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

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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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Institutional Repository of the University of Leiden

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

fa m ily p r o t e in s b in d

t o a n d a c t iv a t e

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

(JBC. 2006 Jan 13;281(2):858-866)

Alrik P. Los

F a b ia n P. V in ke

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 4

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A bstract

The retinoblastoma protein p

R B

is a tu mou r su ppressor

and key regu lator of the cell cycle. W e have previou sly

show n that p

R B

interacts w ith phosphatidylinositol-4 -

phosphate 5-kinases (

P I P 5K

), lipid kinases that can regu -

late

P I

( 4 ,5)

P 2

levels in the nu cleu s. H ere, w e investigated

p

R B

binding to another lipid kinase in the phospho-

inositide cycle: diacylglycerol kinase (

D G K

) that phos-

phorylates the second messenger diacylglycerol (

D A G

)

to yield phosphatidic acid (

P A

). W e fou nd that

D G K

c , bu t

not

D G K

_ or

D G K

e , interacts w ith p

R B

in vitro and in

vivo. B inding of

D G K

c to p

R B

is dependent on the phos-

phorylation statu s of p

R B

, since only hypophosphorylat-

ed p

R B

interacts w ith

D G K

c .

D G K

c also binds to the

p

R B

-related pocket proteins p1 0 7 and p1 3 0 in vitro and

in cells. W hile

D G K

c did not affect the ability of p

R B

to

regu late

E 2 F

-mediated transcription, w e fou nd that p

R B

,

p1 0 7 and p1 3 0 potently stimu late

D G K

c activity in vitro.

F inally, overex pression of

D G K

c in p

R B

-nu ll fibroblasts

reconstitu tes a cell cycle arrest indu ced by a -irradiation.

These resu lts su ggest that

D G K

c may act in vivo as a

dow nstream effector of p

R B

to regu late nu clear levels of

D A G

and

P A

.

The retinoblastoma family proteins bind to and activate diacylglycerol kinase-c

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Introduction

Diacylglycerol (DAG) regulates many cellular processes including prolif eration, dif f erentiation and cell migration by modulating the activity of several proteins, such as protein kinase C (PKC) ,Ras guanyl nucleotide-releasing proteins (Ras- GRP) , chimaerins, and Munc-1 3 (Brose et al., 20 0 4 ) . DAG can be produced by the action of several dif f erent signal transduction pathways including phospholipase C (PLC) -mediated hydrolysis of phosphoinositides (PI) or phosphatidylcholine, phospholipase D-mediated hydrolysis of phosphatidylcholine f ollowed by dephos- phorylation of phosphatidic acid (PA) , and during de novo synthesis of phospho - l ipids (van Blitterswijk et al., 1 9 9 4 ) .

DAG is not only produced at the plasma membrane but at other intracellular sites as well, including the nucleus. Nuclear DAG levels are increased in liver as a conseq uence of two thirds partial hepatectomy (Banf ic et al., 1 9 9 3 ) and in cell cultures treated with insulin-like growth f actor 1 (I GF-1 ) , which stimulates prolif - eration (Divecha et al., 1 9 9 1 ; Martelli et al., 1 9 9 2) . This suggests that nuclear DAG levels are intimately linked with cell cycle progression, but a causal relationship has not been f irmly established. An attractive hypothesis is that nuclear DAG sti mulates cell cycle progression via a DAG-binding protein such as PKC (Nishiz uka, 1 9 9 5 ;Brose et al., 20 0 4 ) . I ndeed, DAG in the nucleus recruits and activates PKC in response to I GF-1 stimulation of Swiss 3 T3 cells, which is req uired f or G1 to S-phase transition (Divecha et al., 1 9 9 1 ; Neri et al., 1 9 9 8 ) . H owever, the role of PKC in regulating the cell cycle is complex , with dif f erent PKC isof orms inducing a cell cycle arrest or stimulation of cell cycle progression. Furthermore, the same PKCisof orm is able to induce both an arrest and progression through the cell cycle when ex - pressed in dif f erent cell types (Livneh and Fishman, 1 9 9 7 ) .

I n the nucleus, DAG kinase (DGK) controls the levels of DAG generated f rom PI-PLC-mediated hydrolysis of PI(4 ,5 )P₂ (D’Santos et al., 1 9 9 9 ) and nuclear DGK activity can be stimulated in response to both growth f actor (Martelli et al., 20 0 0 ) and peptide-hormone treatment (Bregoli et al., 20 0 1 ) . DGKc (Fig. 1 A) is one of ten dif f erent DGK isof orms identif ied to date (Luo et al., 20 0 4 ; van Blitterswijk and H oussa, 20 0 0 ) . DGKc contains a nuclear localiz ation signal (^NLS) (Goto and Kondo, 1 9 9 6 ; Topham et al., 1 9 9 8 ) and DGKc has indeed been shown to be nuclear in some cell types (H oz umi et al., 20 0 3 ; H ogan et al., 20 0 1 ) . The NLS seq uence in DGKc overlaps with a motif similar to the PKC phosphorylation-site domain (PSD) within the myristoylated alanine-rich C-kinase substrate (MARCKS) protein (DGKc-MARCKS- PSD) . The DGKc-MARCKS-PSD can be phosphorylated by PKC, which prevents nuclear accumulation of DGKc (Topham et al., 1 9 9 8 ) . Furthermore, PKC-medi ated phosphorylation of the DGKc-MARCKS-PSD also inhibits DGKc activity (Luo et al., 20 0 3 ) . I mportantly, overex pression of DGKc within the nucleus inhibits cell cycle progression (Topham et al., 1 9 9 8 ) . Thus, the levels and activity of DGKc in the nucleus are subject to regulation by PKC, while, conversely, DGKc may regulate nuclear DAG levels and conseq uently PKC activity.

