Diacylglycerol kinase theta and zeta isoforms: regulation of activity,
protein binding partners and physiological functions
Los, Alrik Pieter
Citation
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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I s t h e r e a r o le fo r
d ia c y lg ly c e r o l k in a s e - c
in c e ll c y c le r e g u la t io n ?
Alrik P. Los
J oh n d e W id t
W im J . v a n B lit t e rsw ijk
N u llin D iv e c h a
Chapter 6
A b stract
W e p rev iously show ed that diacylglycerol kinase- c
(
D G Kc ) b inds to and is activ ated b y the retinob lastom a
p rotein (p
R B) and its fam ily m em b ers. In search of a
p hysiological function of the interaction b etw een
D G Kc
and p
R Bfam ily m em b ers, w e inv estigated a p ossib le role
of
D G Kc in regulating the cell cycle. H ow ev er, ov erex -
p ression of
D G Kc in different cell lines did not affect the
cell cycle, neither did
D G Kc affect the p
R B-dep endent
G 1-arrest induced b y v arious stim uli. In addition,
D G Kc -
deficient m ouse em b ryonic fib rob lasts (
M E Fs) show ed
the sam e cell cycle p rofile as control cells. O ur results
indicate that ov erex p ression or deletion of
D G Kc neither
influences ex p onential cell grow th nor
G 1-arrest. In our
p rev ious studies,
D G Kc could reconstitute a
G 1-arrest in
a -irradiated p
R B -/-M E Fs, indicating that the inv olv em ent
of
D G Kc in cell cycle regulation is only ap p arent under
som e sp ecific conditions.
Is there a role for diacylglycerol kinase-c in cell cycle regulation?
118
Introduction
The retinoblastoma tumour suppressor protein (pRB) and its close relatives p10 7 and p13 0 are master switches in the reg ulation of developmental processes, in- cluding cell cy cle reg ulation, dif f erentiation and apoptosis (Ng uy en and M cC ance, 2 0 0 5 ; C hau and Wang , 2 0 0 3 ; C obrinik, 2 0 0 5 ) . During the cell cy cle, pRB reg ulates G1 to S-phase transition by inhibiting the E2 F f amily of transcription f actors, thereby repressing E2 F targ et g enes and keeping cells in G1 or G0 (Seville et al., 2 0 0 5 ) . When conditions are optimal f or a cell to divide, cy clin-dependent kinases (C DKs) phosphory late pRB which liberates E2 F. I n G1, cy clin D in complex with C DK4 or C DK6 , and cy clin E-C DK 2 reg ulate pRB phosphory lation (Sherr and Roberts, 2 0 0 4 ) . C y clin-C DK complex es, in turn, are reg ulated by C DK inhibitors (C K I) . The C I P/K I P f amily of C K Is, including p2 1WAF 1/C I P1, p2 7K I P1 and p5 7K I P2, inhibit cy clin E-C DK com- plex es, whereas they stabilise and activate cy clin D-C DK4 /6 complex es. The I NK f amily of C K Is, including p16I NK 4 a, p15I NK 4 b, p18I NK 4 c and p19I NK 4 d, specif ically interact with C DK4 /6 , thereby blocking D-ty pe cy clin binding to C DK4 /6 and preventing C DK activation (Sherr and Roberts, 19 9 9 ) . The activity of cy clin-C DK complex es and ex pression of C K Is are reg ulated by many sig nalling pathway s to tig htly reg ulate pRB phosphory lation and, with that, G1 to S-phase transition (M assag ue, 2 0 0 4 ) .
