Diacylglycerol kinase theta and zeta isoforms: regulation of activity, protein binding partners and physiological functions

Los, Alrik Pieter

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Los, A. P. (2007, January 25). Diacylglycerol kinase theta and zeta isoforms: regulation of

activity, protein binding partners and physiological functions. Retrieved from

https://hdl.handle.net/1887/9451

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P r o t e in k in a s e C

in h ib it s b in d in g o f

d ia c y lg ly c e r o l k in a s e - c

t o t h e r e t in o b la s t o m a

p r o t e in

(BBA Molecular Cell research, in press)

Alrik P. Los

J oh n d e W id t

M a t t h e w K . T op h a m

W im J . v a n B lit t e rsw ijk

N u llin D iv e c h a

Chapter 5

A bstract

W e prev iou sly show ed that the retinoblastoma protein

(p

) , a key regu lator of

to

-phase transition of the

cell cycle, binds to and stimu lates diacylglycerol kinase- c

(

c ) to phosphorylate the lipid second messenger

diacylglycerol into phosphatidic acid. p

binds to the

phosphorylation-site domain of

c that can

be phosphorylated by protein kinase

(

) . H ere, w e

report that activ ation of

by phorbol ester inhibits

c binding to p

.

o 3 1-8 2 2 0, a specific inhibitor of

, allev iated this inhibition of binding. M imicking of

phosphorylation of serine residu es (by

/

bu t not

/

mu tations) w ithin the

c -

phosphoryla-

tion-site domain also prev ented

c binding to p

,

su ggesting that

phosphorylation of these residu es

negativ ely regu lates the interaction betw een

c and

p

. I n

ov erex pression stu dies, it appeared that

activ ation of particu larly the (w ild-type)

_ isoform

inhibits

c binding to p

, w hereas dominant-nega-

tiv e

_ neu traliz ed this inhibition.

_ activ ation

thu s prev ents

c regu lation by p

, w hich may hav e

implications for nu clear diacylglycerol and phosphatidic

acid lev els du ring the cell cycle.

Protein kinase C inhibits binding of diacylglycerol kinase-c to the retinoblastoma protein

1. Introduction

The second messenger diacylglycerol (DAG) participates via its targets, including PKC,Ras-GRP, chimaerins, and Munc-1 3 , in the regulation of several cellular processes, such as prolif eration, dif f erentiation and cell migration (Brose et al., 2 0 0 4 ). Several growth f actors stimulate DAG production b y activating phospholi- pase C, which hydrolyses phosphatidylinositol 4 ,5 -b isphosphate. DAG is not only present at the plasma memb rane b ut also in the nucleus where it can also b e generated b y a nuclear phospholipase C (D’Santos et al., 1 9 9 9 ) that is activated independently f rom a cytosolic phospholipase C (X u et al., 2 0 0 1 ), suggesting that nuclear DAG has distinct f unctions.

A well-characterised target of DAG is PKC. The PKC f amily consists of ten dif f erent isof orms, of which only the conventional (_, `I , `I I and a) and novel (b, -¡, -d, and -e)PKC isof orms respond to DAG (or phorb ol ester) (Newton, 2 0 0 3 ). O ne poorly def ined f unction of PKC relates to the cell cycle. Depending on the cell system, PKC isof orm, and the timing of PKC activation within the cell cycle, PKC can stimulate or inhib it the cell cycle (reviewed in Black, 2 0 0 0 ). PKC regulates phosphorylation of the retinob lastoma-f amily of pocket proteins (pRB, p1 0 7 or p1 3 0 ), which regulate G1 to S-phase transition and cell cycle entry and ex it b y seq uestering and inactivating the E 2 F f amily of transcription f actors (DiCiommo et al., 2 0 0 0 ; Classon and Dyson, 2 0 0 1 ). I n addition, PKC`2 was f ound to directly phosphorylate pRB in retinal endothelial cells, causing E 2 F release and prolif era- tion (Suz uma et al., 2 0 0 2 ). I n many cell types, PKC induces the ex pression of p2 1WAF / CI P1, a cyclin/CDK inhib itor that prevents phosphorylation of pocket proteins, causing a cell cycle arrest (reviewed in Black, 2 0 0 0 ).

