CDKIs), which interact with each other to form complexes that are essential for controlling normal cell cycle progression. A variety of other proteins, e.g. the retinoblastoma protein (pRb) and members of the E2F family of transcription factors, also can interact with, and modulate the activities of, these complexes. Despite the major role that these proteins play in other cell types, little was known until recently about their existence and activities in immature (proliferating) or mature (non-proliferating) cardiac myocytes. The reason(s) why cardiac myocytes lose their ability to divide during development remains unknown, but if strategies were developed to understand the mechanisms underlying cardiac myocyte growth, it could open up new avenues for the treatment of cardiovascular disease. In this article, we shall review the function of the cell cycle machinery and outline some of our recent findings pertaining to the involvement of the cell cycle in modulating cardiac myocyte growth and hypertrophy.

The role of 80K/MARCKS, a specific substrate of protein kinase C, in cell growth and tumour progression

Since its discovery more than a decade ago [Wu et al., 1982; Rozengurt et al., 1983], the 80-87 kDa myristoylated a lanine-rich C-kinase substrate (80K/MARCKS) protein has attracted a great deal of attention from researchers interested in cell growth and tumour progression. However, despite its ubiquitous distribution, a definitive functional role for 80K/MARCKS has not been found. The purpose of this review is to describe the properties, distribution and regulation of 80K/MARCKS and to discuss some of the most recent findings, both from our laboratory and from others, that have suggested a functional role for this protein in modulating cell growth and tumour progression. Furthermore, I will present data from our laboratory that implicates 80K/MARCKS as a novel tumour suppressor in cells of melanocyte origin.

Roles of the translationally controlled tumour protein (TCTP) and the double-stranded RNA-dependent protein kinase, PKR, in cellular stress responses

Translationally controlled tumour protein (TCTP) is a highly conserved protein present in all eukaryotic organisms. Various cellular functions and molecular interactions have been ascribed to this protein, many related to its growth-promoting and antiapoptotic properties. TCTP levels are highly regulated in response to various cellular stimuli and stresses. We have shown recently that the double-stranded RNA-dependent protein kinase, PKR, is involved in translational regulation of TCTP. Here we extend these studies by demonstrating that TCTP is downregulated in response to various proapoptotic treatments, in particular agents that induce Ca++ stress, in a PKR-dependent manner. This regulation requires phosphorylation of protein synthesis factor eIF2α. Since TCTP has been characterized as an antiapoptotic and Ca++-binding protein, we asked whether it is involved in protecting cells from Ca++-stress-induced apoptosis. Overexpression of TCTP partially protects cells against thapsigargin-induced apoptosis, as measured using caspase-3 activation assays, a nuclear fragmentation assay, using fluorescence-activated cell sorting analysis, and time-lapse video microscopy. TCTP also protects cells against the proapoptotic effects of tunicamycin and etoposide, but not against those of arsenite. Our results imply that cellular TCTP levels influence sensitivity to apoptosis and that PKR may exert its proapoptotic effects at least in part through downregulation of TCTP via eIF2α phosphorylation.

Platelet endothelial cell adhesion molecule-1 regulates collagen-stimulated platelet function by modulating the association of phosphatidylinositol 3-kinase with Grb-2-associated binding protein-1 and linker for activation of T cells

Background: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI–Fc receptor (FcR)-chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3-kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT). An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb-2-associated binding protein-1 (Gab1), which is regulated by binding of the Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses. Platelet endothelial cell adhesion molecule-1 (PECAM-1) functions as a negative regulator of platelet reactivity and thrombosis, at least in part by inhibiting GPVI–FcR-chain signaling via recruitment of SHP-2 to phosphorylated immunoreceptor tyrosine-based inhibitory motifs in PECAM-1. Objective: To investigate the possibility that PECAM-1 regulates the formation of the Gab1–p85 signaling complexes, and the potential effect of such interactions on GPVI-mediated platelet activation in platelets. Methods: The ability of PECAM-1 signaling to modulate the LAT signalosome was investigated with immunoblotting assays on human platelets and knockout mouse platelets. Results: PECAM-1-associated SHP-2 in collagen-stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen-stimulated PI3K signaling. We therefore propose that PECAM-1-mediated inhibition of GPVI-dependent platelet responses result, at least in part, from recruitment of SHP-2–p85 complexes to tyrosine-phosphorylated PECAM-1, which diminishes the association of PI3K with activatory signaling molecules, such as Gab1 and LAT.

