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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I n t r o d u c t io n t o t h e

fa m ily o f r e t in o b la s t o m a

g e n e p r o d u c t s

Chapter 3

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A bstract

T he retinoblastoma gene family consists of three mem-

bers: the retinoblastoma protein ( p

R B

) , p10 7 and p13 0 .

T he most conserv ed part of these three proteins is the

pock et domain that binds to sev eral other proteins,

including the

E 2 F

family of transcription factors. T he

best k now n function of these retinoblastoma family

proteins, also named pock et proteins, is the regulation

of the cell cycle. B y inhibiting

E 2 F

transcription factors,

they prev ent cell cycle entry and mediate cell cycle ex it,

for ex ample during differentiation. P ock et proteins are,

in turn, inhibited by cyclin/

C D K

- mediated phosphoryla-

tion, w hich activ ity is regulated by sev eral signalling

pathw ays. In addition, pock et proteins are req uired for

ex pression of late differentiation mark ers and for cell

surv iv al. T herefore, pock et proteins are master sw itches

during dev elopment as w ill discussed in more detail in

this chapter.

Introduction to the family of retinoblastoma gene products

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1. Introduction

The cell cycle is a highly ordered process in which a cell duplicates its contents and divides into two daughter cells. A cell passes through four seq uential events in one cell division cycle ( Fig. 1) ( N orbury and N urse, 19 9 2) . The cycle starts with the first gap phase (G1) , in which the environment is monitored to determine whether conditions are optimal for a cell to divide. The decision to engage in a new cell division cycle leads to the progression into S-phase and the duplication of cellular DN A .S-phase is followed by a second gap phase (G2) in which the DN A is being checked to ensure that the DN A replication is complete and mistakes are repaired. Finally, cells enter mitosis (M-phase) in which the actual division occurs.

A fter completing a division cycle, a cell will either continue cycling or enter a spe- cialised resting state ( q uiescent state, G0) in which a cell can remain for a long time period before it resumes proliferation.

A ll cell cycle stages are highly controlled at specific checkpoint that control via feed back mechanisms whether a phase is completely succeeded before going into the next phase. A combination of mutations in critical checkpoint proteins and/or in signalling pathways that feed into these checkpoints can cause dysfunc- tion of the cell cycle control system leading to uncontrolled proliferation and cancer ( H anahan and W einberg, 2000) .

The cell cycle contains two major checkpoints: in G1 just before entering S-phase and in G2 at the entry of mitosis. The G1 checkpoint is also called the restriction point, because after passing the G1 checkpoint, the cell will finish the cell cycle independently of environmental conditions. A key protein that inhibits passage through the restriction point is the retinoblastoma protein ( pRB) .

The pRB gene was identified as the gene mutated in retinoblastoma tumours ( Friend et al., 19 8 6; Dunn et al., 19 8 8 ) . It was the first identified tumour suppressor protein and was shown to be mutated in several cancers ( Sherr and McCormick, 2002; Classon and H arlow, 2002; Cobrinik, 2005) . pRB regulates G1 to S-phase transition by binding to and inactivating the E2F family of transcription factors

Fig. 1:

Function of pRB in the cell cycle.

InG0 and early G1, pRB binds and inactivatesE2F. During G1 mitogens inhibitCDK inhibitors and stimulate cyclin/CDK complex formation causing pRB to become highly phosphorylated.E2F is released and activates genes involved in S-phase allowing cells to pass the restriction point (R) and enter into S-phase.

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(Fig. 1) (Seville et al., 2005). Upon phosphorylation of pRB,E2F is released and binds to E2F-binding sites in promotors of several genes that promote S-phase entry. Phosphorylation of pRB is regulated by several cyclin/cyclin-dependent kinase (CDK) complexes that are, in turn, regulated by a complex signalling network. Altogether, this control mechanism allows tight regulation of G1 to S-phase transition, which will be discussed in more detail in this chapter.