When cells are stimulated with mitog ens, cy clin D-C DK4 /6 complex f ormation is stimulated at dif f erent levels. C y clin D transcription is stimulated, assembly of cy clin D-C DK4 /6 complex es is enhanced, and nuclear localisation and protein stability are increased (Sherr and Roberts, 19 9 9 ) . F or ex ample, activation of the Ras-ERK pathway promotes cy clin D1 transcription and cy clin D1-C DK4 complex assembly (Peeper et al., 19 9 7 ; C heng et al., 19 9 8 ) . Accumulation of cy clin D-C DK4 /6 complex es reg ulate cell cy cle prog ression by phosphory lating pRB and by binding to and seq uestering p2 1WAF 1/C I P1 and p2 7K I P1, allowing cy clin E/C DK2 complex es to become active (Sherr and Roberts, 19 9 9 ) . I n late G1, activated cy clin E-C DK2 phos- phory late p2 7K I P1, thereby targ eting it f or deg radation, and allowing f urther phos- phory lation of pRB at specif ic sites. At this point, cells will f inish the cell cy cle independent of mitog ens. H ig hly phosphory lated inactive pRB releases E2 F that subseq uently binds to E2 F binding sites to activate promoters of g enes involved in S-phase, including g enes req uired f or DNA sy nthesis.
U nder several conditions pRB phosphory lation is inhibited, causing a pRB- mediated G1-arrest. F irst, when cells are depleted of mitog ens, assembly of cy clin D-C DK4 /6 complex es is not stimulated any more and complex es are deg raded. The pool of p2 1WAF 1/C I P1 and p2 7K I P1 that was seq uestered by cy clin D-C DK4 /6 is released and inhibit cy clin E-C DK2 , causing a pRB-mediated G1-arrest (Sherr and Roberts, 19 9 9 ) . Second, several g rowth inhibiting sig nals, including members of the TGF` superf amily and contact inhibition, induce sy nthesis of p15I NK 4 b, p2 1WAF 1/C I P1 and p2 7K I P1 that maintain pRB in its active low phosphory lated state (Poly ak et al., 19 9 4 ; Rey nisdottir et al., 19 9 5 ; Li et al., 19 9 5 ) . Third, in replicative senescence p16I NK 4 a as well as p2 1WAF 1/C I P1 are induced (Alcorta et al., 19 9 6 ; Noda et al., 19 9 4 ) . U preg ulation of p16I NK 4 a also occurs in primary f ibroblasts ex pressing oncog enic Ras, causing a cell cy cle arrest that resembles senescence (Serrano et al., 19 9 7 ) .
In accord with a role of p16INK4a in pRB-signalling, overexpression of p16INK4a causes aG1-arrest that is dependent on functional pRB proteins (Bruce et al., 2000).
Fourth, DNA replication errors and DNA damage induced by radiation or chemical agents causes a pRB-mediated cell cycle arrest to allow a cell to repair its DNA. Recognition of DNA damage leads to the activation of the p53 tumour suppressor protein that induces many proteins including p21WAF1/CIP1 that inhibit proliferation.
pRB is essential in radiation-induced G1-arrest, since pRB-null MEFs keep prolifer- ating (Harrington et al., 1998; Brugarolas et al., 1999).
We have previously shown that pRB, p107 and p130 bind to and stimulate dia- cylglycerol kinase c (DGKc), one of the ten mammalian DGK isoz ymes identified that phosphorylate the lipid second messenger diacylglycerol (DAG) to produce phosphatidic acid (PA).DAG participates via its targets, such as protein kinase C (PKC),Ras guanyl nucleotide-releasing protein (Ras-GRP), chimaerins and Munc- 13, in the regulation of many cellular processes, including cell division, differen- tiation, actin-remodeling and vesicular trafficking (Brose et al., 2004). Recent evi- dence also suggest that the DGK product PA acts as a second messenger to acti vate mTO R and can regulate transcription factors (Loewen et al., 2004; Avila-Flores et al., 2005). O verexpression of DGKc in T cells inhibits Ras activation by inhibiting DAG-mediated activation of Ras-GRP, a Ras exchange factor that activates Ras (Z hong et al., 2002; Topham and Prescott, 2001). Conversely, in T cells derived from DGKc-null mice, stimulation of T-cell receptors causes prolonged Ras-ERK acti- vation and enhanced T-cell receptor response (Z hong et al., 2003). In addition, T cell receptor-induced proliferation was enhanced in DGKc-deficient T cells com- pared to wild-type cells ex vivo. In contrast, overexpression of DGKc in HEK293 cells increased cell doubling time and, in CO S-7 cells, DGKc arrested cells in G1 (Topham et al., 1998). These results indicate that modulation of DGKc expression can have strong effects on cellular signalling.
