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

Los, Alrik Pieter

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

activity, protein binding partners and physiological functions. Retrieved from

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

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

s ig n a llin g fu n c t io n s o f

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

a n d - e is o fo r m s

Chapter 1

A b stract

D iacylglycerol kinases (

s) p hosp horylate the second

messenger diacylglycerol (

) yielding p hosp hatidic

acid (

). T en different mammalian

isoforms hav e

b een describ ed and in this rev iew w e w ill focus on the

c and

e isoforms.

c is ub iq uitously ex -

p ressed, w hereas

e ex p ression is limited to certain

sp ecific tissues. B oth isoforms and their activ ities hav e

sev eral sub cellular locations, b eing regulated b y lip ids,

p rotein interactions and grow th factors. T his comp lex

regulation serv es to temp orally and sp atially restrict

c and

e activ ity. A ccumulating ev idence suggest

that

c and

e hav e a dual role in signalling b y reg-

ulating

-b inding p roteins as w ell as

-b inding p ro-

teins. T he former include p rotein kinase

(

),

Ras guanyl nucleotide-releasing p rotein (

as-

),

chimaerins, and M unc-13 , w hereas the

-b inding p ro-

teins include p hosp hatidylinositol-5 -kinases (

)

and mammalian target of rap amycin ( m

). T herefore,

c and

e may p lay a crucial role in determining

the correct b alance b etw een

and

signalling

p athw ays to regulate p hysiological p rocesses.

Introduction to the signalling functions of diacylglycerol kinase-c and -e isoforms

Contents

1. Introduction...13

2 . C h a ra cte ris tics , ce ll/ tis s ue dis trib ution a nd s ub ce llula r loca lis a tion of D G Kc...15 2 .1. C h a ra cte ris tics of D G Kc...15

2 .2 . C ons e rv a tion a m ong s p e cie s...15 2 .3 . C e ll/ tis s ue dis trib ution of D G Kc...17 2 .4 . S ub ce llula r loca lis a tion of D G Kc...18

2 .4 .1. D e p e nde nce on ce ll ty p e , e nv ironm e nta l conditions a nd de v e lop m e nt...18 2 .4 .2 . D e p e nde nce on D G Kc s tructura l dom a ins...19

3 . C h a ra cte ris tics , ce ll/ tis s ue dis trib ution a nd s ub ce llula r loca lis a tion of D G Ke...20 3 .1. C h a ra cte ris tics of D G Ke...20

3 .2 . C ons e rv a tion a m ong s p e cie s...20

3 .3 . C e ll/ tis s ue dis trib ution a nd s ub ce llula r loca lis a tion of D G Ke...22 4 . Re g ula tion of D G Kc a nd D G K e a ctiv ity...22

4 .1. S ub s tra te s p e cif icity a nd re g ula tion b y lip ids...22 4 .2 . Re g ula tion b y p rote ins a nd p rote in m odif ica tions...24 4 .3 . T ra ns loca tion a nd a ctiv a tion induce d b y g row th f a ctors...24 5 . S ig na lling f unctions of D G K c a nd D G Ke...25

5 .1. D G Kc a s ne g a tiv e re g ula tors of P K C s ig na lling...25 5 .2 . D G Ke a s ne g a tiv e re g ula tors of P K C s ig na lling...27 5 .3 . Role of D G K c in th e ^Ra s -E RK p a th w a y...27

5 .4 . Role of D G K c in a ctin cy tos k e le ton re a rra ng e m e nt...29 5 .5 . Role of D G K e in a ctin cy tos k e le ton re a rra ng e m e nt...33 5 .6 . D G Kc a nd D G Ke in ne urona l f unctioning...33

5 .7 . D G Kc in ce ll cy cle re g ula tion...35

5 .8 . S ig na lling f unctions of P A g e ne ra te d b y D G K c a nd/ or D G K e...36 6 . C oncluding re m a rk s...38

7 . O utline of th is th e s is...38

1. Introduction

The lipid second messenger diacylglycerol (DAG) is produced upon stimulation with a variety of hormones that regulate many cellular processes including cell division, differentiation, cytoskeletal reorganisation and vesicular transport.