2. The Retinoblastoma family proteins

2 . 1 . C h a r a c t e r i s t i c s o f pR B a n d i t s f a m i l y m e m b e r s p 1 0 7 a n d p 1 3 0 Human pRB cDNA encodes a protein of 928 amino acids with a molecular weight of 105-110 kDa (Fig. 2). Many of the mutations observed in tumours are in the small pocket domain of pRB, consisting of an A and b domain. Structure analysis revealed that the A domain is required for the correct folding of the B domain (L ee et al., 1998). The B domain contains a highly conserved groove that bind to proteins containing an L X CX E motif, which is present in several cellular proteins and viral oncoproteins. The C-terminus contains a nuclear localisation signal and cyclin binding motifs that are required for pRB phosphorylation by cyclin/CDK complexes (Adams et al., 1999). The C-terminus regulates the small pocket as phosphorylation of the C-terminal residues disrupts the binding of L X CX E-containing proteins with the small pocket (Harbour et al., 1999). As both the C-terminus and the small pocket domain are required for E2F binding and biological activity (Q in et al., 1992; Rubin et al., 2005), this part of the protein is also called the large pocket region. The function of the N-terminus is not known, but it binds, like the small pocket and the C-terminus, to several cellular proteins (Morris and Dyson, 2001).

The small pocket domain of pRB and related pocket proteins p107 and p130 is highly conserved (Fig. 2) (Classon and Dyson, 2001). p107 and p130 are more similar to each other (50% sequence identity) than to pRB (30-50% identity each).

The spacer between the A and B domain is longer in p107 and p130 compared to pRB and contains a high affinity binding site for cyclin/CDK complexes that form stable complexes with both proteins. In addition, p107 and p130 contain a region near the N-terminus that has CDK inhibitor activity. L ike pRB, p107 and p130 bind to E2F, are phosphorylated by cyclin/CDK complexes, have growth suppressive functions and are inactivated by viral oncoproteins.

2 . 2 . E x p r e s s i o n o f p o c k e t p r o t e i n s a n d p h e n o t y p e s o f k n o c k- o u t m i c e

All three pocket proteins are expressed in many, if not all tissues. However, each of them is characteristically expressed during the cell cycle and differentiation.

pRB is expressed in quiescent, differentiated and cycling cells (Classon and Dyson, 2001). Expression of pRB is quite constant and changes only slightly when cells re-enter the cell cycle after being quiescent (small increase) or exit the cell cycle (small decrease). p107 levels are low in differentiated cells, increase when

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quiescent cells enter the cell cycle, but drop dramatically when cells exit the cell cycle. In contrast, p130 is highly expressed in quiescent and differentiated cells, whereas its levels decrease when cells re-enter the cell cycle. The differences in expression of the pocket proteins already suggest specific functions of pocket proteins.

Analysis of knock-out mice revealed overlapping and unique functions of each pocket protein (Wikenheiser-Brokamp, 2006). pRB, but not p107 and p130, is essential for development. pRB knock-out embryos die after 13-15 days of gestation with defects in the nervous system, haematopoietic system and lens formation.

However, embryonic lethality was caused by defects in the placenta, since pRB^-/- embryo’s supplied with a wild-type placenta died after birth. p107 and p130 knock- out mice develop normally. However, p130^-/- mice in a Balb/cJ genetic background are embryonically lethal with defects in neuronal, muscle and heart development, whereas p107^-/- mice in the same background have impaired growth and myelopro- liferative disorders. The distinct phenotypes of knock-out mice with a single pocket protein depletion indicate that each pocket protein has unique functions in development. However, pocket proteins also have overlapping functions and can compensate for each other. pRB^-/-/p107^-/- or pRB^-/-/p130^-/- double knock-out embryos die 2 days earlier than pRB^-/- mice and showed, more severe defects in the nervous and haematopoietic systems, indicating that p107 and p130 can partially compensate for pRB. p107^-/-/p130^-/- double knock-out mice die at birth with defects in bone and epidermal development. Since single knock-out mice develop normally, the p107^-/-/p130^-/- double knock-out mice phenotype suggests overlapping functions for p107 and p130.

Fig. 2:

Schematic representation of the domain organisation of pocket proteins.

Pocket proteins are characterised by anA and B domain that together form the small pocket domain that binds to viral oncoproteins and cellular proteins containing a L X CX E motif. p107 and

p130 contain a bipartite B domain and the spacer region between the A and B domain contains a binding site for cyclin A-CDK2 and cyclin E-CDK2 complexes.

In addition, p107 and p130 contain a

domain with CDK inhibitor activity. atinoblastome gne productef rioIntroductn o to the familys

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The defects in pocket protein knock-out mice are consequences of failures in cell cycle regulation, differentiation and apoptosis, which will be discussed below in more detail.