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We previously demonstrated that, in vivo, the level of nuclear PI(4,5)P₂ can be modulated by the interaction of Type I PIP-kinases (PIP5K, enzymes that syn- thesize PI(4,5)P₂) with pRB (Divecha et al., 2002). Together with its family members p107 and p130, pRB regulates cell cycle progression by interacting with and atten- uating the activity of the E 2F transcription factor family (DiCiommo et al., 2000;

Classon and Dyson, 2001). Since PI(4,5)P₂ is hydrolysed by phospholipase C which generatesDAG, and since this nuclear DAG was shown to be subsequently phosphorylated by a DGK (D’Santos et al., 1999; Martelli et al., 2000), we questioned whether pRB may act as a nuclear scaffold to regulate PI signalling and DAG phosphorylation.

In this study, we show that GST-pRB fusion proteins can bind and extract DGK, PIP-kinase and PI-kinase activities from cell lysates. We identify DGKc as the ^DGK isoform that specifically interacts both in vit r o and in vivo with pRB and its family members p107 and p130, and show that this interaction potently enhances DGK activity. Finally, we demonstrate that DGKc likely lies downstream of pRB signalling in a DNA damage signalling pathway. O ur data would imply that disruption of pRB function, which frequently occurs in human cancers, may lead to enhanced nuclear DAG levels and, in turn, to uncontrolled nuclear PKC activity.

M aterials and methods

E x p r e s s i o n p l a s m i d s

Wild-type DGKc, catalytic inactive DGKc, the DGKc-MARCKS-PSD deletion mutant and CO O H-terminal FLAG-tagged DGKc were published previously (Topham et al., 1998; Topham and Prescott, 2001). NH₂-terminal HA-tagged DGKc and GFP-DGKc were cloned via 3-point ligations into respectively pMT2SM-HA and pE GFP-C2 using respectively internal N deI and X m a I sites. The 5’ fragment was generated by PCR and the 3’ fragment was digested from wild-type DGKc.GST-DGKc and VSV-DGKc (both CO O H-terminus) were cloned by inserting DGKc in respectively pMT2SM-GST and pMT2SM-tag via a 3-point ligation using the internal S p h I site. The 3’ fragment was generated by PCR and the 5’ fragment was derived from wild-type DGKc. The GST-pRB C-terminus (amino acids 767-928) was generated by PCR and inserted into pGE X-4T-2. Wild-type and kinase-inactive DGKc were cloned into pBabe by PCR.

C e l l c u l t u r e a n d t r a n s f e c t i o n

CO S-7,HE K293T, Phoenix, MCF7,ME F,ME L,SAO S-2 and C33A cells were grown in Dulbecco’s modified E agle’s medium containing 8% heat-inactivated fetal calf serum, 2 mM glutamine and antibiotics. CO S-7 cells were transfected using the DE AE-dextran method, HE K293T, Phoenix and C33A cells using the calcium phosphate precipitation method, and SAO S-2 cells using Fugene (Roche) according to manufacturer’s instructions.

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C e l l u l a r l y s a t e s a n d i m m u n o p r e c i p i t a t i o n s

Rat brain lysates were prepared as described (Divecha et al., 2002). Cells were lysed 48 hours after transfection in 1% NP40 lysis buffer (50 mM Tris pH 8.0, 50 mM KCl, 10 mM EDTA, 1% NP40, complete protease inhibitor cocktail (Roche)). Immu- noprecipitations were performed overnight using an anti-DGKc polyclonal antibody (Bunting et al., 1996), anti-pRB polyclonal antibody C-15 (Santa Cruz) anti-FLAG monoclonal antibody M2 (Sigma), or anti-HA monoclonal antibody 12CA5 (Roche).

Antibodies were captured using Protein A or G Sepharose beads (Amersham) and washed with 1% NP40 lysis buffer. Endogenous immunoprecipitates were then washed once with PIPkinase buffer (25 mM Tris pH 7.4, 10 mM MgCl₂, 80 mM KCl, 1 mM EGTA) and 15% or 20% was resuspended in 20 +l of 10 mM Tris (pH 7.4) for DGK activity assay, while 85% or 80% was analyzed by Western blotting. Immuno- precipitates or total lysates were separated by SDS-PAGE, transferred to nitrocellulose, and incubated with anti-DGKc polyclonal antibody, anti-pRB monoclonal antibody G3-245 (Pharmingen), anti-p107 polyclonal antibody C-18 (Santa Cruz), anti-p130 polyclonal antibody C-20 (Santa Cruz), or anti-cyclin E monoclonal antibody sc-248 (Santa Cruz). Blots were stained with secondary antibodies (DAKO) and visualized using \ (Amersham) or super signal (Pierce).

A f f i n i t y - p u r i f i c a t i o n s

GST-DGKc (expressed in COS-7 cells) and GST fusion proteins of pRB, p107, and p130 (expressed in bacteria and induced with 200 +M IPTG) were purified using glutathione-sepharose 4B beads (Amersham) according to manufacturer’s instructions. Approximately 200 +g of cell lysate was incubated with 4 +g of immobilized GST fusion proteins for 2 hours at 4°C and beads were then washed with 1% NP40 lysis buffer. For DGK activity assays, equal amounts of GST-protein complexes were washed once in PIPkinase buffer, resuspended in 20 +l of 10 mM Tris (pH 7.4), and assayed for DGK activity. For Western blotting, affinity-purified proteins were separated by SDS-PAGE, transferred to nitrocellulose and probed with an anti- DGKc polyclonal antibody, or anti-VSV monoclonal antibody P5D4 (Roche).

MCF7 lysate (450 +g) or 250 ng eluted GST-pRB or GST-Cdc42 was incubated overnight with 100 +g of biotinylated TAT-DGKc-^MARCKS peptide (Y ARAAARQ ARA- GKASKKKKRASFKRKSSKK) or TAT control peptide (Y ARAAARQ ARAG), which were immobilized on streptavidin agarose (Sigma) and washed with wash buffer (50 mM Tris pH 7.4, 140 mM NaCl, 10 mM MgCl2, 0.1% Tween20). Affinity-purified pRB or GST-fusion protein was visualized by immunoblotting.