The diverse role of DAG in signal transduction is mediated by an array of DAG- binding proteins including protein kinase C (PKC) ,Ras guanyl nucleotide-releas- ing proteins (Ras-GRP) , chimaerins, and Munc-13 (Brose et al., 20 0 4) . DAG is generated by G-protein coupled receptor-mediated activation of phospholipase C-` (PL C`) or tyrosine kinase-linked receptor-mediated activation of PL Ca.PL C hydrolyses phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) yielding DAG and inositol-1,4,5-trisphosphate (IP₃) . In addition, DAG is produced by phospholipase D-mediated hydrolysis of phosphatidylcholine (PC) followed by dephosphorylation of phosphatidic acid, and during de novo synthesis of phospholipids and triglyc- erides (van Blitterswijk et al., 19 9 4) .

DAG is involved in regulating many signalling pathways and its levels likely need to be tightly regulated. U ncontrolled synthesis of DAG may contribute to cellular transformation as tumours often show elevated levels of DAG (Kato et al., 19 87; Kato et al., 19 88) . Attenuation of DAG is achieved by five possible mecha- nisms: phosphorylation by diacylglycerol kinases yielding phosphatidic acid (PA) , hydrolysis by DAG lipase generating monoacylglycerol and fatty acids, conversion into PC by CDP-choline:DAG choline phosphotransferase or into phosphatidyletha- nolamine by CDP-ethanolamine:DAG ethanolamine phosphotransferase, or conversion by transphosphatidylation into bisphosphatidic acid (van Blitterswijk et al., 19 9 4) . It is thought that DGKs are involved in rapid attenuating of DAG produced after phosphatidylinositol hydrolysis.

The importance of DGKs in signalling is underlined by the siz e of the DGK family; recently, the tenth DGK isoform, DGKg has been described (Imai et al., 20 0 5) .DGK isoforms can be divided into five classes, based on their structural domains. They all contain two or three cysteine-rich domains (CRD) that are thought to bind DAG, and a catalytic domain that is conserved among all DGKs.

In addition, each class of DGKs contains different signalling domains that link them to signal transduction pathways. In addition to the mammalian DGK iso- forms, DGKs have been identified in D r os op h ila m ela nog a s t er , C a enor h a b dit is eleg a ns , D ic t y os t eliu m dis c oideu m , and A r a b idop s is t h a lia na (Topham, 20 0 5) . The complex ity of the family is further increased by the ex istence of splicing variants of DGKs.

W hile the DGK family and characteristics and functions of each isoform were described in several reviews (van Blitterswijk and H oussa, 20 0 0 ; Kanoh et al., 20 0 2; L uo et al., 20 0 4b; Goto and Kondo, 20 0 4; Topham, 20 0 5) , this chapter is limited to recent progress on DGKc and DGKe isotypes only, focusing on their domain structure, (sub) cellular localisation and mode of regulation and activation and their role in signal transduction.

Introduction to the signalling functions of diacylglycerol kinase-c and -e isoforms

Fig. 1.

Schematic representation of the domain organisation of DGKc and DGKe and their homologs.

H umanDGKc and DGKe and their homologues contain a conserved cata- lytic domain, respectively two and three cysteine-rich domains (CRD) of which the most C-terminalCRD is extended with a stretch of 15 amino acids (E xt).

In addition, DGKc contains a domain that is homologue to the phosphoryla- tion site domain of the MARCKS protein (MARCKS -PS D) and four Ankyrin re- peats. Caenorabditis elegansDGKc (ceDGKc, gene 2G748, accession number N P_ 495301) lacks ankyrin repeats, whereas, D rosop h ila m elanogaster DGK2 lacks a MARCKS -PS D.DGKe con- tains a proline- and glycine-rich domain (Pro) and a Pleckstrin homology (PH ) domain overlapping with a Ras asso- ciation (RA) domain. D ic ty osteliu m dis- c oideu m DGKA (ddDGKA) lacks a PH domain, but contains a large poly-aspar- agine stretch (Asp).