3. Pocket proteins and regulation of the cell cycle

3 . 1 . R e g u l a t i o n o f t h e E 2 F f a m i l y o f t r a n s c r i p t i o n f a c t o r s

The best known function of pocket proteins is their regulation of the cell cycle.

Ectopic expression of individual pocket proteins in several cell types induces a G1-arrest. Pocket proteins inhibit G1 to S-phase transition by binding to and inactiva ting members of the E2F family of transcription factors. E2F forms het- erodimers with DP1 to activate transcription of genes involved in S-phase entry.

All pocket proteins bind to E2F, but they bind to different members of the E2F family. pRB associates with E2F1, -2, and -3, whereas p107 and p130 bind almost exclusively to E2F4 and E2F5 (Cobrinik, 2005). E2F1-3 are more potent activators of transcription than E2F4 and E2F5 and are therefore called activator E2Fs, where- asE2F4 and E2F5 are repressor E2Fs (see below).

Pocket proteins not only sequester E2F and prevent transactivation activity, but also act together with E2F as transcriptional repressors at E2F-responsive promotors. Transcription repression by pocket proteins is mediated by recruiting chromatin-modifying enz ymes, including histone deacetylases (HDAC), histone methyltransferases, and nucleosome remodelling complexes (Frolov and Dyson, 2004).HDACs remove acetyl groups from histones H3 and H4, thereby compacting the chromatin structure, making it less accessible for transcription factors.

Recruitment of SUV 39H1 histone H3 methyltransferase by pocket proteins promotes methylation of histone H3 lysine 9, which attracts the transcription repressor protein HP1. Pocket proteins also bind to the Brahma (Brm) ATPase and its related product BRG1 that are part of the hSWI/SNF complex that mediates chromatin remodelling.

Since pocket proteins are differently expressed during the cell cycle and bind specific E2F family members, transcription of E2F-responsive genes is differently regulated at specific phases of the cell cycle. In quiescent cells and early in G1, p107 and p130 in conjugation with E2F4 and E2F5 and chromatin modifying enz ymes bind to E2F-responsive promotors and repress transcription and, with that, block S-phase entry. pRB also inactivates E2F1-3 at promotors or by sequestration. In late G1, when pocket proteins become phosphorylated, E2F4 and -5 translocate to the cytoplasma and E2F1-3 activate E2F responsive genes to start S-phase. Results from p107^-/-/p130^-/- double knock-out MEFs revealed that many E2F responsive genes are regulated by p107 and p130 repressor complexes, whereas only a subset of genes, including cyclin E and p 1 0 7 , are deregulated in pRB-deficient MEFs, indicating the importance of p107 and p130 transcription repressor complexes.

Transcription repression is involved in determining reversible and permanent cell cycle arrest (Cobrinik, 2005). In serum starved cells, all cell cycle regulated E2F

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responsive promotors bound E2F4 in complex with p107 or p130 and relatively few promotors bound E2F1-3 or pRB. Thereby S-phase entry is prevented. By regulating the amount of histone H3 lysine 9 methylation, pocket proteins are thought to determine whether a cell becomes quiescent or permanently exit the cell cycle. In senes- cent or differentiated cells, more histone H3 lysine 9 methylation of E2F responsive promotors was found than in quiescent cells, which was dependent on pocket proteins.

3 . 2 . R e g u l a t i o n o f p o c k e t p r o t e i n p h o s p h o r y l a t i o n b y c y c l i n /C D K c o m p l e x e s a n d C D K i n h i b i t o r s

Pocket proteins are phosphorylated when conditions are optimal for a cell to divide (Sherr and Roberts, 2004). In early and mid G1, mitogenic signals stimulate cyclin D1 expression which forms a complex with CDK4 or CDK6 that phosphorylate pocket proteins. In late G1, cyclin E is induced, which acts with CDK2 to further phosphorylate pocket proteins, causing E2F release. Active E2F stimulates E2F responsive genes, causing S-phase entry. Pocket proteins remain in their inactive hyperphosphorylated state until protein phosphatase-1 removes the phosphates after mitosis (Tamrakar et al., 2000).