D G K a c t i v i t y a s s a y

Immunoprecipitates, GST-protein complexes, or purified proteins were assayed for DGK activity as described by Divecha et al. (Divecha et al., 1991). Lipid vesicles were prepared by sonicating 1 nmol of dioleoylglycerol (DAG; from Sigma), 1 nmol of PIP (Biomol), 1 nmol of PI (Sigma), and 3 nmol of PA (Sigma) in 10 mM Tris (pH 7.4). Reactions were performed at 30°C for 10 minutes (GST pull-downs and immunoprecipitates) or 5 minutes (purified proteins) in PIPkinase buffer containing 10 µM cold ATP and 5 µCi of [ a-³²P]ATP in a final volume of 100 µl. Lipids were extracted

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with 0.5 ml of chloroform:methanol [1:1, v:v], followed by the addition of 125 µl of 2.4M HCl and phase separation. Lipid extracts were dried and separated by thin layer chromatography (silica gel 60 TLC plates (Sigma) soaked in 1 mM EDTA and 1 mM potassium oxaloacetate and heat-activated) using chloroform:methanol:water:

ammonia (45:35:7.5:2.5, v:v:v:v). Lipids were visualized by autoradiography and quantified using a phosphorimager.

E 2 F l u c i f e r a s e a c t i v i t y a s s a y

C33A cells were seeded in 6-well plates and transiently transfected with the indicated plasmids using the calcium phosphate precipitation method. 48 hours after transfection cells were lysed in 250 µl 1 x passive lysis buffer (Promega) and 50 µl was used to measure first firefly luciferase and then Renilla luciferase activity using the Dual-luciferase reporter assay system (Promega). Luciferase activity was detected using a Wallac 1420 multilabel counter (PerkinElmer) according to manufacturer instructions. To control for transfection efficiency, firefly luciferase activity was corrected for Renilla luciferase activity.

P r e p a r a t i o n o f p u r i f i e d p r o t e i n s f o r D G K a c t i v i t y a s s a y

In order to determine DGK activation as a result of direct protein-protein interaction (DGKc and GST-fusion proteins of pocket proteins), the following purification steps were performed: HA-DGKc was immunoprecipitated from 600 +g of COS-7 lysate and eluted from the beads using elution buffer (50 mM Tris pH 8.0, 300 mM NaCl) containing 1 mg/ ml HA peptide (YPYDVPDYA). Purified GST fusion proteins were eluted using GST-elution buffer (50 mM Tris pH 8.0, 300 mM NaCl, 20 mM glutathione), and quantified using a BSA standard on a Coomassie-stained gel.

Purified HA-DGKc was incubated with GST fusion proteins at 4°C for 1 hour. All samples contained equal amounts of GST-elution buffer and 5 +g of BSA.

G e n e r a t i o n o f pR B^{- / -} M E F s s t a b l y e x p r e s s i n g D G K

c

, a n d c e l l c y c l e a n a l y s i s a f t e r

a

- i r r a d i a t i o n

To immortalize mouse embryonic fibroblasts (MEFs) and to generate cells stably expressing DGKc,^{LZ RS}-TBX2-iresGFP (kindly provided by M. van Lohuizen in our Institute) or DGKc-pBabe constructs were transfected in Phoenix packaging cells and used to transduce MEFs. Ecotropic retroviral supernatants were collected 48 hours after transfection, filtered through a 0.45 µm filter and incubated with 4 µg/ ml of polybrene (Sigma) before adding to the cells. Viral supernatants were diluted 6 hours after transduction. After immortalizing cells with TBX2, cells were transduced with DGKc and, after 48 hours, were selected with 200 µg/ ml of puro- mycin (Sigma). For irradiation experiments, 50.000 cells were plated per well (6-well plates). Two days after seeding, cells were a-irradiated using a 137Cs radiation source. 30 minutes after irradiation, cells were treated with nocodazole (1 µg/

ml; Sigma) for 30 hours. For cell cycle analysis, cells were trypsinized, fixed in ice-cold 70% ethanol and resuspended in 200 µl of phosphate-buffered saline containing 50 µg/ ml propidium iodide and 50 µg/ ml RNase. Cell cycle distribution was determined by FACScan analysis and quantified using FCS express 2.

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Results

D G K

c

i n t e r a c t s w i t h pR B, p 1 0 7 a n d p 1 3 0 i n v i t r o

To determine which lipid kinases interact with pRB, we used three different GST- pRB fusion proteins (Fig. 1B): the large pocket region that includes the small pocket domain and the C-terminus (GST-pRB), the small pocket domain (GST-pRB(A+B)), and the pRB C-terminus (GST-pRB(C)). These GST fusion proteins were used to affinity-purify lipid kinases from lysates of rat brain, murine erythroleukemia (MEL) cells, or human MCF7 breast cancer cells. GST-Cdc42 served as a negative control for non-specific binding. Figure 2A shows that, in each cell lysate, each of the three pRB constructs bound three different lipid kinase activities, i.e. PI-kinase (yielding PIP),PIP-kinase (yielding PIP₂), and DGK (yielding PA), as assessed by ³²P incor- poration into the respective lipid products. The ability of pRB to interact with PIP- kinases is in agreement with our previous data (Divecha et al., 2002). Here, we focus on the novel finding that pRB binds to DGK.

Figure 2A shows that rat brain contains much more pRB-binding lipid kinase activities than MEL and MCF7 cells. In each of the cell lysates, GST-pRB(A+B) (the small pocket of pRB; lanes 2, 6 and 10) shows slightly less binding of each of lipid kinase activities than the other two pRB constructs (large pocket and C-terminus) (for rat brain, this is better seen at the lower exposure; lanes 1^*-4^*). Thus, in three different cell lysates, three different lipid kinases of the canonical PI-cycle, i.e. a DGK,PI-kinase and a PIP-kinase all associate with GST-pRB fusion proteins.

These data suggest that pRB may act as a scaffold protein in order to regulate nuclear PI signalling.

To define which DGK isoform interacts with pRB, we used GST-pRB to extract DGKs from lysates of COS-7 cells overexpressing VSV-tagged DGK_ -^DGKc or -DGKe. Interaction between ^DGKs and GST-pRB was assessed using in vitroDGK activity assays and by Western blotting. GST-pRB extracted 30-fold more DGK activity from lysates expressing DGKc compared to those expressing DGK_ or

Fig. 1.