Domain Function Re ference

CRDs

DAG binding?

Essential for DGK activity Localisation

(Bunting et al., 1996) (Santos et al., 2002) (Santos et al., 2002)

MARCKS-PSD Localisation

Phosphorylated by PKC

(Topham et al., 1998) (Luo et al., 2003b) pRB (pocket protein) binding (Los et al., 2006)

Catalytic domain Essential for DGK activity (Topham et al., 1998; Santos et al., 2002)

Ankyrin repeats Localisation

Leptin receptor (Ob-Rb) binding

(Goto and Kondo, 1996; Hozumi et al., 2003) (Liu et al., 2001)

PDZ-binding motif Syntrophin binding Localisation

(Hogan et al., 2001)

(Hogan et al., 2001; Abramovici et al., 2003) T ab l e 1 . _DGKc structural domains and their functions

2. Characteristics, cell/tissue distribution

and subcellular localisation of

c

2 . 1 . C h a r a c t e r i s t i c s o f D G K

c

In 1996, DGKc was independently identified by two groups: Prescott and co-work- ers cloned DGKc from human umbilical vein endothelial cells, whereas Goto and Kondo isolated DGKc (DGK-IV) cDNA from cDNA libraries of rat retina and brain (Bunting et al., 1996; Goto and Kondo, 1996). Two years later, Prescott and co-work- ers also cloned DGKc from a mouse brain cDNA library (Ding et al., 1998). Human and rat/mouse DGKc cDNAs encode a protein of, respectively, 928 and 929 amino acids and share 88% identity at the nucleic acid level and 95.5% at the amino acid level. DGKc is classified in class ^{IV DGK}s that also include DGKf, based on some characteristic domains (Fig. 1A; Table 1). The class IV DGKs contain a region that is similar to the phosphorylation-site domain (PSD) of myristoylated alanine-rich C-kinase substrate (MARCKS) (Bunting et al., 1996). The DGKc-MARCKS-PSD contains several putative PKC phosphorylation sites and overlaps with a nuclear localisation signal. In addition, DGKc and DGKf are characterised by the presence of four tandem ankyrin repeats that are involved in protein-protein interactions.

Furthermore, DGKc contains a functional PDZ-binding motif at its C-terminus (Fabre et al., 2000), and two PEST seq uences that may regulate protein degradation (Bunting et al., 1996). The catalytic domain and the two CRDs are essential for DGKc activity, since point-mutations in these domains inactivated the enzyme (Topham et al., 1998; Santos et al., 2002). In contrast, the ankyrin repeats and the PDZ-binding site are not essential for DGK activity.

The human DGKc gene is localised on chromosome 11p11.2 and spans approx- imately 50 kb genomic seq uence containing 32 exons (Ding et al., 1997). An alter- native splice variant of DGKc was isolated from a human skeletal muscle cDNA library. It encodes a DGKc variant of 1.117 amino acids (130 kDa) that contains the same functional domains, but has a uniq ue N-terminus in which no additional known domains could be identified (Ding et al., 1997). The alternative N-terminus of the DGKc splice variant was encoded by an exon between the first two exons of the endothelial DGKc form. The existence of tissue-specific ^DGKc variants suggests that DGKc has tissue-related functions and/or is specifically regulated in different tissues.

2 . 2 . C o n s e r v a t i o n a m o n g s p e c i e s

Homologues of DGKc have been found in at least two other species (Fig. 1A).