Here we investigated if DGKc is involved in pRB-mediated cell cycle regula- tion. To this end, we overexpressed wild-type DGKc or a kinase-inactive DGKc mutant in different cell systems to determine whether DGKc could affect cell cycle distribution or pRB-mediated G1-arrest. Furthermore, we checked for possible cell cycle abnormalities in DGKc-null MEFs.
Materials and methods
C e l l c u l t u r e , t r a n s f e c t i o n s a n d t r a n s d u c t i o n s
HEK293,U2O S,MCF7,Swiss3T3,MEFs and Phoenix cells were cultured in Dulbecco’s modified Eagle’s medium containing 8% heat-inactivated fetal calf serum, 2 mM glutamine and antibiotics. HEK293, Phoenix and U2O S cells were transfected using the calcium phosphate precipitation method. To transduce Swiss3T3 and MCF7 cells, phoenix packaging cells were transfected with DGKc-pBabe-puro or LZ RS- DGKc-Z eo. Retroviral supernatants were harvested 48 hours after transfection, filtered through a 0.45 +m filter and incubated with 10 +g/mlDO TAP (Roche) for
Is there a role for diacylglycerol kinase-c in cell cycle regulation?
120
10 minutes on ice before adding it to the cells. Cells were selected with 2 +g/ml puromycin or 300 +g/ml zeocin to generate cells stably expressing DGKc. Expres- sion of all transfected or transduced cells was checked by Western blotting using aDGKc-specific antibody.
C e l l c y c l e a s s a y s
To analyse the cell cycle profile of HEK293,U2OS,MCF7,Swiss3T3,DGKc-/- and wild- type MEFs, exponentially growing cells were labelled with BrdU and analysed (see below). To determine cell cycle re-entry after release from confluency, 1 million MCF7 cells were seeded in 6-well plates, and after 48 hours, medium was refreshed, and 24 hours later, cells were split 1:6 in 6 cm dishes. Cells were BrdU-labelled at different time-points after splitting. For confluency-induced cell cycle arrest, 1 million MCF7 cells were seeded in 6-well plates and fixed at 1 day and 2 days of confluency. As a control, 200,000 cells were seeded in 6 cm dishes to determine cell cycle profiles of exponential growing cells. a-Irradiation-induced G1-arrest of MCF7 cells was performed as described (Los et al., 2006). For serum starvation-induced G1-arrest, 100,000 Swiss3T3 cells were seeded in 6 cm dishes. 48 hours after seed- ing, cells were serum-starved and fixed after different time-points. To determine
p16INK4A-mediated G1-arrest, 200,000 U2OS cells were seeded in 6 cm dishes and
transfected after 24 hours. 48 hours after transfection, cells were treated with 1 +g/ml nocodazole for 30 hours and fixed.
F l o w c y t o m e t r y
For cell cycle profile analysis, cells were labelled for 1 hour with 10 +M 5-Bromo- 2'-deoxy-uridine (BrdU) (Roche) to detect cells that replicate their DNA. Cells were trypsinised and fixed in ice-cold 70% ethanol. BrdU staining was performed in 96-well plates. Samples were RNase-treated and incubated with DNA-denatur- ing solution (5 M HCl, 0.5% Triton) for 20 minutes at room temperature. For neutra- lisation, cells were washed twice with 1 M Tris-HCl pH 8.0. After washing the cells twice withPBS/0.5% Tween-20, cells were resuspended in anti-BrdU solution (1:40 anti-BrdU(DAKO), 1% BSA in PBS/Tween) and incubated for 30 minutes. Cells were washed twice with PBS/Tween and incubated for 30 minutes with secondary antibody solution (1:20 FITC-conjugated anti-mouse antibody (DAKO), 1% BSA in PBS/Tween). After subsequent washing, DNA was stained with 50 +g/ml propidium iodide (Sigma) in PBS. Samples were analysed by flow cytometry and quantified using FCS express 2.