Domain structure of DGKc and pRB. A , Conserved domains of DGKc. N ext to a catalytic domain and two cysteine -rich domains (CRD), common to all DGK iso - types,DGKc contains a MARCKS phosphorylation- site domain (-PS D) overlap - ping a nuclear localiz ation signal (N L S), four ankyrin repeats and a PDZ -binding motif for protein-protein interactions.

B, S tructural domains of pRB and the GS T-pRB fusion proteins used in this study.

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Fig. 2.

DGKc associates with the retinoblastoma gene product (pRB)in vitro. A, Endogenous DGK,PIP-kinase, and PI-kinase activities associate with GST- pRB fusion proteins. GST fusion pro - teins of the pRB large pocket region (GST-pRB), pRB small pocket domain (GST-pRB(A+B)), pRB C-terminus (GST-pRB(C)), and Cdc42 (GST-Cdc42;

used as a control) were isolated from bacteria and eq ual q uantities were incubated for 2 hours with cell lysates of rat brain,MEL cells, or MCF7 cells (500 +g each) and assayed for lipid kinase activity by adding lipid vesicles composed ofDAG, PI, PIP, and PA together with [a-³²P]ATP and cold ATP, as detailed in the Experimental procedures. The radi- olabelled lipid products, PIP2,PIP and PA, were separated by TLC and visualized by autoradiography (positions indicated). Lanes marked 1* to 4* represent a short exposure of lanes 1 to 4 of the sameTLC plate. B,GST-pRB fusion proteins specifically bind to DGKc.V SV- taggedDGK_, -c, and -e (or empty vector) were overexpressed in CO S-7 cells and lysates were incubated for 2 hours with the denoted GST fusion proteins.

15% of affinity-purified proteins was assayed for DGK activity as described in A. Autoradiographs of the PA spots on the TLC plates are shown (upper panels).DGK activity associated with theGST proteins is shown in lanes 1-8.

DGK activity in the total lysates (2.5%

of input lysate) is shown in lanes 9-12.

85% of the affinity-purified proteins was separated by SDS-PAGE, transferred to nitrocellulose and visualized using the V SV-tag specific antibody P5D4 (lower panels). Data are representative of 3 experiments. C,DGKc has highest affinity for the large pocket domain and the C-terminus of pRB. Increasing amounts ofGST-pRB,GST-pRB(A+B),GST- pRB(C) or GST-Cdc42 (negative control) were incubated with lysates of DGKc- or vector- overexpressing CO S-7 cells.

AssociatedDGKc was detected by Western blotting with a DGKc- specific antibody (upper panel). Blots were reprobed with a GST- specific antibody (lower panel). Total lysates represent 1/10 of input lysate.

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DGKe (Fig. 2B, upper panel, compare lane 3 with lane 1 and 5) even though less DGKc activity was present in the lysates compared to DGK_ or DGKe activity (compare lane 10 with lane 9 and 11). Similarly, more DGKc bound to ^GST-pRB than DGK_ or ^DGKe, as revealed by Western blotting, despite the lower expression of DGKc in cell lysates (Fig. 2B, lower panel). These data indicate that DGKc is the predominant isotype that binds to pRB.

Fig. 3.

DGKc binds to pRB, p107 and p130 in vitro and in cells.

A, Lysates from COS-7 cells transfected withGFP-taggedDGKc or empty vector were incubated for 2 hours with indi- catedGST proteins. Bound DGKc was visualized by Western blotting with an anti-DGKc antibody (upper panel).

A ponceau staining of the GST proteins shows the amount of GST proteins used (lower panels). B, C, D,HEK293T cells were transfected with FLAG-DGKc and pRB (B), p107 (C), or p130 (D) constructs and lysed 48 hours after

transfection.FLAG-DGKc was immunoprecipitated using an anti-FLAG antibody. Immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose and visualized using antibodies specific for either pRB (G3-245), p107 (C-18), or p130 (C-20) (in respectively panel B, C, and D).

Blots were stripped and reprobed with an anti-DGKc antibody (lower panels).

Total lysates represent 1/50, 1/250 and 1/50, of input lysate for, respec - tively, pRB, p107 and p130.

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To assess if DGKc activity was required for interaction with pRB we mutated a conserved glycine residue within the ATP binding site. This mutant showed less than 1% of the activity in vitro but was still able to interact with GST-pRB as well as the wild-type enzyme (data not shown).

To more carefully examine the region of pRB where DGKc binds, we performed a titration experiment in which increasing amounts of GST fusion proteins were used to bind DGKc from COS-7 lysates. DGKc has similar affinities for GST-pRB and GST-pRB(C) (Fig. 2C, compare lanes 3 and 8), whereas GST-pRB(A+B) only weakly bound to DGKc. The substantial DGK activity bound to GST-pRB(A+B) from endogenous cell systems (Fig. 2A) compared to the small amounts of DGKc bound to pRB(A+B) found on Western blot may be due to differences in the assays utilized.

Based on Western blot analysis we conclude that the C-terminus of pRB is the major binding site for DGKc.

Since pRB is a member of the family of pocket proteins that also include p107 and p130, we assessed if p107 and p130 also interact with DGKc. As GST-p130 and DGKc have the same molecular weight we used lysates expressing GFP-DGKc to discriminate between them on SDS-PAGE. Like GST-pRB,GST-p107 and GST-p130 also bound to GFP-DGKc (Fig. 3A). However, GST-p130 extracted less DGKc compared to GST-pRB and GST-p107, which might reflect the lower amount of GST protein used, or that p130 has a lower affinity for DGKc. These data show that DGKc can interact with all members of the pRB pocket protein family.

D G K

c

b i n d s pR B, p 1 0 7 a n d p 1 3 0 i n c e l l s

To demonstrate that DGKc binds to pRB also in cells, we expressed pRB and FLAG- tagged DGKc (FLAG-DGKc), alone or in combination, in HEK293T cells and immunoprecipitated the lipid kinase from cell lysates using a specific anti-FLAG antibody. pRB and DGKc were expressed in total lysates of transfected HEK293T cells (Fig. 3B lower panels) and FLAG-DGKc could be immunoprecipitated. However, pRB was only detected in the immunoprecipitates from lysates expressing both pRB and DGKc (Fig. 3B, upper panels). We also tested the pRB family members p107 and p130 for DGKc binding in cells. Total lysates revealed that DGKc, p107 and p130 were expressed in transfected HEK293T cells (lower panels of Fig. 3C and 3D).