Drosophila melanogaster DGK2, which is encoded by the rdgA locus, is 42% iden- tical and 61% similar to human DGKc (Masai et al., 1993; Bunting et al., 1996). DGK2 has an elongated N-terminus with unknown function and does contain the four tandem ankyrin repeats but no MARCKS-PSD. A Caenorhabditis elegansDGK, encoded by gene 2G748 (also known as K06A1.6 or Y K1670), is most similar to the DGKc isoform (39% identity at the amino acid level). It also contains two ^CRDs, a catalytic domain and a domain that is similar to the MARCKS-PSD with four serines and a stretch of positively charged residues, but lacks ankyrin repeats.

Introduction to the signalling functions of diacylglycerol kinase-c and -e isoforms

Tissue Cell type Subcellular localisation References

Embryogenesis: (Ding et al., 1998)

– Somites – Limb buds – Interdigital regions – dorsal root ganglia – vibrissa follicles

– rat lung (Katagiri et al., 2005)

Lung Various epithelial cells nucleus (Katagiri et al., 2005)

Alveolar type II cells nucleus

Heart Cardiomyocytes nucleus (Takeda et al., 2001)

Skeletal muscle Myoblasts sarcolemma (Abramovici et al., 2003)

Ovary follicles (theca cells) (Toya et al., 2005)

corpora lutea interstitial cells

Placenta labyrinthine zone (all cell types) (Toya et al., 2005)

Lymphoid system T cells cytosol (Santos et al., 2002)

Brain: (Goto and Kondo, 1996;

Hozumi et al., 2003)

– Cerebellum Purkinje cells nucleus+ proximal

dendritic shaf t

granule cells nucleus

– Hippocampus Pyramidal cells nucleus

dentate granular cells nucleus scattered interneurons nucleus

– cerebral cortex most neurons nucleus

– olf actory bulb and tubercle

hypothalamic nuclei (Liu et al., 2001)

L`T2 gonadotrope cells nucleus/cytosol (Davidson et al., 2004) DDT1-MF2 ductus def erence

smooth muscle nucleus/cytosol (Fukunaga-Takenaka et al.,

2005)

IIC9 fibroblasts cytosol (Bregoli et al., 2001)

MEF,U2-OS,MCF7,MDCK,

N1E-115,N2A cytosol/nucleus our unpublished

observations

HL60 human leukemia cells (Batista, J r. et al., 2005)

Table 2 . Tissue distribution and subcellular localisation of DGKc:

However, this protein has not been characterised. The presence of DGKc in lower organisms indicates that DGKc is evolutionally conserved, with essential func- tions that are already required for more primitive organisms.

2 . 3 . C e l l / t i s s u e d i s t r i b u t i o n o f D G K

c

DGKc is already expressed during embryogenesis in diverse developmental struc- tures as is summarised in Table 2. Intriguingly, DGKc was not only expressed in tissues in which massive proliferation and differentiation occurs, including limb buds, but also in areas with extensive apoptosis. For example, DGKc is expressed in interdigital regions of the limbs in which apoptosis is required to shape the digits. The broad distribution of DGKc suggests a diverse role in embryogenesis (Ding et al., 1998). However, DGKc knock-out mice did not show any defects during development as they do not have a phenotype (G. Koretzky, personal communi- cations), indicating that DGKc is not essential for development.

DGKc is broadly expressed throughout the adult body and in diverse cell types as is shown in Table 2. DGKc mRNAs could be detected in all human and rat tissues that were tested, but highest expression was found in brain (Bunting et al., 1996;

Liu et al., 2001). The alternatively spliced DGKc variant identified in skeletal muscle was also expressed in heart, bladder and intestine muscle and in the lymphoid system (Ding et al., 1997; Zhong et al., 2002). Although not all of these tissues have been checked for DGKc protein expression, the presence of DGKc mRNAs in all tested tissues suggests that DGKc plays a role throughout the whole body.

