Protein kinase C (PKC) level is increased in PC12 cells overexpressing transfected liver-type phosphofructokinase

Biol Cell (1994) 81, 23-29

23

© Elsevier, Paris

Original article

Protein kinase C (PKC) level is increased in PC12 cells overexpressing transfected liver-type phosphofructokinase Ari Elson, Yael Weiss, Yoram Groner* Department of Molecular Genetics and Virology, The Weizmann Institute of Science, Rehovot 76100, Israel (Received 4 January 1994; accepted 29 March 1994)

Summary - PC I2 cells which overexpress transfected liver-type phosphofructokinase (PFKL) have previously been described as a model system for PFKL overexpression in Down's syndrome and have been shown to perform glycolysis at enhanced rates. Here we report that levels of protein kinase C (PKC) in PC I2-PFKL cells were almost doubled, as estimated from in vitro activity and phorbol ester binding experiments and from an increase found in PKC-alpha mRNA levels. Most of the added PKC was found to be associated with the cellular membrane while the cytoplasmic levels of PKC were barely increased. The steady-state levels of 1,2-sn-diacylglycerol in PC12-PFKL cells were found to be unaltered, suggesting that enhanced glycolysis in these cells did not influence PKC by altering the amounts of this compound. PFKL is one of several genes known to be overexpressed in Down's syndrome. Upregulation of PKC due to PFKL overexpression could result in widespread disturbances of gene expression and play a part in causing some of the many symptoms of the disease. gene-dosage ofphosphofructokinase / protein kinase C I Down's syndrome/ transfected cells

Introduction Protein kinase C (PKC) is an ubiquitous Ca 2÷- and phospholipid-dependent serine/threonine kinase which is a major component in the mechanism of generation of cellular responses to a wide variety of extracellular signals [36, 38]. Binding of ligands to certain extracellular receptors results in the breakdown of membranal phosphoinosi~tide bisphosphate and generates the intracellular second messengers inositol trisphosphate and diacylglycerol (DAG); the former acts to mobilize calcium [4], while DAG activates PKC by increasing its affinity for Ca 2÷ and phospholipids [36, 38]. Activation of PKC by this mechanism is transient and results in translocation of the enzyme from the cellular cytoplasm to the membrane. Cases of constitutive and prolonged increases in DAG whose source is not the breakdown of membrane lipids have been described in, among others, cultured cells transfected with the ras, sis, src, abl and fms oncogenes [9, 19, 30, 42 50, 51]. Enhancement of glucose concentrations led to similar observations in rat adipocytes [12, 21, 26], capillary endothelial cells [31, 32] and pancreatic islets [41], as did treatment of adipocytes and solei muscles with insulin [21 ]. In most of these cases, elevation of DAG amounts led to translocation of PKC which was usually followed by down-regulation of the enzyme [8, 49, 51]. In other cases, such as treatment of BC3H-1 myocytes or rat diaphragm with insulin, increased DAG concentrations resulted in an increase in the total amount of PKC present ([I 1, 18, 47] reviewed in [17]). The source of DAG in these cases has been shown to be mostly increased de novo synthesis from phosphatidic acid [18] generated from the glycolytic intermediates dihydroxyacetone-phosphate and glycerol-3-phos-