Cyclin/CDK complexes, in turn, are regulated by CDK inhibitors (CKI) that are divided into two classes (Sherr and Roberts, 1999). The first class includes INK4 proteins that prevent cyclin D binding to CDK4 and CDK6. This class comprises four proteins: p16ÎNK4a, p15ÎNK4b, p18ÎNK4c and p19ÎNK4d. The CIP/KIP family, including p21^WAF1/CIP1, p27^KIP1 and p57^KIP2, inhibit cyclin E- and A-dependent kinases, but stimulate cyclin D-dependent kinases. All together, diverse stimuli activate different signalling pathways that feed into the complex network of cyclin/CDK complexes and CDK inhibitors to regulate pocket protein phosphorylation and proliferation (Blagosklonny and Pardee, 2002; Massague, 2004). For example, growth factor stimulation of the Ras/MEK/ERK pathway induces cyclin D expression to promote proliferation. In contrast, TGF` stimulation stimulates the same pathway to induce p21^WAF1/CIP1 and p15ÎNK4b and inhibit cell cycle progression. PKC can also both stimulate cell cycle progression and cell cycle arrest by regulating cyclin D1 and CKIs, including p21^WAF1/CIP1.DNA damage induces a G1-arrest by downregulating cyclin D and inducing p21^WAF1/CIP1, whereas growth factor withdrawal induces p27^KIP1 to arrest cells in G1.

Some molecules in the G1 to S-phase transition regulatory network are frequently mutated in tumours (Malumbres and Barbacid, 2001). p16^INK4a, cyclinD, CDK4/CDK6 and pRB highlight a pathway, also referred to as the pRB pathway that control G1 to S-phase transition. In many if not all tumour tissues one component of this pathway is mutated or amplified leading to uncontrolled S-phase entry and proliferation (Sherr and McCormick, 2002).

In contrast to pRB, p130 is less frequently mutated and p107 inactivation has not been reported in tumours (Classon and Dyson, 2001). However, p130 levels are frequently downregulated in tumours, whereas p107 levels are upregulated.

It is not clear whether these changes in expression are cause or consequence of tumour progression. The fact that viral oncoproteins of DNA tumour viruses,

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including SV40 large T antigen, adenoviral E1A and human papilloma virus E7 inactivate all three pocket proteins indicate that inactivation of all pocket proteins has an advantage in tumour formation.

4. Pocket proteins and regulation of differentiation

In addition to cell cycle regulation, pRB is involved in regulating differentiation.

The role of pRB in differentiation is independent of its role in the cell cycle. Pocket mutants that were unable to bind E2F and to repress transcription were unable to induce a G1-arrest, but still could promote differentiation of SAO S-2 cells (Sellers et al., 1998). In muscle differentiation, pRB is required for terminal cell cycle arrest, but it is not required for maintaining cells in the post-mitotic state. However, ablation of pRB in myotubes reduced tissue-specific gene expression (Camarda et al., 2004).

Muscle differentiation is a highly ordered well characterised process that is regulated by basic helix-loop-helix proteins (MyoD,Myf5, myogenin and MRF4) in collaboration with the MEF2 family of transcription factors (Weintraub, 1993).

Ectopic expression of MyoD in fibroblasts deprived of mitogens induces the muscle differentiation program. Muscle differentiation starts with the expression of myogenin that induces p21 to permanently arrest cells in a pRB dependent way.

When cells are post-mitotic they start to express late differentiation markers, including myosin heavy chain and muscle creatine kinase (MCK). Finally, cells fuse into multinucleated myotubes. pRB^-/-MEFs overexpressing MyoD do express p21 and the early differentiation marker myogenin, but are unable to express myosin heavy chain (Novitch et al., 1996). This effect was specific for pRB, as p107^-/- and p130^-/- MEFs differentiate normally and express myosin heavy chain. pRB is required for MyoD transactivation of MCK and MEF2 promotors and for MEF2-dependent gene transcription (Novitch et al., 1996; Novitch et al., 1999). Defects in differentiation of pRB^-/-MEFs to muscle could be partially rescued by p107 (Schneider et al., 1994).

In contrast, p130 inhibits differentiation of myoblasts and is involved in the deter- mination of reserve cells that remain quiescent and undifferentiated, but have the capacity to self-renew and give rise to differentiated myoblasts (Carnac et al., 2000).