The localisation of DGKc in brain is extensively investigated. In brain, highest expression was found in Purkinje cells of the cerebellum and in pyramidal neurons of the hippocampus that integrate signals and are responsible for the output of the cerebellar cortex and hippocampus (Goto and Kondo, 1996; Hogan et al., 2001;

Hozumi et al., 2003). DGKc is present in the cell bodies and dendrites of Purkinje cells but not in axons, suggesting that DGKc is involved in signal receiving and integration. Since DGKc is present in hippocampus, cerebral and cerebellar cortex, and olfactory bulb, which are brain areas known to be involved in synaptic plasticity, it is thought that DGKc is involved in learning and memory function (Hozumi et al., 2003). Interestingly, DGKc expression in brain is increased by 2-fold in adults compared to newborn mice (Ding et al., 1998), which may imply a role for DGKc in neuronal development and learning. DGKc is lower expressed in spinal cord and medulla that have autonomous functions, and in thalamus and corpus callosum that receive, process and distribute information and connect different areas (Ding et al., 1998), suggesting that it is less important for those brain functions.

In addition to its potential role in learning, DGKc may also regulate energy balance as it is involved in leptin signalling (Liu et al., 2001). DGKc is expressed in neurons of hypothalamic nuclei that regulate food uptake and body weight.

Interestingly, hypothalamic DGKc expression was increased in mice that consumed a high-fat diet, whereas its levels then declined when the mice were maintained on the high-fat diet and became obese. The function of DGKc in leptin signalling needs to be further investigated.

Introduction to the signalling functions of diacylglycerol kinase-c and -e isoforms

The DGKc homologue in Drosophila,DGK2, is localised in photoreceptors of the retina, but was not detectable in brain or in other parts of the body (Masai et al., 1993).DGKc was also detected in the rat eye, but in different cell types (Goto and Kondo, 1996). DGKc is localised in the inner granular layer of the retina in Mü ller’s supporting cells and in bipolar, horizontal and amacrine cells that are involved in transducing signals from the photoreceptors to ganglion cells, the output neurons of the retina. Since DrosophilaDGK2 and mammalian DGKc are differently local- ised they may have different functions in the retina.

2 . 4 . S u b c e l l u l a r l o c a l i s a t i o n o f D G K

c

In addition to the broad tissue distribution, DGKc can have several subcellular locations. Depending on cell-type and environmental conditions, it is localised within the nucleus (most often), cytosol and/or plasma membrane (Table 2).

DGKc localisation is regulated by its signalling domains and by binding to other proteins.

2 . 4 . 1 . D e p e n d e n c e o n c e l l t y p e , e n v i r o n m e n t a l c o n d i t i o n s a n d d e v e l o p m e n t

In many, but not all cell types, DGKc is found in the nucleus. In COS-7 cells, HEK293 cells and cultured hippocampal neurons, overexpressed full-length DGKc is local- ised in the nucleus (Goto and Kondo, 1996; Hozumi et al., 2003; Avila-Flores et al., 2005). Alternatively spliced muscle-specific DGKc is also localised in the nucleus (Ding et al., 1997). In skeletal muscle, on the other hand, DGKc is localised at the sarcolemma (Abramovici et al., 2003). Fractionation analysis of IIC9 fibroblasts revealed that DGKc is present in the cytosol, but not in the nucleus (Bregoli et al., 2001). In different cell types in lung and in cardiac myocytes, DGKc is localised in the nucleus (Katagiri et al., 2005; Takeda et al., 2001). Overexpression of DGKc-GFP fusion proteins in L`T2 murine gonadotrope cells or DDT1-MF2 hamster ductus deferens smooth muscle cells also showed high expression in the nucleus and mod- erate cytosolic expression (Davidson et al., 2004; Fukunaga-Takenaka et al., 2005).

We also examined localisation of overexpressed DGKc in ^COS-7 and HEK293 cells and also found DGKc to be present in the nucleus, but only in a small percentage of cells. Other cells showed cytosolic and plasma membrane staining. We also examined DGKc localisation in various types of cells that stably express DGKc at low levels and observed that in (fixed) mouse embryonic fibroblasts, U2-OS osteosarcoma, MCF7 breast adeno carcinoma, N115 and N2A neuroblastoma, and Madin-Darby caning kidney cells, the majority of DGKc is localised in the cytosol, whereas faint staining is present in the nucleus (our unpublished data). In a minority of cells, a stronger nuclear staining was visible, but that was especially in cells expressing high levels of DGKc. Therefore, localisation of highly expressed DGKc may be artificial to some extent.