* Correspondence and reprints

phate brought about by an increase in glycolytic rates ([9, 41] reviewed in [8]). Down's syndrome (DS or trisomy 21) is a human genetic abnormality caused by the triplication of the distal part of chromosome 21, band 21q22 [28, 43]. The symptoms of the disease include a wide variety of structural and functional abnormalities, most prominent of which are mental retardation, morphogenetic abnormalities, a host of metabolic and e n d o c r i n o l o g i c a l aberrations and the d e v e l o p m e n t of Alzheimer's-type pathology in the brains of patients who survive into their forties [16, 45]. Genes present on the triplicated part of chromosome 21 are generally thought to be expressed as their counterparts in the rest of the genome, leading to a situation where abnormal amounts of the products encoded by these genes are synthesized. It is believed that the imbalance caused by this series of overexpressions is the basic cause of the symptoms of DS [16]; however, the precise mechanisms by which overexpression of individual genes contributes to specific s y m p t o m s of the disease remain largely unknown. The gene for the liver-type subunit of the key glycolytic enzyme phosphofructokinase (PFKL) is located in band 21q22 and is known to be overexpressed in DS erythrocytes (summarized in [16]) and fibroblasts [2], leading to an increase of 30-60% in the total catalytic activity of phosphofructokinase in these tissues. In the course of our efforts to create model systems for analysis of the role PFKL overexpression may play in the pathogenesis of DS we have previously cloned and characterized both the cDNA [33] and gene [15] of the human PFKL. The PFKL cDNA was stably transfected into rat PC12 cells and clones with increases of 40-60% in their phosphofructokinase activity were isolated [ 14]. The PC 12-PFKL cells were shown to perform glycolysis some 40% faster than controls and the biochemical-regulatory properties of PFK activity isolated from these cells was shown to be altered; both phenomena have been shown to have parallels in DS (discussed in [14]).

24

A Elson et al

The connections previously found between enhanced glycolytic rates, D A G accumulation and PKC activation raised the possibility that the amounts and subcellular distribution of PKC and amounts of D A G were perturbed in PC 12-PFKL cells due to their increased rate of glycolysis. The experiments presented here show that PC12-PKFL cells contain elevated amounts of PKC and that most of the increase in PKC activity can be found in association with the cellular membrane. No changes were found in the steady-state levels of DAG suggesting that the alterations found in PKC were not the result of increased DAG production due to enhanced glycolysis.

Materials and methods Tissue culture and RNA analysis Isolation of PCI2 clones overexpressing PFKL and the precise conditions of growth and maintenance were described in [14], as were cytoplasmatic RNA preparation and analysis. All PFKLoverexpressing and control clones were G-418 resistant although the experiments described here were not carried out in the presence of this drug. ' Isolation of PKC from whole cell homogenates 2-5 x 106 cells were grown on 9- or 15-cm dishes for 2-3 days prior to analysis. Dishes were washed twice with PBS and once with buffer A (20 mM Tris--Cl (pH 7.5), 2 mM EDTA, 0.5 mM EGTA, 2 mM PMSF and 25/.tg/ml aprotonin) and resuspended in 1 ml of buffer A. Cells were lysed by 25 strokes with a glass-glass Dounce homogenizer. Triton X-100 was added to a final concentration of 1% and the mixture was incubated on ice for 10 min. The lysate was spun at 100000 g for 30 min at 4°C, and I-2 mg protein of the surpernatant fraction were loaded on a 0.5 x 2 cm DE-52 column which had been pre-equilibrated with buffer B (20 mM Tris-CI (pH 7.5), 2 mM EDTA and 0.5 mM EGTA). The resin was washed with 8 ml of buffer B and elution was performed in 2 ml of buffer B containing 100 mM NaCI. PKC prepared in this manner was used for activity measurements on the same day. Isolation of cytoplasmic and membrane-bound fractions of PKC This was performed essentially as described by Thomas et al [44]. Cells were washed twice in PBS and once again in l ml buffer A to which 330 mM sucrose had been added. Cells were resuspended in 2 ml buffer A with sucrose and lysed with 25 strokes of a Dounce homogenizer followed by centrifugation at 100000 g for 30 min at 4°C. The supematant fraction was collected and saved on ice. The pellet was resuspended in 2 ml buffer A without sucrose to which 1% Triton X-100 had been added and resuspended by Douncing as above. After 60 min of incubation on ice the fraction was centrifuged as above and the supematant saved. Both supematants were purified on DE-52 columns and assayed for PKC activity as described below. PKC activity assay Calcium- and phospholipid-dependent protein kinase C activity was assayed basically as described by Thomas et al [44], using a reaction mixture consisting of 20 mM Tris-C! (pH 7.5), 20 mM Mg(OAc) 2, 270/.tg/ml histone III-S (Sigma), 50 uM ATP, 0.12 mCi/ml of [7 -32p] ATP (5 000 Ci/mmol, Amersham), 1.25 mM CaCI 2, 0.25 mg/ml phosphatidylserine (Sigma) and 5/.tg/ml diolein (l,2-dioleoyl-sn-glycerol, Sigma). 40/.tl of column eluate were placed in Eppendorf tubes in ice-water, and 200/.tl of the above reaction mix were dispensed into each tube using a repeating dispenser. The tube contents were mixed and placed in a water bath at 30°C for 10 min, at the end of which the tubes were again put in ice-water. Aliquots of 120/.tl were spotted on Whatman 3MM paper, washed four times in 10% TCA, dried and counted, using 25% Lumax scintillation fluid in xylene. PKC activity was calculated from the difference in radioactivity