Since DAG activates PKCs, its sub cellular levels need to b e tightly controlled.

O ne mechanism b y which this can b e achieved is through diacylglycerol kinases that phosphorylate DAG into phosphatidic acid. Ten dif f erent DGK isof orms have b een identif ied that act in dif f erent signal transduction pathways (van Blitterswijk and H oussa, 2 0 0 0 ; I mai et al., 2 0 0 5 ). O ne isof orm, DGKc, is characteriz ed b y a domain homologous to the PKC phosphorylation-site domain (PSD) within the myristoylated alanine-rich C-kinase sub strate (MARCKS) protein (MARCKS-PSD).

The DGKc-^MARCKS-PSD contains a nuclear localisation signal and was shown to b e phosphorylated b y PKC_ (Goto and Kondo, 1 9 9 6 ; Topham et al., 1 9 9 8 ; Luo et al., 2 0 0 3 b ).

Although DGKs are thought to attenuate PKC activation, regulation of PKC_ activity b y DGKc is more complicated since these two enz ymes interact and regu- late each others activity. U nder b asal conditions, DGKc b inds to and inhib its PKC_ activity (Luo et al., 2 0 0 3 a), b ut upon activation of PKC_, the interaction with DGKc is disrupted. PKC_, in turn, phosphorylates DGKc, which causes nuclear ex port and inhib ition of DGKc activity (Topham et al., 1 9 9 8 ; Luo et al., 2 0 0 3 b ). These f eed- b ack/ f eed-f orward mechanisms allow tight regulation of DAG levels as well as DGKc and ^PKC_ activity.

We previously f ound that DGKc b inds to pRB and that the DGKc-MARCKS-PSD is suf f icient f or pRB b inding (Los et al., 2 0 0 6 ). Since PKC phosphorylates the DGKc-

MARCKS-PSD, we questioned whether PKC-mediated phosphorylation of DGKc might regulate DGKc binding to p^RB. In this study we show that this is indeed the case. We show that PMA pre-treatment decreases the interaction between DGKc and pRB and that this is attenuated by pre-treatment with the PKC inhibitor Ro 31-8220. Mutation of critical serine residues within the DGKc-MARCKS-PSD, previ- ously shown to be phosphorylated by PKC, to aspartate which mimics phosphor- ylation, inhibits DGKc binding to pRB. Finally, using overexpression of different PKC isofoms, we provide evidence that PKC_ is the isoform that is involved in regulating the interaction between DGKc and pRB. Our data suggests that PKC mediated phosphorylation of the DGKc-MARCKS-PSD not only leads to nuclear exit (Topham et al., 1998), but also to a decrease in the nuclear DGKc activity by inhibiting its interaction with pRB, a potent stimulator of DGKc activity.

2. Materials and methods

2 . 1 . M a t e r i a l s , c e l l s , p l a s m i d s a n d t r a n s f e c t i o n

Dulbecco’s modified Eagle’s medium (DMEM) was from Invitrogen. [³²P] orthophos- phate was from Amersham Biosciences. Ro 31-8220 was from Calbiochem. PMA and 4_-phorbol-12,13-didecanoate (4_-PDD) were obtained from Sigma. COS-7 and MCF7 cells were grown in DMEM containing 8% heat-inactivated fetal calf serum, 2 mM glutamine and antibiotics. COS-7 cells were transfected using the DEAE-dextran method or TransIT-LT1 Transfection Reagent (Mirus) according to manufacturer’s instructions. Wild-type- and FLAG-tagged DGKc, as well as the DGKc-MARCKS-PSD mutants, in which serines (Ser²⁵⁸,Ser²⁶⁵,Ser²⁷⁰ and Ser²⁷¹) were mutated to asparagines (S/N) or aspartates (S/D) (see Figure 2A) were generated as described previously (Topham et al., 1998; Luo et al., 2003b). Human PKC isotypes, including PKC_ and its dominant-negative version (dnPKC_), in which a lysine in the ATP-binding site was replaced by an aspartate (K368D), were subcloned into pcDNA3 plasmid (Invitrogen).