Similar to muscle differentiation, pRB^-/-MEFs only induce early differentiation markers when stimulated to differentiate into adipocytes. Expression of late genes require cooperation between pRB and the differentiation-specific transcription factor CCAAT/enhancer-binding proteins (C/EBP) (Chen et al., 1996). O verexpres- sion of C/EBP and a second differentiation-specific transcription factor, peroxisome proliferator-activated receptor-a (PPARa) in pRB^-/-3T3 cells in combination with differentiation medium did induce some differentiation, but reconstitution of pRB- stimulated adipocyte differentiation. In contrast, p107^-/-/p130^-/- double knock-out 3T3 cells differentiated already in the absence of PPARa and C/EBP, whereas cells lacking p107 or p130 have an intermediate phenotype and showed some differen-

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tiation. Reintroduction of p107 in p107^-/-/p130^-/- double knock-out 3T3 cells inhibited differentiation, indicating that p107 inhibits adipocyte differentiation. Therefore, p107 and pRB have opposing roles in adipocyte differentiation (Classon et al., 2000).

The requirement of pocket proteins in regulating differentiation is also illus- trated by phenotypes of knock-out mice. In pRB^-/- mice, erythroid, neuronal and lens development is initiated and early differentiation markers are expressed, but they were unable to fully differentiate (Lipinski and Jacks, 1999). Some of the defects in pRB^-/- mice were caused by severe disruptions of the architecture of the placenta, because of enhanced proliferation of trophoblasts and a failure of trophoblast differentiation, and therefore are caused by non-cell autonomous functions of pRB (Wu et al., 2003; de Bruin et al., 2003). For example, erythropoiesis occurred normally in pRB^-/- embryo’s supplied with a wild-type placenta. However, these rescued pRB^-/- embryo’s still showed increased mitosis in neurons and, like in non-rescued pRB^-/- embryo’s, they showed defects in cell cycle exit and elevated apoptosis in the lens. Rescued pRB^-/- embryo’s died after birth because of severe skeletal muscle dysplasia that probably causes inability to respire. Because of defects in muscu- lature the vertebral column is also misshaped. These observations also indicate an important role for pRB in muscle differentiation.

p107^-/- and p130^-/- mice were normal and fertile (Cobrinik et al., 1996; Lee et al., 1996). In contrast, p107^-/-/p130^-/- double knock-out mice have shortened limbs and are embryonically lethal because of defects in endochondral bone development caused by a failure in terminal cell cycle exit in chondrocytes. These results suggest that p107 and p130 are able to substitute for each other when only one of these pocket proteins is absent.

All together these results indicate that pocket proteins are required for terminal cell cycle exit and the expression of tissue-specific genes, and they have cell-lineage specific functions.

5. p

RB

as negative regulator of apoptosis

In addition to defects in cell cycle regulation and differentiation, pRB^-/- embryo’s also showed increased apoptosis in the central nervous system, peripheral nervous system, lens and liver, suggesting a role of pRB in regulating apoptosis (Chau and Wang, 2003). However, in pRB^-/- embryo’s supplied with a wild-type placenta no apoptosis in the nervous system and liver was observed, indicating that apoptosis in those tissues is caused by non-cell autonomous functions of pRB. In contrast, apoptosis in lens fibers still occurred in rescued mice. pRB inhibits apoptosis by sequestering E2F that stimulates transcription of a number of proteins involved in apoptosis, including apoptosis protease activating factor-1 (Apaf-1), p19Arf and several caspases. p19Arf is involved in stabilising p53, which stimulates Apaf-1 activity causing activation of the caspase pathway that promotes apoptosis. pRB itself is also cleaved by caspases during apoptosis, which amplifies the apoptosis

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signal since E2F-mediated transcription of apoptotic genes is enhanced. Degra- dation-resistant pRB in which caspase cleavage sites are mutated was shown to attenuate apoptosis. Therefore, pRB is, in addition to its regulatory role in the cell cycle and differentiation important, for cell survival.