retained on the filters between reactions run as above and control reactions, where phosphatidylserine, Ca -'+ and diolein were replaced by EDTA (0.3 mM) and EGTA (0.075 mM). Histone phosphorylation was linear with respect to the amount of column eluate used and incubation time. Activity was expressed as pmol 32p incorporated into histone per minute per microgram of column eluate protein. Protein was measured according to Bradford [7] with BSA as a standard. Phorbol ester binding to cell homogenates This was performed following protocols described in [10, 24, 27]. For the purpose of analysis of binding at several concentrations, two 15-cm plates were seeded with 2-5 x 106 cells each and grown for 2-3 days. Cells were then washed twice in PBS and once in buffer C (25 mM Tris-CI (pH 7.5), 0.25 M sucrose, 2.5 mM Mg(OAc) 2, 2.5 mM EGTA and 2.5 mM DTT). Cells were scraped into 2 ml of buffer D and disrupted by sonication. Protein concentration was adjusted to 0.2 mg/ml and the crude lysate was used for binding experiments. Binding was performed using 20-[3H] phorbol-12,13-dibutyrate ([3H] PDBU, 20 Ci/mmol, New England Nuclear). A series of two-fold dilutions from 100 to 1.56 nM [3H]PDBU was prepared by diluting the labeled PDBU with binding buffer (50 mM Tris-CI (pH 7.5), 22.5 mM Mg(OAc) 2 and 1.5 mg/ml BSA). Binding reactions contained 50/11 (10/.tg total protein) of crude lysate, 200 t.tg/ml phosphatidylserine (freshly sonicated), 50,ul of [3H]PDBU of the appropriate concentration and 1 mM CaC12 in a final volume of 160/.d. The reactions were incubated for 15-25 rain in a 37°C water bath, placed in ice-water and 20/11 were removed for measurement of total radioactivity present. To the remainder of the sample, 50/.d of 12 mg/ml goat IgG and 150/.d of 33.6% PEG (M r 8000, in 50 mM Tris-CI (pH 7.5)) were added followed by immediate vigorous vortexing and another 5 rain of incubation on ice. The reaction mix was then filtered on GF/C filters (Whatman), and washed twice with 5 ml of filtering solution (20 mM Tris-CI (pH 7.5), 10 mM MgC12, I mM CaCI2). The filters were air-dried and counted at 36% efficiency using Lumax scintillation fluid (40% in xylene). Non-specific binding was estimated from identical reactions to which a 200-fold excess of non-radioactive PMA was added. Total binding was I-5% of the total radioactivity present in the samples. For the purpose of binding at a single, representative concentration of PDBU to PKC, 250000 cells were plated on 6-cm plates and processed as above. Binding experiments were performed as above except that a single representative concentration (50 or 100 nM) of PDBU was used. Measurement of diacylglycerol concentrations in PCI 2 cells 1-2 x 106 cells were seeded in 9-cm plates and grown for 2-3 days prior to analysis. Cells were washed twice with PBS, resuspended in l ml of PBS and an aliquot was removed for protein measurement to allow comparison between samples. Lipids were extracted from the remainder of the cells using methanol and chloroform according to the method of Bligh and Dyer [5]. The DAG content of the lipids was measured by phosphorylating the DAG with [7-32p]ATP using E coli DAG kinase, using the reagents system supplied by Amersham according to the method of Preiss et al [42]. The radioactively-labeled phosphatidate was purified on Amprep silicon-hydroxide mini-columns (Amersham) and counted with lumax scintillation fluid (40% in xylene).