2 . 2 . C e l l u l a r l y s a t e s a n d a f f i n i t y - p u r i f i c a t i o n s

48 Hours after transfection, cells were lysed in 1% NP40 lysis buffer (50 mM Tris, pH 8.0; 50 mM KCl; 10 mM EDTA; 1% NP40; complete protease inhibitor cocktail (Roche)). Affinity purifications were performed using the following GST fusion proteins: GST-pRB, consisting of the large pocket region of pRB that includes the small pocket domain and the C-terminus, GST-pRB(A+B), consisting of the small pocket domain of pRB, and GST-Cdc42. GST fusion proteins were expressed in bacteria and, after induction with 200 +M IPTG, were purified using glutathione- sepharose 4B beads (Amersham) according to manufacturer’s instructions.

Approximately 4 +g of immobilized GST fusion proteins were incubated with 200 +g of cell lysate for 2 h at 4°C and washed with 1% NP40 lysis buffer. All sam- ples within an assay contained the same amount of beads. For peptide compe- tition assays, 1 or 10 +g of peptide was incubated together with COS-7 lysate and

Protein kinase C inhibits binding of diacylglycerol kinase-c to the retinoblastoma protein

GST-pRB. Affinity-purified proteins were separated by SDS-PAGE, transferred to nitrocellulose and probed with an anti-DGKc polyclonal antibody (Bunting et al., 1996). Blots were stained with secondary antibodies (DAKO) and visualised using ECL (Amersham) or Super Signal (Pierce).

For peptide affinity-purifications, 100 +g of biotinylated TAT-DGKc-MARCKS- PSD peptides (wt: Y ARAAARQ ARAGKASKKKKRASFKRKSSKK,S/D:Y ARAAAR- Q ARAGKADKKKKRADFKRKDDKK, or S/N:Y ARAAARQ ARAGKANKKKKRANFKRKNN- KK) or TAT control peptide (Y ARAAARQ ARAG) were incubated overnight with MCF7 lysate (450 +g protein). Peptides were captured on streptavidin agarose (Sigma) and washed with wash buffer (50 mM Tris pH 7.4, 140 mM NaCl, 10 mM MgCl₂, 0.1%

Tween-20). Associated pRB was visualised by immunoblotting using anti-pRB monoclonal antibody G3-245 (Pharmingen).

2 . 3 . I n v i v o p h o s p h o r y l a t i o n

Transfected COS-7 cells were serum-starved overnight and incubated for 2 hours with phosphate-depleted DMEM containing 250 +Ci [³²P]orthophosphate per 6 cm dish. Cells were maintained as controls or were stimulated with PMA (100 nM) for 20 minutes. Cells were lysed in 1% NP40 buffer including protease and phosphatase inhibitors and FLAG-DGKc was immunoprecipitated overnight using monoclonal anti-FLAG M2 antibody (Sigma). Antibodies were captured on protein G-Sepha- rose beads (Amersham) and washed with 1% NP40 lysis buffer. Beads were boiled in SDS-sample buffer and proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and [³²P]phosphorylated proteins were analysed by autoradiography. Blots were stained with a FLAG-specific antibody to detect total DGKc.

Fig. 1.

DGKc binding to pRB is inhibited by PKC activation.