Similar to pRB, p107 and p130 were only detected in the anti-FLAG immunoprecipitates when they were co-expressed with FLAG-DGKc (upper panels of Fig. 3C and 3D). The interaction of p130 with DGKc was lower in both the in vitroGST pull-downs as well as in the co-immunoprecipitations suggesting that p130 binds DGKc with a lower affinity than pRB and p107.

To demonstrate that endogenous DGKc and pRB interact with each other, we immunoprecipitated DGKc from MEL lysates and subjected the precipitate to immunoblotting with a pRB-specific antibody. pRB was present in the DGKc immunoprecipitate, but not in a control precipitate (Fig. 4A, upper panel). Figure 4A (lower panels) confirms the presence of DGK activity and the presence of DGKc protein in the DGKc immunoprecipitate but not in the control precipitate. To suc- cessfully immunoprecipitate pRB and determine DGKc co-immunoprecipitation, we used MEL lysates of differentiated cells that contain pRB predominantly in the

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hypophosphorylated status, the status of pRB that binds DGKc (see Fig. 6). A small fraction of pRB was immunoprecipitated (Fig. 4B, upper panel) and DGKc protein and DGK activity was specifically co-immunoprecipitated (Fig. 4B, lower panels).

These results indicate that endogenous pRB and DGKc interact with each other.

T h e M A R C K S-P S D o f D G K

c

i s a pR B- b i n d i n g s i t e

The MARCKS-PSD of DGKc has previously been shown to be a major determinant for the localization of DGKc in the nucleus (Topham et al., 1998). We therefore postulated that the MARCKS-PSD may be important in the interaction of DGKc with pRB. To test this hypothesis, we tested the interaction between pRB and a DGKc mutant in which the MARCKS domain was deleted (DGKc-6MARCKS). While both wild-type (wtDGKc) and DGKc-6MARCKS were equally expressed (Fig. 5A, compare lane 17 with lane 18), DGKc-6^MARCKS was hardly detectable compared to wtDGKc in the GST-pRB fusion protein precipitates (lanes 5-8). Since the MARCKS-PSD contains a large number of basic amino acids that might be important for electro- static interaction between pRB and DGKc, we tested a DGKc-MARCKS-PSD mutant Fig. 4.

pRB co-immunoprecipitates with endogenous DGKc, and vice versa.

A,DGKc was immunoprecipitated from 2 mg of MEL lysate using a DGKc-pecific antibody. Pre -immune serum (control) was used as a control. Immunoprecipi- tates were split: 85% was used for Western blotting with a pRB- specific antibody (upper panel); 15% of the DGKc precipitate was assayed for DGK activity and [³²P]PA was separated by TLC and visualized by autoradiography (lower panel). The lanes marked ‘total lysate’ represent 1/2000 of input lysate and were present on the same blot as the immunoprecipitates. The Western blot in the middle panel shows that

DGKc is indeed immunoprecipitated.

This blot is from a different immuno - precipitate, because pRB and DGKc have almost the same molecular weight.

Total lysate represents 1/25 of input lysate. B, pRB was immunoprecipitated from 2.5 mg of lysate of differentiated MEL cells. 20% of the immunoprecipitate was assayed for DGK activity and 80%

was analyzed on Western blot. First, blots were stained for DGKc using a DGKc- specific antibody. Second, blots were stripped and reprobed for pRB. The lanes marked ‘total lysates’ contain 1/1000 of input lysate for pRB and 1/500 forDGKc and were on the same blot.

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Fig. 5.

DGKc associates with pRB via its MARCKS phosphorylation-site domain (PSD).

A,HEK293T cells were transfected with wild-typeDGKc (wtDGKc), a MARCKS - PSD deletion mutant (DGKc-6MARCKS), aMARCKS -PSD mutant in which all basic amino acids are substituted for alanines (DGKc-K/RAA), or empty vector as indicated. Lysates were incubated with the indicated GST fusion proteins and associated proteins were analyzed by immunoblotting using a DGKc- specific antibody. GST fusion protein precipitates are shown in lanes 1-16 and total lysates in lanes 17-20. B, DGKc-MARCKS -PSD peptide specifi - cally binds to pRB. Biotinylated DGKc- MARCKS -PSD peptide or biotinylated control peptide were incubated overnight with MCF7 cell lysates and immo - bilized on streptavidin agarose beads.

Associated pRB was visualized by immunoblotting using an anti-pRB antibody.

Total lysates contain 1/25 of input lysate. C, Full-length DGKc binding to pRB is blocked by a DGKc-MARCKS - PSD peptide. 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 visualized by immunoblotting using an anti-DGKc antibody (lanes 1-6). 10% of input lysates are shown in lanes 7 and 8.

D, Purified eluted GST-pRB directly binds to DGKc-MARCKS -PSD peptide.

250 ng of purified eluted GST-pRB or GST-Cdc42 was incubated overnight with 100 +g biotinylated DGKc-MARCKS - PSD or control peptide. Peptides were immobilized on streptavidin agarose beads and associated GST-proteins were analyzed by Western blotting using a GST- specific antibody. The lanes marked

‘input’ contain 1/10 of GST-fusion pro - teins used in the assay.

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in which all basic amino acids of the nuclear localization signal were substituted for alanines (DGKc-K/RAA) for pRB binding. Similar to the MARCKS-PSD deletion mutant, the DGKc-K/RAA mutant failed to interact with GST-pRB (lanes 9-12), even though the protein was expressed at a higher level compared to wtDGKc (compare lane 19 with lane 17).