Subcellular localisation of endogenous DGKc is extensively studied in brain and appeared to be highly regulated under specific conditions. Endogenous DGKc was shown to be nuclear localised in most neurons, including pyramidal cells in the cerebral cortex, all neurons in the hippocampus and in Purkinje and granule

cells in de cerebellum (Hozumi et al., 2003). Interestingly, DGKc was translocated from the nucleus to the cytosol in hippocampal CA1 pyramidal neurons in ischemic rat brain, whereas such translocation was not observed in other hippocampal cells or other brain areas (Ali et al., 2004). In addition, in cerebral infarction, DGKc levels rapidly decreased in ischemic cortical neurons, which occurred prior to neuronal degeneration (Nakano et al., 2006). The functional consequence of regu- lating DGKc levels or translocation is not known. Finally, localisation of DGKc in cerebellar Purkinje cells is dependent on the developmental stage of the cere- bellum (Hozumi et al., 2003). During cerebellar development DGKc relocalises from the perinuclear cytoplasm and axon hillock to the nucleus and dendrites, suggesting that it has a function during cerebral development.

2 . 4 . 2 . D e p e n d e n c e o n D G K

c

The nuclear localisation signal (NLS) within the MARCKS-PSD is sufficient for nuclear localisation of DGKc, as a MARCKS-GFP fusion protein was localised in the nucleus. In addition, a rat DGKcNLS-GFP fusion protein was also localised in the nucleus of cultured hippocampal neurons (Hozumi et al., 2003). Deletion ofthe MARCKS-PSD or inactivation of the NLS by substitution of all basic into neutral amino acids dislocalised DGKc from the nucleus to the cytosol.

The N-terminal part of DGKc including the CRDs is also involved in regulating DGKc localisation. In T-cells, DGKc is localised in the cytosol (Santos et al., 2002).

However, when both CRDs of DGKc are removed, it is mainly found in the nucleus.

In contrast, mutation of the first histidine of the CRD core structure that is suffi- cient to inactivate the enzyme, still results in localisation in the cytosol, suggest- ing that the N-terminus keeps DGKc in the cytosol, but that functional CRDs are not required.

Removal of the C-terminus up to the catalytic domain (DGKc-6C) showed a nuclear localisation especially in intranuclear granules in COS-7 cells (Goto and Kondo, 1996). In contrast, in hippocampal neurons DGKc-6C is localised in the cytosol (Hozumi et al., 2003). Also localisation of the expressed C-terminus is different in COS-7 cells and hippocampal neurons, i.e. nucleus or cytosol respec- tively. The difference in localisation may reflect the difference in cell type or expression levels, which is extremely high in COS-7 cells. The cytosolic expression in hippocampal neurons of the DGKc-6C mutant, in which the NLS domain remained unaffected, suggests a role for the C-terminus in nuclear localisation. Indeed, the C-terminal PDZ-binding motif is involved in regulating subcellular localisation of DGKc by binding to a1-syntrophin (Hogan et al., 2001). The adapter protein

a1-syntrophin keeps DGKc in the cytosol, whereas DGKc recruits a1-syntrophin to the nucleus in HeLa cervix carcinoma cells. Inhibiting the interaction by over- expressing DGKc-FLAG or a PDZ-binding motif mutant of DGKc, disrupts the balanced localisation of both proteins and caused DGKc to accumulate in the nucleus and a1-syntrophin in the cytosol. Also in muscle, DGKc is partly relocal- ised from the plasma membrane to the nucleus in cells with lower syntrophin levels or when the PDZ domain is masked by a FLAG tag (Abramovici et al., 2003).