6. p

RB

regulation independent of

E2F

In addition to induction of late differentiation genes, pRB mutants revealed more E2F-independent functions of pRB. A mutant that is unable to bind E2F and LXCXE- motif containing proteins (pRB-R661W) was shown to inhibit G1 to S-phase transition and was able to induce a terminal cell cycle arrest in SAOS-2 cells, though not as efficiently as wild-type pRB (Whitaker et al., 1998). The ability of pRB-R661W to arrest cells was at least in part mediated by the pRB C-terminus, since a pRB-R661W mutant in which the pRB C-terminus was inactivated by mutations, was unable to arrest cells in G1 or to induce terminal cell cycle exit. Since a C-terminal fragment of pRB was also able to inhibit G1 to S-phase progression, these results suggest that pRB can inhibit cell growth via two independent mechanisms: (1) via its C-terminus and (2) via the pocket domain in an E2F and LXCXE independent way. However, E2F and/or LXCXE binding is required for a complete terminal cell cycle arrest.

Indeed, LXCXE-motif binding is required for pRB-mediated terminal cell cycle arrest. A mutant that is able to bind E2F but is unable to bind LXCXE proteins (pRB- N757F) is as efficient as wild-type pRB in inducing a G1-arrest in SAOS-2 cells, but is unable to terminally arrest differentiated myocytes (Chen and Wang, 2000).

The same mutant was also unable to arrest pRB-negative C33A cells in G1 upon UV-irradiation, whereas wild-type pRB could mediate a G1-arrest (Pennaneach et al., 2001). The pRB-N757F was also unable to protect SAOS-2 cells from apoptosis induced by UV-mediated DNA damage, whereas wild-type pRB promoted cell survival after UV-irradiation (Pennaneach et al., 2001).

All together these studies suggest that E2F-independent mechanisms exist that arrest cells in G1. Furthermore, E2F binding is not sufficient to induce a terminal cell cycle arrest or to stimulate cell survival, but also require LXCXE-motif containing proteins. It is thought that HDACs that contain a LXCXE-motif are required for sustained growth suppression, but also other proteins may be involved.

7. Concluding remarks

Pocket proteins are key regulators in development as they regulate cell cycle, differentiation and apoptosis. DNA tumour viruses have developed oncoproteins that inactivate all pocket proteins to force cells to proliferate. In addition, in many tumours the pRB pathway is inactivated, which contributes to uncontrolled proliferation. On the other hand, pRB is required for cell survival. As pRB inactivation

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can stimulate proliferation and apoptosis via E2F-mediated transcription, pRB activity has to be differentially regulated. It is not entirely known how the balance between proliferation and apoptosis is regulated, but it is important to know since understanding may give an idea how tumours with inactive pRB may be triggered for apoptosis. In differentiation, pocket proteins have a dual function as they regulate cell cycle exit and expression of tissue-specific genes.

Thus, pocket proteins are central players in an enormous signalling network that regulate cell division, differentiation and apoptosis. However, it is far from clear how pRB suppress tumorigenesis in different tissues and cell types. There- fore, it is not possible yet to predict what effect a deregulated pRB pathway will have on tumorigenesis in a given tissue. As pRB mutants unable to bind E2F or LXCXE binding sites still have tumour suppressive activity, discovering new interacting proteins is likely to give us insight into how this may occur. In the next Chapters, I describe a novel pRB-interacting protein, diacylglycerol kinase-c, and investigate the relevance of this interaction for the cell cycle and in muscle cell differentiation.

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Adams, P.D., Li, X., Sellers, W.R., Baker, K.B., Leng, X., Harper, J.W., Taya, Y ., and Kaelin, W.G., Jr. (1999).

Retinoblastoma protein contains a C-terminal motif th at targ ets it f or ph osph ory lation by cy clin-cd k complex es. Mol Cell Biol. 19, 1068-1080.

Blagosklonny, M.V. and Pardee, A.B. (2002). T h e restriction point of th e cell cy cle. Cell Cy cle 1, 103-110.

Camarda, G., Siepi, F., Pajalunga, D., Bernardini, C., Rossi, R., Montecucco, A., Meccia, E., and Crescenzi, M. (2004). A pRb-ind epend ent mech a- nism preserv es th e postmitotic state in terminally d if f erentiated sk eletal mu scle cells. J Cell Biol.

16 7, 417-423.

Carnac, G., Fajas, L., L’honore, A., Sardet, C., Lamb, N.

J., and Fernandez, A. (2000). T h e retinoblastoma- lik e protein p1 3 0 is inv olv ed in th e d etermination of reserv e cells in d if f erentiating my oblasts.

Curr. Biol. 10, 543-546.