To test whether the DGKc-MARCKS-PSD was sufficient to mediate interaction with pRB, we used a biotinylated DGKc-MARCKS-PSD peptide to affinity-purify pRB from MCF7 cell lysates. The DGKc-^MARCKS-PSD peptide bound to pRB, while a biotinylated control peptide was unable to bind pRB (Fig. 5B). Furthermore, the interaction between full-length DGKc and GST-pRB was inhibited by the DGKc-MARCKS- PSD peptide (Fig. 5C, lane 3) but not by the control peptide (Fig. 5C, lane 4). Together, these results indicate that the DGKc-MARCKS-PSD is important in mediating the interaction between pRB and DGKc.

T h e M A R C K S-P S D o f D G K

c

b i n d s t o pR B d i r e c t l y

To determine whether pRB directly binds to DGKc, we tested whether purified and eluted GST-pRB could be extracted by the biotinylated DGKc-MARCKS-PSD peptide.

Purified GST-pRB specifically bound to the DGKc-^MARCKS-PSD peptide coupled to streptavidin agarose (Fig. 5D), whereas GST-Cdc42 did not bind. GST-pRB was not extracted by a control peptide. These results suggest that pRB binds to the MARCKS-PSD of DGKc directly.

D G K

c

b i n d s h y p o p h o s p h o r y l a t e d pR B

pRB regulates cell-cycle progression through its interaction with the transcripti on factor E2F. During G1, pRB exists in a hypophosphorylated state and can bind to and inactivate E2F. When cells progress to S-phase, pRB becomes highly phosphorylated by cyclin/CDK complexes, which leads to the release of E2F, enabling E2F- mediated transcription of genes required for S-phase progression (Mittnacht, 1998).

In total cell lysates, pRB is present in both the low and highly phosphorylated state (see the doublet in Fig. 3B, lower panel), while only one band is detectable in the DGKc immunoprecipitate (Fig. 3B, upper panel), suggesting that the inter-

Fig. 6.

DGKc binds to hypohosphorylated pRB. GST-DGKc was isolated from COS-7 cells and incubated with lysates of SAOS -2 cells that were transfected with pRB and/or cyclin E as indicated constructs.

Affinity-purified proteins (lanes 1-6) and total lysates representing 1/20 of input lysates (lanes 7-9) were subjected to Western blotting using a pRB- specific (upper panel), GST- specific (lower left panel) or cyclin E- specific antibody (cycE, lower right panel).

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action between DGKc and pRB may be dependent on the phosphorylation status of pRB. To further test this, we used the osteosarcoma cell line SAOS-2 that lacks functional pRB. When pRB is overexpressed in SAOS-2 cells it is not phosphorylated (Fig. 6, lane 7) and causes a cell cycle arrest. Co-expression of pRB with cyclin E, however, leads to hyperphosphorylation of pRB (lane 8), which attenuates the pRB- mediated cell cycle arrest (Hinds et al., 1992). We purified GST-DGKc from COS-7 cells and used it to affinity-purify pRB from lysates of SAOS-2 cells expressing pRB alone, or co-expressing pRB and cyclin E. Hypophosphorylated pRB (lane 1) specifically bound to GST-DGKc, but in the presence of cyclin ^E when pRB is hypophosphorylated (lane 2), binding was almost undetectable (the minor amount of pRB in lane 2 is hypophosphorylated). These results indicate that, similar to E2F, DGKc preferentially binds to the hypophosphorylated form of pRB.

D G K

c

d o e s n o t a f f e c t s e q u e s t e r i n g a n d i n a c t i v a t i o n o f E 2 F b y pR B Since DGKc interacts specifically with hypophosphorylated pRB, we questioned ifDGKc might influence pRB-mediated regulation of E2F transcriptional activity.

Therefore, we used a reporter construct with a promoter containing six E2F binding sites upstream from the firefly luciferase reporter coding region. Binding of E2F to the promoter drives transcription of the luciferase reporter. C33A cells were co-transfected with the E2F luciferase reporter construct, a control Renilla luciferase receptor construct, E2F1 and DP1, in the absence or presence of pRB and/or DGKc.E2F and DP1 caused a 4.5-fold stimulation of E2F promoter activity compared to background E2F activity. This stimulation was 50% and 75% reduced by co-transfection of respectively 100 and 250 ng of pRB expression plasmid (Fig. 7). Addition of DGKc plasmid did not affect basal E2F activity or pRB-mediated inhibition of E2F activity, indicating that DGKc does not affect the regulation of E2F activity by pRB.

pR B, p 1 0 7 a n d p 1 3 0 s t i m u l a t e D G K

c

a c t i v i t y

To explore the function of the interaction between DGKc and p^RB, we tested whether pRB could regulate DGKc activity. Therefore, we compared the activity of HA-DGKc immunoprecipitated with an anti-HA antibody with HA-DGKc affinity-

Fig. 7.

DGKc does not aff ect regulation of E2F by pRB.

C33A cells were co -transfected with indicated constructs. Cells were lysed 48 hours after transfection and assayed for firefly luciferase and Renilla luciferase activity using a luminometer.

The firefly luciferase data were corrected for Renilla luciferase activity and plotted in the histogram shown as means ± the range of the duplicates (n= 2).

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Fig. 8.

pRB stimulates DGKc activity.

A,COS-7 cells were transfected with eitherHA-DGKc or vector as indicated and lysed after 48 hours. HA-DGKc was immunoprecipitated from 20 +g (lane 1) and 40 +g (lane 2) of cell lysate, using a fixed amount of anti-HA antibody; or affinity-purified by 1, 2, or 3 +g of GST- pRB (lanes 4-6) from 80 +g of cell lysate.

Negative controls are included in lanes 3, 7 and 8. Immunoprecipitates and affinity-purified proteins were split and 20% was assayed for DGK activity, using substrate and [a-³²P]ATP. [³²P]PA was separated by TLC and quantified using a phosphorimager. The data were plotted in the histogram shown as means ± S.D. (n=3). The other 80% was used to visualize the amount of DGKc affinity-purified by either the HA- specific antibody or GST-pRB by immunoblotting using a DGKc- specific polyclonal

antibody (lower panel). Note that lane 2 and lane 6 contain the same amount ofDGKc protein, whereas the activity ofHA-DGKc bound to GST-pRB was 7.5-fold higher than the immunoprecipi- tatedHA-DGKc. B,HA-DGKc overexpressed in COS-7 cells was immuno - precipitated using an anti-HA antibody and eluted from the beads using HA peptide. Eluted HA-DGKc was incubated with indicated amounts of GST fusion proteins that were isolated from bacte - ria, eluted from the beads using glutathione, and quantified relative to BSA standards on a Coomassie - stained gel.