Results The cells used throughout this work were PC12 clones independently isolated from stable transfections with the human PFKL cDNA and which exhibited, on the average, increases of 50% in their total in vitro phosphofructokinase catalytic activity [14].

PKC in PC12 cells

25

Table I. Relative specific activities and [3H]PDBU binding of PKC in PC I2 clones and pools and in HeLa pool.

Cell type

Clone #

PKC activity (-fold increase)

[3H]PDBU binding (-fold increase)

PC 12-PFKL clones

PC 12-PFKL pool

59 133 139 239 59, 139, 239

1.49 __.0.08 ~ 1.78 -+ 0.10 a 2.03 -+ 0.19 a 2.14 _+0.09 a 1.83 +0.19 a

1.82 -+ 0.18 a nd 2.36 _ 0.36 a 1.71 _+0.10 a 1.71 +_0.10 a

13 77 84 146

1.08 0.72 0.97 1.22 1.21

_+0.03 _+0.05 + 0.10 __.0.07 _+0.12

1.13 __.0.18 0.86 - 0.05 nd i .07 __.0.20 nd

13, 77, 146

1.04 _+0.09

1.00 _ 0.06

PC 12-Cont clones

Parental PC 12 PC 12-Cont pool HeLa -PFKL pool

1 . 0 0 __. 0 . 1 2

nd

Each parameter was measured in PKFL-expressing and -non-expressing cells simultaneously; the values obtained were compared with the average of those measured in the PKFL non-expressors. Activity values are the mean +_ SE of 1-3 separate experiments each performed in quadruplicate; binding results are from 3-8 separate measurements at 50 or 100 nM [3H]PDBU. Representative specific activity values for non-PFKL expressing pools were: 1508 -+ 302 (PC12) and 2003 +__76 (HeLa) pmol/min per mg protein of column eluate. PDBU binding to PC l2-control pool was 2.49 _ 0.14 pmol/mg protein, aStatistically significant (P < 0.001) from control values by t-test analysis; nd, not done.

k.•

PC12-PFKL cells exhibit enhanced PKC catalytic activi~ and phorbol ester binding

o]

0.12" • •

T h e c a t a l y t i c a c t i v i t y o f P K C p r e s e n t in p a r t l y - p u r i f i e d homogenates of PC 12-PFKL clones and controls was determined in vio'o using histone-III-S as an exogenous substrate for phosphorylation by PKC. The results, summarized in table I, showed that PKC activity was increased in individual PC12P F K L clones by an average of 86% when compared to controls. In order to determine whether enhanced PKC activity was due to an increase in the amounts of PKC present or to modifications which rendered it more active without increasing its amounts, the amount of PKC present in the different PC I2 clones was estimated also by quantification of phorbol ester binding to these cells, a technique independent of activity measurements. Fifty or 100 nM of tritiated phorbol-12,13dibutyrate ([3H]PDBU) was allowed to bind to homogenates of PC 12-PFKL or control clones and the amount bound quantiffed; the results (table I) indicated that the individual PFKLexpressing clones tested bound on the average 96% more PDBU than did controls. Similar increases in PKC catalytic activity and PDBU binding were measured when clones of PC 12-PFKL or control cells were separately pooled and used instead of individual clones (table I); all subsequent work was therefore performed using pooled clones. Binding of [3H]PDBU to PKC in the above system was fiarther i n v e s t i g a t e d in u n p u r i f i e d w h o l e - c e l l l y s a t e s by repeating the binding experiments at PDBU conceng-ations ranging from 100 nM to 1.56 nM and subjecting the results to Scatchard-type analysis [24]. From analysis of the data obtained in the course o f these experiments (fig I) it was c a l c u l a t e d that the P C 1 2 - P F K L pool c o n t a i n e d (3.73 _ 0.26) x 1012 binding sites per mg protein vs (2.26 _+ 0.08) x 1012 in the control pool, an increase o f 65%. The data also indicated that both pools contained binding sites which displayed the same, single affinity towards PDBU with a dis-