Cells that were not labelled with BrdU were trypsinised and fixed in ice-cold 70% ethanol. After removing the ethanol, cells were incubated for 20 minutes in PBS containing 50 +g/ml propidium iodide and 50 +g/mlRNase, prior to flow cytometry analysis.
We s t e r n b l o t t i n g
DGKc expression was determined by Western blotting as described in (Los et al., 2006).
Results
To determine whether or not DGKc could affect cell cycle distribution, we over- expressed DGKc in different cell lines. Human embryonic kidney (HEK293) cells and the human osteosarcoma cell line U2OS were transiently transfected, and Swiss 3T3 fibroblasts and MCF7 breast cancer cells were retrovirally transduced and selected. Both transiently overexpressing wild-type DGKc (wtDGKc) cell lines (Fig. 1A and B) and stable DGKc-expressing cells (Fig. 1C and D) showed the same number of cells in either G1,S or G2/M phases of the cell cycle compared to vector- transfected control cells. Overexpression of kinase-inactive DGKc (kdDGKc) also did not affect the number of cells in each phase of the cell cycle (Fig. 1B and 1D).
These results indicate that neither overexpression of wtDGKc nor kdDGKc affect cell cycle distribution.
To investigate whether or not DGKc overexpression would affect pRB-medi- ated cell cycle regulation, we determined the effect of DGKc on cell cycle entry after release from contact inhibition and on pRB-mediated cell cycle arrest. To inves- Fig. 1.
Exponentially growing cell lines over- expressing wild-type or kinase-dead DGKc have the same cell cycle profile as control cells.
DGKc- or vector-transfected HEK293 cells (A),U2OS cells (B),MCF7 (C) and Swiss3T3 cells (D) were labelled with BrdU 4 8 hours after seeding and fixed.
DN A was stained with propidium iodide,
whereas incorporated BrdU was detect- ed by immunostaining. Cell cycle pro - files were analysed by flow cytometry (upper panels). Data are means ± S.E. (n= 3 ) (A), means ± the range of the duplicates (n= 2) (D) or from single samples (B and C).DGKc expression was checked by Western blotting (lower panels).
Is there a role for diacylglycerol kinase-c in cell cycle regulation?
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tigate whether DGKc influences S-phase entry after releasing cells from contact inhibition-mediated G0/G1-arrest, we used MCF7 cells stably expressing wtDGKc or vector that were kept confluent for three days and were arrested in G0/G1.
Cells were split to allow them to re-enter the cell cycle and the number of cells entering S-phase was measured by BrdU incorporation. wtDGKc- and vector-over- expressing MCF7 cells showed essentially the same kinetics for entry into S-phase after release from confluency (Fig. 2). These results suggest that DGKc did not affect the number of cells re-entering the cell cycle nor did it affect the kinetics of re-entry.
To investigate whether DGKc is involved in pRB-mediated G1-arrest, we treat- ed cells overexpressing DGKc with growth-inhibiting signals that block the cell cycle in a pRB-dependent way, namely contact inhibition, DNA damage, serum starvation, and p16INK4A overexpression. As for contact inhibition, equal amounts of MCF7 cells were seeded that stably expressed wtDGKc or vector and cell cycle distribution was analysed when cells were just confluent (day 0), and 1 day or 2 days post-confluency. Both cell lines showed the same increase in cells in G1 at increasing confluencies (Fig. 3A), suggesting that DGKc does not affect G1-arrest upon contact inhibition.