In vector controls, 100 ng of indicated GST fusion proteins was added. All samples contained 5 +g of carrier BSA. Mixtures were assayed for DGK activity as in A. Results are the means ± the range of the duplicates (n=2) and repre - sentative of 3 experiments.

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purified by GST-pRB. Immunoprecipitation with the anti-HA antibody yields DGKc that is not bound to pRB, while affinity-purification with GST-pRB ensures that all of the DGKc present on the beads interacts with p^RB. The amount of DGKc protein on the beads was determined by immunoblotting, while DGK activity was assessed by an in vitro assay. As shown in Figure 8A, the amount of immunoprecipitated HA-DGKc in lane 2 was comparable with the amount of HA-DGKc affinity-purified by GST-pRB in lane 6. However, 7.5-fold more DGK activity was associated with GST-pRB-bound HA-DGKc compared to HA-immunoprecipitated DGKc. Since the HA-tag antibody did not interfere with HA-DGKc activity (data not shown), these results suggest that DGKc is more active when in a complex with pRB.

To further verify that all the pocket protein family members could stimulate DGKc activity, we purified ^GST-pRB, -p107 and -p130 and assessed their effects on DGKc activity in vitro.^HA-DGKc was immunoprecipitated from ^COS-7 cell lysates and eluted from the beads using an HA-peptide. Purified GST-pRB,GST-p107, and GST-p130 were eluted from the beads using glutathione. Purified HA-DGKc and GST fusion proteins were combined on ice and complexes were allowed to form prior to the DGK activity assay. GST-pRB,GST-p107, and GST-p130 enhanced DGKc activity 5-, 3.5-, and 4.5-fold respectively in a concentration dependent manner, whereas a GST-Cdc42 control did not affect DGKc activity (Fig. 8B). Together, these results indicate that pRB family members activate DGKc in vitro.

O v e r e x p r e s s i o n o f D G K

c

c a n p a r t i a l l y r e s c u e t h e l o s s o f a G1 - a r r e s t a f t e r

a

- i r r a d i a t i o n i n pR B- n u l l M E Fs

In order to establish a physiological role for DGKc in pRB-dependent signalling pathways, we studied the G1-arrest induced by a-radiation. Cell cycle arrest in response to ionizing radiation is a well established tumour suppressive pathway that is dependent on the growth suppressive activity of pRB. This pathway is com- pletely blocked in pRB-null mouse embryonic fibroblasts (MEFs) (Harrington et al., 1998). We postulated that, if DGKc kinase activity is enhanced by pRB, then overexpression of DGKc may partially substitute for the loss of pRB in a-irradiation- induced cell cycle arrest. MEFs isolated from pRB-null mice were transduced with viral constructs encoding vector (pRB^-/- vector), kinase-inactive DGKc (p^RB^-/- kdDGKc) or wild-type ^DGKc (p^RB^-/- wtDGKc). As a control, ^MEFs were isolated from wild-type mice. In all cases, MEFs were immortalized by prior transduction with TBX2 which blocks passage-induced senescence. MEFs were a-irradiated and, after 30 minutes, treated with nocodazole to arrest them in G2/M. Cells arrested in G1 were assessed by FACS analysis. As shown in Figure 9, a-irradiation of wild-type MEFs (pRB^+/+) led to a dose-dependent increase in the number of cells arrested in G1. As expected, irradiation of pRB-null MEFs (pRB^-/- vector) did not lead to any increase in the number of cells in G1. Consistent with a role for DGKc in pRB signalling, overexpression of DGKc in pRB-null MEFs led to a partial rescue of the arrest defect at all doses tested. The rescue was dependent on the activity of DGKc, as it was not observed in cells transduced with the kinase-inactive DGKc.

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Discussion

In this study we show that the retinoblastoma gene product (pRB) specifically interacts with DGKc in vitro and in vivo. The C-terminus of pRB is required for the interaction with DGKc while the MARCKS-PSD of DGKc is sufficient to mediate the interaction. Furthermore, the interaction between pRB and DGKc is dependent on the phosphorylation status of pRB, since DGKc only binds active hypophosphorylated pRB. The interaction between DGKc and pRB does not appear to modulate the repression of E2F transcriptional activity by pRB. However, we show that pRB and other pocket protein family members are potent activators of DGKc activity in vitro.

We previously demonstrated that PIP5K, an enzyme that converts PI(4)P into PI(4,5)P_2, interacts with, and is activated by pRB (Divecha et al., 2002). In this paper, we now demonstrate that a PI 4-kinase that will generate PI(4)P, and a DGK that phosphorylates DAG to PA, can also interact with pRB. Furthermore, the p55 kDa subunit of PI 3-kinase which can generate PI(3,4,5)P₃ via the 3’ phosphorylation of PI(4,5)P₂ was also shown to interact with pRB (Xia et al., 2003). We suggest that pRB may act as a scaffold protein to integrate nuclear PI signalling and may provide a link between cell cycle regulation and changes in nuclear signalling lipids. For example, as cells progress from G1 to S-phase, nuclear levels of DAG, PA,PIP, and PIP₂ have been shown to change (D’Santos et al., 1999; Clarke et al., 2001).

Fig. 9.

DGKc reconstitutes a cell cycle arrest induced by a-irradiation in pRB-null MEFs.

pRB^+/+ and pRB^-/- mouse embryonic fibroblasts (MEFs) were immortalized byTBX 2 and pRB^-/-MEFs were transduced with empty vector, wtDGKc, or kdDGKc. Stable cell lines were irradiated with the indicated doses of a-irradiation and 30 minutes after irradiation cells were treated with noco - dazole (1 µ g/ml) for 30 hours. Cells arrested in G1 phase were quantified using flow cytometry. A significant radiation-induced increase in the per- centage of cells in G1 phase above basal levels (subtracted in the figure) is apparent only in pRB^+/+ cells and in wtDGKc-transduced pRB^-/- cells (black bars). Data are means ± S.E. (n=3) and representative of three separate experiments. Significance: *, p < 0.05; **, p < 0.01 (Student’s t-test).