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20

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SPECIFICALLY-BOUND

0.4 3H-PDBU

0.$

0.6

(nM)

Fig 1. Scatchard plot of [3H]PDBU binding to PCI2 extracts. The plot drawn is representative of a total of four separate experiments. Calculated maximal number of PKC binding sites per mg protein was: PC12-PFKL pool: (3.73 _ 0.26) x 10 t2, control pool: (2.26 _+ 0.08) x 101L (PCI2- PFKL/Control = 1.65, P < 0.01). Dissociation constant of PDBU from PKC in both pools was 4.46 _+ 0.77 nM. Shaded circles, PC12-PFKL pool; open circles, control pool. Inset, binding curve of [3H]PDBU to control extract as measured in a representative experiment. TB, total bound; SB, specifically bound; NSB, non-specifically bound.

sociation constant o f 4.46 _+ 0.77 nM. The fact that PC12 cells displayed a single class of PDBU binding sites as well as the amount and measured affinity o f these sites towards PDBU were in agreement with data previoulsy published regarding PKC in PC12 cells [29, 35]. Taken together, these results indicated that transfection of PC12 cells with P F K L resulted in an increase in the a b s o l u t e amounts o f P K C

26

A Elson et al

PC12:

-

+ O

L.

¢1,

p=0.002 4000

[ ] Control Pool [ ] (+) Pool

P=0.053

o~

30o0

¢1

~41!

¢¢ 28S

~

2o00

eL E

1000

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PCI2 Cyt.

PCI2 Memb.

HeLA Cyt.

HeLa Memb.

Fig 3. Distribution of PKC enzymatic activity between the cytoplasm and membrane in PCI2 and HeLa pools. Data represent the mean and standard error of two to three separate experiments with five repeats each. Data were compared by t-test analysis.

18S

BB Fig 2. Northern analysis of total cytoplasmatic RNA from PCI2

pools hybridized with the PKC-a cDNA fragment. Bottom panel, hybridization of same blot with a rat actin probe for comparison of RNA amounts on blot. (+) = PC I2-PFKL; (-) = PC l2-control.

present in the cells. The observation that similar results were obtained from several independently-derived clones supported the claim that PFKL overexpression was the cause for the upregulation of PKC observed here. To estimate the steady-state levels of PKC-~ mRNA, which is one of the major types of PKC present in PC12 cells, Northern blots of total RNA prepared from both types of cells were hybridized with a bovine P K C - a cDNA probe. It was found that the levels o f PKC-(x m R N A were s i g n i f i c a n t l y increased in the PC12-PFKL pool as compared to the control PC12 pool (fig 2). Measurements of PKC catalytic activity were also conducted using pools of HeLa clones transfected with mouse PFKL, and in which the total phosphofructokinase activity was elevated by 50-125% relative to controls (Elson A, Levanon D, unpublished results). No differences in the PKC activity levels were found between HeLa-PKFL and control cells (table I); this indicated that the correlation between enhanced PFK and PKC activities might be linked to a property characteristic of PC12 cells which is missing from HeLa cells. The majority of the added PKC in PC12-PFKL cells is membrane-bound The subcellular localization of PKC in P C 1 2 - P F K L and control cells was examined by lysing the cells under condi-

tions where the membrane-bound and cytoplasmic fractions of PKC could be separated. PKC present in each fraction was partly purified by DE52 column chromatography and the activities of each fraction from both pools were compared. The results (fig 3) showed that PKC activity present in the m e m b r a n a l f r a c t i o n of P C 1 2 - P F K L cells was increased by some 97% (P = 0.002), while that of the cytoplasmic fraction was increased by only 23%, a value of borderline statistical significance (P = 0.053). These results indicated that while m o s t of the added PKC in the P C 1 2 - P F K L clones was associated with the membranal fraction, there was no evidence to show that translocation from the cytoplasm to the membrane had taken place. No alterations in the amounts of PKC isolated from either fraction were observed in the pool of PFKL-overexpressing HeLa clones (fig 3), in line with the previous finding that unfractionated PKC levels were unchanged in this pool. 1,2-diacyl-glycerol levels in PC12-PFKL cells are not changed The steady-state amounts of DAG in P C 1 2 - P F K L cells were determined using E coli DAG kinase in a sensitive enzymatic assay specific for 1,2-sn-diacyl-glycerols, the type of D A G s which activate PKC [42]. E x p e r i m e n t s showed that the PC12-PFKL pool contained 3.56 _ 0.17 pmol DAG//.tg protein (n = 5) while the pool of control clones contained 3.90 _+ 0.11 picomol/pg protein (n = 6). The differences were shown to be statistically insignificant by t-test analysis. These results showed that while PKC levels were significantly increased in PFKL-overexpressing PC12 cells, the biochemical reason underlying the increase is not merely the higher level of DAG resulting from the enhanced glycolysis of PC 12-PFKL.