(A)CO S-7 cells overexpressing DGKc were stimulated with PMA (100 nM; 20 min) or its inactive analogue 4_-PDD. Cell lysates were subjected to affinity- purifications using GST-pRB or GST- Cdc42 (as a control). DGKc binding to GST fusion proteins was assessed by Western blotting using a DGKc- specific antibody (lef t panel). T otal cell lysates were immunoprobed using a DGKc- specific antibody to demonstrate eq ual expression (rig ht panel). (B)CO S-7 cells overexpressing DGKc were pre - treated with Ro 31-8220 (5 +M) or vehi- cle for 20 minutes prior to PMA stimu- lation. L ysates were assayed for DGKc binding to GST-pRB or GST-pRB(A+B).

Data are representatives of three exper- iments with similar results.

3. Results

3 . 1 . A c t i v a t i o n o f P KC i n h i b i t s D G K

c

Since the DGKc-MARCKS-PSD was shown to be phosphorylated by PKC and we have shown that the retinoblastoma gene product (pRB) binds to the DGKc-MARCKS- PSD, we assessed the role of phosphorylation on the interaction between pRB and DGKc.COS-7 cells overexpressing DGKc were treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) to activate PKC and lysates were used for affinity-purifications using GST-pRB or GST-Cdc42 as a control. In untreated cells, a substantial amount of DGKc bound to GST-pRB, whereas GST-pRB extracted less DGKc from cells pre-treated with PMA (Fig. 1A). DGKc binding to pRB was not

Fig. 2.

DGKc binding to pRB is inhibited by phosphorylation site mimics of serine residues within the MARCKS-phosphor- ylation-site domain of DGKc.

(A) Schematic representation of DGKc and the DGKc-MARCKS -PSD mutants.

Substituted residues are indicated in bold. (B)COS-7 cells were transfected with wild-type DGKc (wt-DGKc) or DGKc-MARCKS -PSD S/D or S/N point- mutants and were stimulated with (+) or without (-) PMA (100 nM; 20 min).

Cell lysates were used for affinity-purifi- cation of DGKc using equal amounts ofGST-pRB or GST-Cdc42 as a negative control. Affinity-purified DGKc was analysed by Western blotting using a DGKc- specific antibody (upper panel), and quantified relative to wt-DGKc (non- stimulated control; set 100% ) (b ot- tom panel). Black bars, non- stimulated;

gray bars, PMA- stimulated. Total cell lysates were immunoprobed using a DGKc- specific antibody to demonstrate equal expression (m id d le panel).

affected when cells were treated with the inactive phorbol ester analogue 4_-phorbol-12,13-didecanoate (4_-PDD). These results suggest that activation of PKC by (active) phorbol ester inhibits DGKc binding to pRB.

To implicate PKC activity in PMA-mediated regulation of the interaction between DGKc and pRB, we pre-treated COS-7 cells overexpressing DGKc with Ro 31-8220, a specific PKC inhibitor, prior to PMA treatment. Pre-treatment with Ro-31-8220 completely rescued the PMA-mediated inhibition of DGKc binding to pRB (Fig. 1B), indicating that PMA-mediated activation of PKC regulates DGKc binding to pRB. As a control, DGKc shows weak or no binding to the small pocket region (A+B) of pRB (Fig. 1B), like we described previously (Los et al., 2006).

3 . 2 . M i m i c k i n g o f P KC- m e d i a t e d p h o s p h o r y l a t i o n o f s e r i n e r e s i d u e s i n t h e D G K

c

c

To investigate whether PKC-specific serine phosphorylation sites in the DGKc- MARCKS-PSD are involved in regulating DGKc binding to pRB, we used DGKc- MARCKS-PSD mutants that either mimic phosphorylation (serine to aspartate substitution, S/D) or cannot be phosphorylated (serine to asparagine substitution, S/N) (Fig. 2A). Wild-type DGKc and both types of serine mutants were overexpres- sed in COS-7 cells and lysates were used for affinity-purification using GST-pRB fusion proteins. Whereas wild-type- and S/N-DGKc bound to ^GST-pRB, binding of S/D-DGKc to p^RB was impaired (Fig. 2B), suggesting that, indeed, phosphorylation of serines in the DGKc-MARCKS-PSD inhibits the interaction between DGKc and pRB. Treatment of cells with PMA reduced the pRB binding of wild-type DGKc

Fig. 3.