Why DGKc-6C is localised in the cytosol of neurons remains an open question,

Introduction to the signalling functions of diacylglycerol kinase-c and -e isoforms

since this mutant cannot be kept in the cytosol by syntrophin. Since this mutant also lacks ankyrin repeats, these particular domains may also be involved. Alter- natively, the NLS in the DGKc-6^C mutant might be cryptic in the tertiary structure, and molecules interacting with the C-terminal region might determine whether or not the NLS is exposed to allow nuclear transport (Hozumi et al., 2003).

In conclusion, the NLS is not the only structural domain that regulates DGKc subcellular localisation. Both C-terminal and N-terminal sequences may co-regu- late DGKc localisation, depending on interactions with proteins such as a1-syn- trophin and exposure to environmental signals, in a cell type-dependent fashion.

3. Characteristics, cell/tissue distribution

and subcellular localisation of

e

3 . 1 . C h a r a c t e r i s t i c s o f D G K

e

DGKe was cloned in 1997 in our lab (Houssa et al., 1997). Human DGKe cDNA encodes a protein of 941 amino acids with a molecular mass of 110 kDa and is the only known isoform that belongs to the class V DGK category (Fig. 1B; Table 3).

DGKe is the only DGK isoform that contains three instead of two CRDs. The third CRD of DGKe is most homologous to the second CRD of other DGK isotypes and is extended with a stretch of 15 amino acids that is conserved among all DGKs (Houssa and van Blitterswijk, 1998). Mutational analysis showed that the CRD extension is essential for DGK activity (Los et al., 2004). DGKe contains a catalytic domain and a proline- and glycine-rich domain with a putative SH3 domain binding site. Furthermore, it has a pleckstrin homology (PH) domain that overlaps with aRas association domain. Truncation studies have shown that the entire primary sequence of the protein is required for full activity (Los et al., 2004). Even a minor 33 amino acid truncation at the C-terminus or removal of the N-terminal proline- and glycine-rich domain rendered the protein inactive. Only an N-terminal trun- cation that includes the first CRD yields some residual activity, 10-20% activity of wild-type DGKe (Los et al., 2004).

3 . 2 . C o n s e r v a t i o n a m o n g s p e c i e s

DGKe homologues have been found in Dictyostelium discoideum and Caenorhab- ditis elegans (Fig. 1B). DictyosteliumDGK, which is encoded by the dgk A gene, was first thought to be a myosin II heavy chain kinase. However, the protein did not have a protein kinase domain, but a DGK catalytic domain (De la Roche et al., 2002). It consists of 887 amino acids and is most closely related to DGKe sharing 40% identity and 56% similarity with human DGKe. In addition to three CRDs, with the third being extended, and a catalytic domain, it has a short proline- and glycine-rich domain and an asparagine-rich domain, but lacks a PH domain.

DGK activity analysis showed that the protein indeed phosphorylates DAG (De la Roche et al., 2002; Ostroski et al., 2005). Southern blot analysis and BLAST searches revealed that dgk A is probably the only DGK in Dictyostelium (De la

Domain Function Reference

Pro/Gly-rich motif Essential for DGK activity (Los et al., 2004)

CRDs

DAG binding?

Essential for DGK activity Localisation

(Houssa and van Blitterswijk, 1998) (Los et al., 2004)

(van Baal et al., 2005)

PH/Ras unknown

Catalytic domain Essential for DGK activity RhoA binding

(Los et al., 2004)

(Los et al., 2004; McMullan et al., 2006)

Tissue Cell type Subcellular localisation References

Small intestine Duodenum Liver

(Houssa et al., 1997)

rat small arteries vascular smooth muscle,

endothelial cells nucleus (Walker et al., 2001)

IIC9,MDA-MB-453, MCF7, PC12,

A431,HeLa nucleus/cytosol

(Bregoli et al., 2001;

Tabellini et al., 2003;

van Baal et al., 2005) U2-OS,MDCK,COS,CHO,HEK293,

N1E-115,Jurkat-J6 (van Baal et al., 2005)

Brain: (Houssa et al., 1997)

– Cerebellum Purkinje cells granule cells – Hippocampus

– Cerebral cortex – olf actory bulb – brain stem nuclei

C. elegans neurons cell bodies (peri-nuclear)

and axons (Nurrish et al., 1999)

excretory canals

Roche et al., 2002). It would be interesting to know what function dgkA has in this organism, as it may help to understand the function of mammalian DGKe.