DNA-damage-induced cell cycle arrest was also investigated in MCF7 cells stably expressing wtDGKc or vector. Cells were irradiated with different doses of a-irradiation and, after 30 minutes, treated with nocodazole for 30 hours to arrest cycling cells in G2/M. As is shown in Figure 3B, DGKc and vector-expressing cells showed the same dose-dependent increase in G1-phase cells, suggesting that overexpression of DGKc does not influence radiation-induced G1-arrest.
When cells are deprived of growth factors, they arrest in G0/G1. Subsequent mitogen stimulation induces pRB phosphorylation, allowing re-entry into the cell cycle. To investigate whether DGKc regulates starvation-induced G1-arrest, exponentially growing Swiss3T3 fibroblasts stably expressing wtDGKc, kdDGKc or vector were serum-starved and after different time-points, cell cycle distri- bution was analysed. Figure 3 C shows that all cell lines have the same kinetics of arrest in G0/G1, regardless of wtDGKc or kdDGKc overexpression.
Finally, we tested whether or not DGKc influences G1-arrest induced by artifi- cially activating the pRB pathway. U2OS osteosarcoma cells are deficient in p16INK4A, a lesion commonly observed in many tumours. Reconstitution of the expres sion
Fig. 2.
DGKc overexpressing MCF7 cells enter S-phase in the same time interval as control cells af ter release from con- f luency.
Cell were kept confluent for 3 days and then released from confluency.
At different time -points after release, cells were labelled with BrdU, fixed and stained for BrdU and analysed by flow cytometry.
Fig. 3.
G1-arrest by diff erent stimuli is not aff ected by overexpression of DGKc.
A. Exponentially growing (day 0) and 1 or 2 days- confluent MCF7 cells stably expressingDGKc or empty vector were fixed in ethanol, stained with propidium iodide and cell cycle profiles were ana- lysed by flow cytometry. B. Stable MCF7 cells were a-irradiated 48 hours after seeding. 30 minutes after irradia- tion cells were treated with 1 +g/ml nocodaz ole for 30 hours and fixed with ethanol. Samples were stained with propidium iodide and analysed. C. Swiss3T3 cells stably expressing wtDGKc, kdDGKc or vector were serum- starved 48 hours after seeding. At dif- ferent time -points of serum starvation, cells were trypsinised, fixed in ethanol and stained with propidium iodide.
The number of cells in G1 were deter- mined by flow cytometry. D.U2OS cells were co -transfected with DGKc con- structs with or without p16INK4a. To select for transfected cells, GFP was co -transfected. 48 hours after transfec- tion, cells were treated with nocodaz ole for 30 hours to arrest cycling cells in G2. Cells were fixed, stained with pro - pidium iodide and cell cycle profiles ofGFP-positive cells were analysed by flow cytometry. Data are representative of three independent experiments with similar results.
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of p16INK4A inhibits cyclin D-CDK mediated pRB phosphorylation, leading to a G1-arrest. We co-transfected U2OS cells with wtDGKc, kdDGKc or vector with or without p16INK4A, treated the cells 48 hours post-transfection with nocodazole to arrest cycling cells in G2/M and analysed the cell cycle profile. As is shown in Figure 3D, p16INK4A causes an increase in cells in G1, which was not affected by overexpression of either wtDGKc or kdDGKc. All together, our results indicate that overexpression of wtDGKc or kdDGKc did not affect pRB-mediated G1-arrest induced by different growth inhibiting stimuli.
Since we were unable to find an affect of DGKc on the cell cycle in overexpres- sion studies, we determined cell cycle profiles of wild-type and DGKc-/- mouse embryonic fibroblasts (MEFs). As depicted in Figure 4, DGKc-/-MEFs showed the same cell cycle profile as wild-type MEFs, indicating that DGKc-deficient MEFs require the same time to pass each phase of the cell cycle compared to wild-type MEFs under exponential growth conditions.