S/D-DGKc-MARCKS-PSD peptide has reduced affinity for pRB.

(A) Biotinylated wild-type -, S/D- or S/N mutantDGKc-MARCKS -PSD peptides were incubated with MCF7 cell lysate for 16 hours. Peptides were immobilised on streptavidin agarose beads and extracted proteins were separated by SDS-PAGE, transferred to nitrocellulose and stained for pRB using a pRB- specific antibody. (B)S/D-DGKc-MARCKS -PSD mutant peptide has reduced capacity to compete with full-length DGKc for GST-pRB binding. Lysates of COS-7 cells overexpressingDGKc were incubated for 2 hours with 1 or 10 +g of indicated peptides together with GST-pRB immo - bilized on glutathione - sepharose 4B beads. Affinity-purified DGKc was visualised by immunoblotting using an anti-DGKc antibody.

(like in Fig. 1) to the level of (non-treated) S/D-DGKc. Furthermore, PMA reduced the binding of wild-type DGKc more than S/N-DGKc (Fig. 2B), in agreement with the lack of PKC-mediated phosphorylation at these S/N mutation sites. We note, how- ever, that TPA further reduced the binding of S/D-DGKc and ^S/N-DGKc mutants to GST-pRB (Fig. 2B), suggesting that other phosphorylation sites within DGKc or pRB are also involved in the negative regulation of DGKc binding to pRB by PKC. We previously used a DGKc-MARCKS-PSD peptide to extract pRB from MCF7 cells (Los et al., 2006). Here, we tested whether biotinylated DGKc-MARCKS-PSD peptides containing the S/D or S/N substitutions would be capable of binding to pRB. Like the wild-type peptide, the S/N mutant peptide was capable of extracting pRB from MCF7 cells, whereas pRB binding to the S/D mutant peptide was impaired (Fig. 3A). Furthermore, both wild-type- and S/N mutant peptides could compete with full-length DGKc for p^RB binding (Fig. 3B, lane 5 and 7), whereas the S/D pep- tide competed less efficiently (lane 6). All together, these results indicate that mimicking of PKC-mediated phosphorylation of the DGKc-MARCKS-PSD reduces DGKc binding to pRB.

Fig. 4.

PKC activation inhibits the interaction between DGKc and pRB.

(A)COS-7 cells were co -transfected with DGKc and PKC isoforms indicated and stimulated for 20 minutes with PMA (100 nM). Lysates were used to affinity-purify DGKc using GST-pRB or GST-pRB(A+B) as a control. Affinity-purified DGKc was visualised by Western blotting using aDGKc- specific antibody (left panel).

Total cell lysates were immunoprobed for DGKc and PKC isotopes to demon- strate expression in the various lysates.

(B) Histogram showing the relative amounts of DGKc bound to pRB. Data are means ± S.E.M. of six independent experiments. (* ) represents the signifi- cantly enhanced PMA-induced inhibition ofDGKcbinding by PKC overexpression (p < 0.05), as determined by analysis of variance.

3 . 4 . P KC

_

c

b i n d i n g t o pR B

To determine which PKC regulates the interaction between DGKc and pRB, we co-expressed DGKc with different PKC isoforms (_, `1, `2, b, ¡) in COS-7 cells, stimulated PKC activity by PMA, and performed affinity-purifications using GST- pRBfusion proteins. In agreement with previous experiments, DGKc binding to p^RB was inhibited when cells were treated with PMA (Fig. 4A andB). However, when PKC_ was co-expressed with DGKc, a further (significant) inhibition of binding of DGKc to pRB was found. The other PKC isoforms (of which only `1 is included in Fig. 4) did not significantly enhance the PMA-induced inhibition of DGKc binding to pRB. This result suggests that activation of PKC_ in particular can regulate the interaction between DGKc and pRB. To support this notion, we show in Figure 5 that transfection of dominant-negative PKC_ counteracts the PMA-induced inhibi- tion of DGKc binding to GST-pRB.