Chau, B.N. and Wang, J.Y .J. (2003). Coord inated reg u - lation of lif e and d eath by RB. Nat Rev Cancer 3, 130-138.

Chen, P.L., Riley, D.J., Chen, Y ., and Lee, W.H. (1996).

Retinoblastoma protein positiv ely reg u lates terminal ad ipocy te d if f erentiation th rou g h d irect interaction w ith C/E B Ps. Genes Dev. 10, 2794-2804.

Chen, T.T. and Wang, J.Y .J. (2000). E stablish ment of I rrev ersible G row th A rrest in M y og enic D if f erentiation Req u ires th e RB L X CX E-B ind ing F u nction.

Mol. Cell. Biol. 20, 5571-5580.

Classon, M. and Harlow, E. (2002). T h e retinoblastoma tu mou r su ppressor in d ev elopment and cancer.

Nat. Rev. Cancer 2, 910-917.

Classon, M. and Dyson, N. (2001). p1 0 7 and p1 3 0: V ersa- tile P roteins w ith I nteresting P ock ets. Experimen- tal Cell Research 26 4 , 135-147.

Classon, M., Kennedy, B.K., Mulloy, R., and Harlow, E.

(2000). O pposing roles of pRB and p1 0 7 in ad ipocy te d if f erentiation.PNAS97, 10826-10831.

Cobrinik, D., Lee, M.H., Hannon, G., Mulligan, G., Bronson, R.T., Dyson, N., Harlow, E., Beach, D., Weinberg, R.A., and Jacks, T. (1996). Sh ared role of th e pRB-related p1 3 0and p1 0 7proteins in limb d ev elopment. Genes Dev. 10, 1633-1644.

Cobrinik, D. (2005). P ock et proteins and cell cy cle control. Oncogene 24 , 2796-2809.

de Bruin, A., Wu, L., Saavedra, H.I., Wilson, P., Y ang, Y ., Rosol, T.J., Weinstein, M., Robinson, M.L., and Leone, G. (2003). Rb f u nction in ex traembry onic lineag es su ppresses apoptosis in th e CN S of Rb-d ef icient mice.PNAS 100, 6546-6551.

Dunn, J.M., Phillips, R.A., Becker, A.J., and Gallie, B.

L. (1988). I d entif ication of g ermline and somatic mu tations a f f ecting th e retinoblastoma g ene.

Science 24 1, 1797-1800.

Friend, S.H., Bernards, R., Rogelj, S., Weinberg, R.A., Rapaport, J.M., Albert, D.M., and Dryja, T.P. (1986).

A h u man D N A seg ment w ith proper ties of th e g ene th at pred isposes to retinoblastoma and osteosar- coma. Nature 323, 643-646.

Frolov, M.V. and Dyson, N.J. (2004). M olecu lar mech a- nisms of E 2 F-d epend ent activ ation and pRB- med iated repression. J Cell Sci 117, 2173-2181.

Hanahan, D. and Weinberg, R.A. (2000). T h e h allmark s of cancer. Cell 100, 57-70.

Harbour, J.W., Luo, R.X., Dei, S.A., Postigo, A.A., and Dean, D.C. (1999). Cd k ph osph ory lation trig g ers seq u ential intramolecu lar interactions th at prog ressiv ely block Rb f u nctions as cells mov e th rou g h G 1. Cell 98 , 859-869.

Lee, J.O., Russo, A.A., and Pavletich, N.P. (1998).

Stru ctu re of th e retinoblastoma tu mou r-su ppressor pock et d omain bou nd to a peptid e f rom H P V E 7. Nature 391, 859-865.

Lee, M.H., Williams, B.O., Mulligan, G., Mukai, S., Bronson, R.T., Dyson, N., Harlow, E., and Jacks, T.

(1996). T arg eted d isru ption of p1 0 7: f u nctional ov erlap bet w een p1 0 7 and Rb. Genes Dev. 10, 1621-1632.

Lipinski, M.M. and Jacks, T. (1999). T h e retinoblastoma g ene f amily in d if f erentiation and d ev elopment.

Oncogene 18 , 7873-7882.

Malumbres, M. and Barbacid, M. (2001). T o cy cle or not to cy cle: a critical d ecision in cancer.

Nat Rev Cancer 1, 222-231.

Massague, J. (2004). G 1 cell-cy cle control and cancer.

Nature 4 32, 298-306.