Discussion

The data presented here show that P C 1 2 - P F K L pools exhibited a 70-80% increase in the amounts of PKC present as compared to PC12-control cells. This result was obtained using two independent experimental techniques - in vitro catalytic activity and phorbol ester binding measurements -

PKC in P C I 2 cells

which yielded similar results. The absolute number of PKC binding sites in PC12-PFKL cells was found to be increased by 65%, a value capable of accounting for most, if not all, of the increase in PKC activity. Additional results showed that the affinity with which PKC bound phorbol esters was the same in PC12-PFKL cells and in controls. The results of the activity measurements indicated that PC12-PFKL clones contained more in vitro PKC activity than control clones, while analysis of the binding experiments showed that this was probably brought about by having more PKC present in these cells rather than by an alteration which rendered PKC more active without changing its amount. The fact that similar results were obtained from several independently-derived clones, each overexpressing PFKL, strongly suggested that the phenomena observed truly resulted from PFKL overexpression. The experiments also demonstrated that the added amount of enzyme was distributed so that the specific activity of the activated, membrane-bound form of PKC was almost doubled while there was only a slight (23%) increase in the unactivated, cytoplasmic form of the enzyme, indicating that no translocation of PKC from the cytoplasm to the membrane had occurred. In parallel, an increase in the steady-state levels of PKC-ct mRNA, one of the major types of PKC present in PC12 cells, was also noted, while no changes in the cellular content of 1,2-diacyl-glycerols were found. The original hypothesis upon which this line of work was based, namely that enhanced rates of cellular glycolysis resulted in more DAG being present which activated PKC, cannot account for all of the experimental findings and is totally contradicted by others. First and foremost, DAG concentrations were found to be unchanged in PC 12-PFKL cells despite their previously-documented increase in glycolytic rates. In cases where enhanced glycolysis resulted in higher levels of DAG, it was indeed demonstrated that PKC was p r e f e r e n t i a l l y bound to the c e l l u l a r m e m b r a n e [26, 31,32, 49-51 ]. However, the lack of decrease in cytoplasmic PKC and the increase in total PKC amounts observed here were not found in the above cases. The connection between enhanced glycolysis and PKC upregulation does not readily explain the increase in PKC-ct mRNA levels either. We, therefore, conclude that the changes found in PKC in PC12-PFKL cells were probably caused by a mechanism which does not involve enhancement of DAG concentrations by glycolysis. HeLa cells, which o v e r e x p r e s s e d PFKL to similar extents as the PC12-PFKL cells, did not display the same alterations in PKC content and distribution (table I, fig 3). PKC can be activated through a number of different mechanisms [17, 39] and not all phenomena in which PKC participates can be detected in all systems. For example, the connection between enhanced rates of DAG synthesis, DAG accumulation and PKC activation has been amply documented in rat adipocytes, but was not detected in rat solei muscles when the latter were incubated in high concentrations of glucose, probably due to the low capability of this organ to metabolize glucose to DAG [21]. It is possible that the connection between PKC amounts and PFK overexpression is dependent upon a factor or metabolic cal~ability present in PCI2 cells but missing from HeLa cells. The results described above did not address the mechanisms by which changes in PKC amounts were brought about. However, several cases have been described in which cells which had undergone various treatments displayed seemingly unrelated alterations in their complement of PKC. Messing et al [35] reported a rise of 50% in the amounts of PKC in ethanol-treated PC12 cells and several