To demonstrate that DGKc is phosphorylated in vivo by ^PKC_,^COS-7 cells were transfected with DGKc, incubated with [^32P]orthophosphate to label the ATP pool, and then stimulated with PMA. Unstimulated cells showed basal levels of DGKc phosphorylation, which was increased 2-fold upon stimulation with

Fig. 5.

Dominant-negative PKC_ counteracts the PMA-induced inhibition of DGKc binding to pRB.

COS-7 cells were co -transfected with DGKc and wild-type PKC_ or dominant- negative (dn)PKC_, where indicated, and stimulated for 20 minutes with PMA (100 nM), where indicated. Cell lysates were used to affinity-purify DGKc using GST-pRB or GST-Cdc42 as a control.

Equal amounts of the GST-fusion pro - teins were used, as shown in the Western blots. Affinity-purified DGKc was visual- ised by Western blotting using a DGKc- specific antibody (upper panel). Total cell lysates were immunoprobed for DGKc and PKC_ content (middle panel).

The histogram (low er panel) represents a quantification of GST-pRB-bound DGKc protein levels of the upper panel.

It depicts the ratios (in %) of the amount of DGKc bound with/without PMA stimulation in control cells and in cells overexpressing PKC_ or dnPKC_.

Fig. 6.

DGKc is phosphorylated by PKC (PKC_).

COS-7 cells were transfected with FLAG- DGKc and/or PKC_ (as indicated) and incubated with [³²P] orthophosphate for 3 hours. Cells were kept as controls, or were stimulated with PMA (100 nM) for 20 minutes, as indicated. FLAG- DGKc was immunoprecipitated using aFLAG- specific antibody and immu- noprecipitated proteins were separated bySDS-PAGE and transferred to nitro - cellulose. Radiolabelled DGKc was visu- alised by autoradiography (upper pan- el). Total DGKc was visualised using aFLAG- specific antibody (lower panel).

PMA (Fig. 6, lane 1 and 2). In cells that co-expressed PKC_,PMA treatment further enhanced the phosphorylation of DGKc by 1.6-fold compared to PMA alone (Fig. 6, upper panel, lane 2 and 3). These results indicate that DGKc is phosphorylated by PKC_ in vivo.

4. Discussion

The present study demonstrates that the direct physical interaction between pRB and DGKc is regulated by PKC, particularly PKC_. Activation of PKC by PMA inhibits DGKc binding to pRB, and this inhibition could be prevented by transfec- tion of dominant-negative PKC_ or by preincubation with a PKC-specific inhibitor.

Furthermore, we have shown that phosphorylation of serine residues in the DGKc-^MARCKS-PSD is sufficient for inhib iting DGKc binding to p^RB, suggesting that PKC_ disrupts the interaction between p^RB and DGKc by phosphorylating the DGKc-MARCKS-PSD. The conclusions and some physiological implications of our results are summarised in the cartoon depicted in Figure 7.

In addition to the regulation of DGKc binding to pRB,PKC_-mediated phosphorylation of the DGKc-MARCKS-PSD has also been shown to disrupt bind- ing of PKC_ to DGKc, even though the catalytic domain of DGKc is the PKC_ binding site (Luo et al., 2003a). In N1E-115 neuroblastoma cells, phos phorylation of the DGKc-MARCKS-PSD was shown to negatively regulate Rac1 binding to the C1 domain of DGKc (Yakubchyk et al., 2005). Furthermore, in COS-7 cells, Jurkat T-cells, C2 myoblasts and N1E-115 neuroblastoma cells, phosphorylation of the DGKc-^MARCKS-PSD regulates DGKc loca lisation (Topham et al., 1998; Santos et al., 2002; Abramovici et al., 2003; Yakubchyk et al., 2005). Thus, DGKc-MARCKS-PSD phosphorylation appears to be a general way of regulating DGKc functioning.