Modulation of pro-survival Akt/protein kinase B and ERK1/2 signaling cascades by quercetin and its in vivo metabolites underlie their action on neuronal viability

Much recent interest has focused on the potential of flavonoids to interact with intracellular signaling pathways such as with the mitogen-activated protein kinase cascade. We have investigated whether the observed strong neurotoxic potential of quercetin in primary cortical neurons may occur via specific and sensitive interactions within neuronal mitogen-activated protein kinase and Akt/protein kinase B (PKB) signaling cascades, both implicated in neuronal apoptosis. Quercetin induced potent inhibition of both Akt/PKB and ERK phosphorylation, resulting in reduced phosphorylation of BAD and a strong activation of caspase-3. High quercetin concentrations (30 microM) led to sustained loss of Akt phosphorylation and subsequent Akt cleavage by caspase-3, whereas at lower concentrations (<10 microM) the inhibition of Akt phosphorylation was transient and eventually returned to basal levels. Lower levels of quercetin also induced strong activation of the pro-survival transcription factor cAMP-responsive element-binding protein, although this did not prevent neuronal damage. O-Methylated quercetin metabolites inhibited Akt/PKB to lesser extent and did not induce such strong activation of caspase-3, which was reflected in the lower amount of damage they inflicted on neurons. In contrast, neither quercetin nor its O-methylated metabolites had any measurable effect on c-Jun N-terminal kinase phosphorylation. The glucuronide of quercetin was not toxic and did not evoke any alterations in neuronal signaling, probably reflecting its inability to enter neurons. Together these data suggest that quercetin and to a lesser extent its O-methylated metabolites may induce neuronal death via a mechanism involving an inhibition of neuronal survival signaling through the inhibition of both Akt/PKB and ERK rather than by an activation of the c-Jun N-terminal kinase-mediated death pathway.

c-Jun N-terminal kinase (JNK)-mediated modulation of brain mitochondria function: new target proteins for JNK signalling in mitochondrion-dependent apoptosis

The molecular mechanisms underlying the initiation and control of the release of cytochrome c during mitochondrion-dependent apoptosis are thought to involve the phosphorylation of mitochondrial Bcl-2 and Bcl-x(L). Although the c-Jun N-terminal kinase (JNK) has been proposed to mediate the phosphorylation of Bcl-2/Bcl-x(L) the mechanisms linking the modification of these proteins and the release of cytochrome c remain to be elucidated. This study was aimed at establishing interdependency between JNK signalling and mitochondrial apoptosis. Using an experimental model consisting of isolated, bioenergetically competent rat brain mitochondria, these studies show that (i) JNK catalysed the phosphorylation of Bcl-2 and Bcl-x(L) as well as other mitochondrial proteins, as shown by two-dimensional isoelectric focusing/SDS/PAGE; (ii) JNK-induced cytochrome c release, in a process independent of the permeability transition of the inner mitochondrial membrane (imPT) and insensitive to cyclosporin A; (iii) JNK mediated a partial collapse of the mitochondrial inner-membrane potential (Deltapsim) in an imPT- and cyclosporin A-independent manner; and (iv) JNK was unable to induce imPT/swelling and did not act as a co-inducer, but as an inhibitor of Ca-induced imPT. The results are discussed with regard to the functional link between the Deltapsim and factors influencing the permeability transition of the inner and outer mitochondrial membranes. Taken together, JNK-dependent phosphorylation of mitochondrial proteins including, but not limited to, Bcl-2/Bcl-x(L) may represent a potential of the modulation of mitochondrial function during apoptosis.