27

reports exist of a rise of 100-200% in cellular PKC in the human promyelocytic leukemia cell line HL60 after treatment with 1,25-dihydroxyvitamin D3, DMSO or retinoic acid [34, 40, 52]. In both cases, as in the work presented here, both the overall in vitro catalytic activity and phorbolester binding capability of PKC were found to be increased, while the affinity of PKC towards phorbol esters and cellular concentrations of DAG were unchanged. Obeid and coworkers [40] also reported an increase in PKC-fl mRNA levels and provided evidence to prove this was the result of increased transcription of this gene. Experimental evidence has been supplied in both of the above cases, showing that the elevated PKC amounts in cells as measured in vitro were also expressed as enhanced PKC activity in vivo [35, 40]. Both works suggested that the endogenous DAG levels in the cell types they worked with - among them PCI2 cells - were high enough to activate the added amounts of PKC, suggesting that the additional PKC activity measured in vitro in the PC12-PFKL cells could result in increased enzymatic activity in vitro. Increases in PKC have also been documented in aflatoxin-transformed C3H 10TI/2 cells [13] and in a multidrug-resistant murine fibrosarcoma cell line [48]. Although the mechanisms behind the alterations in PKC still have to be elucidated, these results show that PKC not only affects, but is also affected by a large and seemingly unrelated variety of cellular phenomena. Glycolysis itself has been shown to be influenced by PKC. Treatment of chicken embryo flbroblasts with PMA or insulin has been shown to stimulate glycolysis by increasing the rate of glucose transport and by elevating concentrations of fructose-2,6-bisphosphate [6], which is the strongest activator of phosphofructokinase known [46]. It was presumed that activation of phosphofructokinase by f r u c t o s e - 2 , 6 - b i s p h o s p h a t e a c c o u n t e d partly for the increased glycolytic flux described [6]. The muscle-type subunit of phosphofructokinase itself, distinct from the liver-type subunit transfected into PC12 cells described here, has been shown to be capable of phosphorylation by PKC in vitro in a manner altering its allosteric properties [20]. It is, therefore, possible that PFKL overexpression might influence glycolysis through its influence on PKC in addition to its direct influence by increasing the amounts of phosphofmctokinase, which is a key rate-limiting enzyme of the pathway. Alterations in PKC activity could have several implications to DS. PKC is prominent in the brain, and has been implicated in regulation of ion channel activity, enhancement of neurotransmitter release, control of growth and differentiation and modification of neuronal plasticity [23, 39]. Aberrant PKC activity could be expected to play a role in causing the structural and functional defects of DS brain leading to mental retardation, which is one of the hallmarks of the DS phenotype. PKC has been shown to influence growth regulation; it has been suggested that constitutively active PKC may relieve the dependency of PKC activity on extracellular signals and promote carcinogenesis [8, 36], possibly connecting PFKL overexpression with the well-known increase in the susceptibility of DS patients to develop leukemia. Incorrect PKC function might impair signal transduction by altering the specificity or rates with which PKC phosphorylates its substrates, thereby altering the nature or strength of the signal it is supposed to mediate and resulting in abnormal cellular responses to extracellular signals. Along this line, DS patients are known to suffer from a host of endocrinological, morphological and developmental defects which presumably involve cell-to-cell signalling and might result

28

A Elson et al

from altered signal transduction. Finally, PKC has been shown to be capable of influencing the activity of many proteins [36] including transcription factors [25]. A situation could therefore arise where P F K L o v e r e x p r e s s i o n could result, through increases in PKC, in a series of widespread overexpressions of additional genes. The sum total damage caused by overexpression of P F K L would then be much larger than could be predicted by a limited analysis of PFK activity or glycolysis.

16 17 18

19

Acknowledgments This work was supported by grants from the National Institutes of Health (USA) HD21229; The Minerva Foundation (Munich, Germany); the Weizmann Institute's Leo and Julia Forchheimer Center for Molecular Genetics and the Ebner Family Biomedical Research Foundation ot the Weizmann Institute.

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