Discussion
In this study we have investigated the role of DGKc in regulating the cell cycle.
Overexpression of DGKc did not affect the cell cycle profile of four different cell lines tested. In addition, DGKc-/-MEFs did not show any difference in their cell cycle profiles compared to wild-type MEFs. Since we have shown that DGKc binds to pRB, p107 and p130 (Los et al., 2006), we determined whether DGKc is involved in pRB-mediated growth arrest. However, we were unable to find effects of DGKc on cell cycle re-entry after release from contact inhibition and on pRB-mediated G1-arrest induced by diverse stimuli in the different cell systems.
Our data are in conflict with data previously obtained by Topham and co- workers. They showed that overexpression of DGKc caused a G1-arrest in COS-7 cells and an increased doubling time in HEK293 cells (Topham et al., 1998). We can not provide a simple explanation for these conflicting results. They may be a consequence of differences in cell culturing or in differences in the original cell lines.
Fig. 4.
DGKc-/-MEFs have the same cell cycle profile as wild-type MEFs.
DGKc-/-MEFs and control MEFs were labelled with BrdU 48 hours after seed- ing and fixed. DNA and incorporated BrdU was stained with PI and an anti- BrdU antibody respectively and cell cycle profiles were analysed by flow cytometry. Data are means ± S.E. (n=5).
Our results, however, do not completely exclude a role for DGKc in the cell cycle, asDGKc may still regulate cell cycle progression under more specific conditions.
For example, we have previously shown that overexpression of DGKc could recon- stitute a cell cycle arrest in pRB-/-MEFs upon a-irradiation (Los et al., 2006). In the present study, however, overexpression of DGKc in MCF7 cells did not affect radiation-induced G1-arrest, indicating that just the overexpression of DGKc in cells that are normally arrested upon irradiation does not have a regulatory effect.
Furthermore, evidence that DGKc is involved in cell cycle regulation was obtained by Koretzky and co-workers: in DGKc-/- mice, T cells from lymph nodes and spleen proliferate faster upon T cell receptor stimulation compared to T cells of wild- type mice (Zhong et al., 2003).
How DGKc regulates the cell cycle specifically in irradiated pRB-/-MEFs and in T cells needs to be further determined. It has been shown that DGKc negatively regulates the Ras-ERK pathway, since DGKc-deficient T cells showed enhanced Ras activation (Zhong et al., 2003), which may explain the enhanced cell prolife- ration. DGKc negatively regulates Ras activation by inhibiting DAG-mediated activation of Ras guanyl nucleotide-releasing protein (Ras-GRP) that stimulates GTP binding to Ras (Topham and Prescott, 2001; Zhong et al., 2002). In addition, pRB-/-MEFs that are unable to arrest in G1 upon irradiation (Harrington et al., 1998), also showed enhancedRas activation (Lee et al., 1999), although the mechanism leading to upregulated Ras activation has not been defined. Whether the inability of pRB-/-MEFs to arrest in G1 upon irradiation is due to elevated levels of active Ras is not known. However, DGKc may reconstitute a cell cycle arrest in pRB-/-MEFs by inhibiting DAG-mediated activation of Ras-GRP, thereby negatively regulating Ras signalling and making pRB-/-MEFs susceptible for a-irradiation.
In summary, we have shown that cells overexpressing DGKc and DGKc-/-MEFs have similar cell cycle profiles as control cells and that DGKc does not affect pRB- mediated G1-arrest induced by different stimuli. However, under more specific conditions, i.e. in pRB-/-MEFs and in stimulated T cells, DGKc was shown to be involved in cell cycle regulation. Further studies are required to clarify the role of DGKc in those cell systems and the role of the Ras-ERK pathway as a downstream effector of DGKc.
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Is there a role for diacylglycerol kinase-c in cell cycle regulation?