Morris, E.J. and Dyson, N.J. (2001). Retinoblastoma protein par tners. Adv. Cancer Res 8 2, 1-54.

Norbury, C. and Nurse, P. (1992). A nimal cell cy cles and th eir control. Annu. Rev Biochem. 6 1, 441-470.

Novitch, B.G., Mulligan, G.J., Jacks, T., and Lassar, A.B. (1996). Sk eletal mu scle cells lack ing th e retinoblastoma protein d isplay d ef ects in mu scle g ene ex pression and accu mu late in S and G 2 ph ases of th e cell cy cle. J Cell Biol. 135 , 441-456.

References

(16)

Novitch, B.G., Spicer, D.B., Kim, P.S., Cheung, W.L., and Lassar, A.B. (1999). pRb is required for MEF2- dependent gene expression as well as cell-cycle arrest during skeletal muscle differentiation.

Curr. Biol. 9, 449-459.

Pennaneach, V., Salles-Passador, I., Munshi, A., Brickner, H., Regazzoni, K., Dick, F., Dyson, N., Chen, T.T., Wang, J.Y., Fotedar, R., and Fotedar, A.

(2001). The large subunit of replication factor C promotes cell survival a f ter DNA damage in an LxCxE motif- and Rb-dependent manner.

Mol. Cell 7, 715-727.

Qin, X.Q., Chittenden, T., Livingston, D.M., and Kaelin, W.G., Jr. (1992). Identification of a growth suppression domain within the retinoblastoma gene product. Genes Dev. 6, 953-964.

Rubin, S.M., Gall, A.L., Z heng, N., and Pavletich, N.P.

(2005). Structure of the Rb C-terminal domain bound to E2F1-DP1: a mechanism for phosphorylation-induced E2F release. Cell 123, 1093-1106.

Schneider, J.W., Gu, W., Z hu, L., Mahdavi, V., and Nadal-Ginard, B. (1994). Reversal of terminal differentiation mediated by p107in Rb^-/- muscle cells.

Science 264, 1467-1471.

Sellers, W.R., Novitch, B.G., Miyake, S., Heith, A., Otterson, G.A., Kaye, F.J., Lassar, A.B., and Kaelin, W.G., Jr. (1998). Stable binding to E2F is not required for the retinoblastoma protein to activate transcription, promote differentiation, and suppress tumor cell growth. Genes Dev. 12, 95-106.

Seville, L.L., Shah, N., Westwell, A.D., and Chan, W.C.

(2005). Modulation of pRB/E2F functions in the regulation of cell cycle and in cancer. Curr. Cancer Drug Targets. 5, 159-170.

Sherr, C.J. and McCormick, F. (2002). The RB and p5 3 pathways in cancer. Cancer Cell 2, 103-112.

Sherr, C.J. and Roberts, J.M. (1999). CDK inhibitors:

positive and negative regulators of G1-phase progression. Genes Dev. 13, 1501-1512.

Sherr, C.J. and Roberts, J.M. (2004). Living with or without cyclins and cyclin-dependent kinases.

Genes Dev. 18, 2699-2711.

Tamrakar, S., Rubin, E., and Ludlow, J.W. (2000).

Role of pRB dephosphorylation in cell cycle regulation. Front Biosci. 5, D121-D137.

Weintraub, H. (1993). The MyoD family and myogen- esis: redundancy, net works, and thresholds.

Cell 75, 1241-1244.

Whitaker, L.L., Su, H., Baskaran, R., Knudsen, E.S., and Wang, J.Y. (1998). Growth Suppression by an E2F-Binding-Defective Retinoblastoma Protein (RB) : Contribution from the RB C Pocket.

Mol. Cell. Biol. 18, 4032-4042.

Wikenheiser-Brokamp, K.A. (2006). Retinoblastoma family proteins: insights gained through genetic manipulation of mice. Cell Mol Life Sci.

Wu, L., de, B.A., Saavedra, H.I., Starovic, M., Trimboli, A., Yang, Y., Opavska, J., Wilson, P., Thompson, J.C., Ostrowski, M.C., Rosol, T.J., Woollett, L.A., Wein- stein, M., Cross, J.C., Robinson, M.L., and Leone, G.

(2003). Extra-embryonic function of Rb is essential for embryonic development and viability. Nature 421, 942-947.

(17)