PKC_-mediated regulation of DGKc binding to pRB adds a new level of complexity by which PKC_ and DGKc regulate each others activity. We have previously shown that pRB binding to DGKc stimulates DGKc activity (Los et al., 2006). Since phos- phorylation of DGKc by PKC_ causes nuclear export of DGKc (Topham et al., 1998), it seems likely that this export is facilitated by the PKC_-mediated dissociation of the DGKc-p^RB complex, leaving ‘free’ pRB in the nucleus (Fig. 7). With this dis- sociation, PKC_ at the same time inhibits p^RB activation of DGKc in the nucleus.

Since DGKc activity can furthermore be inhibited upon phosphorylation by PKC_ (Luo et al., 2003b), it can be concluded that pRB and PKC_ have opposing effects on DGKc activity.

Regulation of DGKc activity by pRB and PKC_ in the nucleus supposedly affects nuclear DAG levels. Since DGKc binds to the hypophosphorylated form of pRB (Los et al., 2006), we hypothesise that DGKc keeps nuclear DAG levels low during the G1 phase of the cell cycle and in quiescent cells. When PKC becomes activated, it phosphorylates DGKc and inhibits binding of DGKc to pRB. Thereby, DGKc becomes less active and is exported from the nucleus, causing nuclear DAG levels to increase (Fig. 7). Increased nuclear DAG levels may further activate PKC or recruit more PKC_ to the nucleus. This feed-forward mechanism might contribute to cell cycle regulation. In accordance with this hypothesis, stimulation of Swiss 3T3 cells with IGF-1 caused an increase in nuclear DAG levels, which recruited PKC_ to the nucleus and increased the number of cells entering S-phase (Neri et al., 1998). In contrast, but along the same lines, nuclear DAG levels decrease in MEL cells that were stimulated to terminally differentiate (Divecha et al., 1995;

D'Santos et al., 1999), whereas artificially increasing nuclear DAG levels (by overexpression of nuclear PLC`) inhibits MEL cell differentiation (Matteucci et al., 1998).

How PKC activation contributes to cell cycle progression needs to be further investigated. PKC can positively regulate proliferation by regulating enzymes involved in DNA synthesis and nuclear structure, including DNA-polymerase `,

Fig. 7.

Model for the PKC-mediated regulation of DGKc binding to pRB.

U nder basal conditions, DGKc binds to pRB and stimulates DGKc activity to keepDAG levels low and PKC (PKC_ inactive. Growth factor-induced DAG formation in the nucleus or addition of PMA activates PKC that now phosphor- ylatesDGKc on serines in the DGKc- MARCKS -PSD. This phosphorylation disrupts the interaction between DGKc and pRB, inhibits DGKc activity and leads to DGKc exit from the nucleus (Topham et al., 1998).

topoisomerase, lamins, and histones (Tokui et al., 1991; Pommier et al., 1990;

Hocevar et al., 1993; Iwasa et al., 1980). In contrast, however, PKC isoforms, includ- ing PKC_, can negatively regulate ^S-phase entry by inhibiting expression of cyc- lins and CDKs and by induction of p21^WAF/CIP1 causing hypophosphorylation of pRB and cell cycle arrest (Frey et al., 1997; Black, 2000). Furthermore, depending on the timing of PKCactivation during G1, cell proliferation can be stimulated or inhibited (Z hou et al., 1993). We suggest that positive and negative regulation of DGKc by pRB and PKC respectively are likely to be important in the spacio-temporal modu- lation of nuclear DAG levels which in turn may be involved in regu lating aspects of cell cycle progression.

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