Inset: Western blot showing DGKc expression in stable cell lines.

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In addition to pRB binding, DGKc also interacts with the pRB-related pocket proteins p107 and p130. pRB, p107 and p130 are highly similar within the pocket region, but also regions beyond the pocket domain are conserved. To date, almost all p107- and p130-binding proteins also bind to pRB, while most pRB-binding proteins have not been tested for binding to p107 and p130 (Classon and Dyson, 2001). All pocket proteins show substantial functional overlaps as well as some unique functions (Classon and Dyson, 2001; Classon and Harlow, 2002). When overexpressed, they all can arrest the appropriate cells in G1-phase of the cell cycle, inhibit E2F-mediated gene transcription, and are all phosphorylated by cyclin/CDKcomplexes.

However, p107 and p130 bind different E2F family members than pRB, and therefore regulate transcription of different sets of genes. Also their expression patterns differ during the cell cycle: the levels of pRB are constant during the cell cycle, whereas p107 expression peaks during S-phase of the cell cycle and p130 is highly expressed in quiescent cells. Furthermore, they appear to have specific functions in differentiation. For example, deletion of pRB attenuates adipocyte differentiation, while deletion of p107 acts to sensitize MEFs to adipocyte differentiation (Classon et al., 2000).

In this study we show that overexpression of DGKc is able to partially rescue a cell cycle arrest defect in response to a-irradiation in pRB-null MEFs. Irradiation ofMEFs is known to induce pRB activity, which leads to a cell cycle arrest required to prevent cells from entering S-phase with damaged DNA (Harrington et al., 1998;

Bartek and Lukas, 2001). The arrest is thought to allow DNA repair and thus ensure the survival of the cell. How irradiation induces a cell cycle arrest is not clear.

In response to irradiation, p53 is induced and upregulates the levels of the cyclin kinase inhibitor p21^WAF1/CIP1. This inhibits phosphorylation of pRB leading to its activation. The cell cycle arrest is thought to be induced by pRB-mediated attenu- ation of E2F transcriptional activity. However, in pRB- negative C33A cells, expression of a mutant of pRB in the LxCxE binding site, whilst maintaining the ability of pRB to interact with and repress E2F activity, is unable to reconstitute a DNA damage arrest, whereas wild-type pRB expression can (Pennaneach et al., 2001).

This suggests that yet another factor besides E2F repression determines induc- tion of the cell cycle arrest in response to DNA damage. This factor could be DGKc, as overexpression of DGKc can partially rescue the loss of a cell cycle arrest in pRB-null cells. This would suggest, that DGKc acts either on a parallel pathway or lies directly downstream of pRB. As our studies also demonstrate that the active (hypophosphorylated) form of pRB interacts with and stimulates DGKc activity, we favour the latter suggestion. Furthermore, as a kinase-inactive DGKc is unable to reinitiate a cell cycle arrest, it appears that either the removal of DAG or the generation of PA is important for the cell cycle arrest.

An interesting possibility by which overexpression of DGKc allows regulation of a cell cycle arrest in response to irradiation may be through the other pocket protein family members, p107 and p130. The use of triple-knock-out MEFs may be useful for testing this possibility.

In accord with a role for DGKc to attenuate DAG as possible (co-)regulator of the cell cycle, previous studies have demonstrated that overexpression of a

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phospholipase C, an enzyme that generates DAG by hydrolysis of PI(4,5)P₂ in the nucleus, increases the number of cells entering S-phase in response to IGF-1 (Faenza et al., 2000; Xu et al., 2001). In contrast, suppression of nuclear phospholipase C activity inhibits entry of Swiss 3T3 cells into S-phase (Manzoli et al., 1997;

Neri et al., 1998). Furthermore, overexpression of phospholipase C in the nucleus inhibits terminal differentiation of MEL cells in response to DMSO treatment, while phospholipase C suppression augments differentiation (Matteucci et al., 1998).

These data are consistent with a role for nuclear DAG in modulating cell cycle progression and differentiation. A more direct study has demonstrated that overexpression of DGKc within the nucleus slows down cell cycle progression with an accumulation of cells in G1 phase (Topham et al., 1998).

How nuclear DAG/PA levels regulate cell cycle progression is not clear. DAG is a potent activator of PKC, an enzyme capable of regulating cell cycle progres - sion indirectly by modulating cyclin-dependent kinase inhibitors, including p21^WAF1/CIP1 (Besson and Yong, 2000) and/or directly via its ability to phosphorylate pRB (Suzuma et al., 2002). In contrast to DAG, there are no clear roles for PA in cell cycle progression. However, it is noteworthy that protein phosphatase PP1a is potently inhibited by its interaction with PA (Jones and Hannun, 2002). Interest- ingly, PP1a is a nuclear protein whose nuclear location is regulated during cell cycle progression (Trinkle-Mulcahy et al., 2003). Furthermore, studies in yeast have shown that PA may act as a regulator of transcription factors (Loewen et al., 2004).

Interestingly, PKC itself is also able to phosphorylate DGKc within the ^MARCKS- PSD, which leads to an inhibition of DGKc nuclear accumulation and an inhibition of its activity (Topham et al., 1998; Luo et al., 2003). This suggests that pRB and PKC have opposing effects on DGKc activity and, by consequence, on nuclear DAG/ PA levels. Such a mechanism would allow tight regulation of nuclear DAG/PA levels.

It will be interesting to determine what role PKC-mediated phosphorylation of the DGKc-MARCKS-PSD plays in modulating the interaction between DGKc and pRB.

Altogether, our data show that DGKc binds to and is activated by pRB, p107 and p130. We suggest that pocket proteins act as scaffolds to regulate nuclear inositide metabolism and to regulate the levels of the second messengers DAG andPA. Our data are consistent with a physiological role for either DAG or PA in modulating a pRB-mediated cell cycle arrest in response to DNA damage.