The C. elegans dgk-1 gene encodes a protein of 950 amino acids that is 38%

identical to human DGKe (Nurrish et al., 1999). DGK-1 also has three CRDs, a catalytic domain and a PH domain. Mutational analysis of dgk-1 revealed that all nine amino acid substitutions found to inhibit DGK-1 function were in the second or third CRD or in the catalytic domain, indicating the importance of these domains (Jose and Koelle, 2005).

Table 3 . DGKe structural domains and their functions

Table 4 . Tissue distribution and subcellular localisation of DGKe:

3 . 3 . C e l l / T i s s u e d i s t r i b u t i o n a n d ( s u b ) c e l l u l a r l o c a l i s a t i o n o f D G K

e

DGKe is less ubiquitously expressed in the body than ^DGKc. Expression of ^DGKe in tissues and cell lines is summarised in Table 4. In mammals and C. elegans, highest expression was found in neurons (Houssa et al., 1997; Nurrish et al., 1999).

In brain, DGKe mRNA was ubiquitously expressed in gray matter, but not in white matter. Highest expression was found in Purkinje and granule cells of the cere- bellar cortex and in hippocampus, while moderate expression was observed in the olfactory bulb, cerebral cortex and brain stem nuclei (Houssa et al., 1997). The wider expression throughout the gray matter, suggest a more general function for DGKe in brain (Houssa et al., 1997).

Subcellular fractionation and immunofluorescence of COS-7 cells overex- pressing DGKe showed ^DGKe localisation in membranes, cytosol and in the peri- nuclear region (Houssa et al., 1999). In rat mesenteric small arteries, DGKe was also found in nuclear extracts (Walker et al., 2001). In addition, DGKe expression was observed in the nucleus and cytosol of several cell lines as is shown in Table 4. In MDA-MB-453, but also in some other cell lines, DGKe was localised in nuclear speckles were it colocalised with PI(4,5)P₂,PLC`1 and specific markers for speck- les including the splicing component SC-35 and RNA polymerase II (van Blitter- swijk and Houssa, 2000; Tabellini et al., 2003). However, detection of DGKe in nuclear speckles was observed with one specific DGKe antibody and could not be reproduced with a different set of DGKe-specific antibodies (Van Baal and Van Blitterswijk, unpublished observations), making it difficult to draw definite conclusions about this nuclear localisation.

4. Regulation of

c and

e activity

Phosphorylation of DAG by DGKs can be regulated in two ways: by regulating catalytic activity of DGKs and by bringing DGKs into close proximity with their substrate (Table 5 and 6). Catalytic activity was shown to be regulated by lipids, protein interactions, and by protein modifications, including phosphorylation.

Translocation of DGKs to their substrate and/or catalytic activity is regulated upon stimulation with different growth factors.

4 . 1 . S u b s t r a t e s p e c i f i c i t y a n d r e g u l a t i o n b y l i p i d s

Like other DGK isotypes, DGKc and DGKe prefer 1,2-diacyl-sn-glycerol as substrate over 1,3-diacyl-sn-glycerol and monoacylglycerol (Bunting et al., 1996; Ding et al., 1997; Houssa et al., 1997). An ether-linkage instead of ester-linkage at the sn-1 position, such as in 1-O-hexadecyl-2-arachidonoyl-sn-glycerol generally lowers the enzymatic rate (Epand et al., 2004). DGKc activity is increased when negatively charged lipids, i.e. phosphatidylserine (PS), phosphatidylinositol (PI),PI(4,5)P₂ and PA, are included in mixed micelle assays (Thirugnanam et al., 2001). The posi- tive effect of PI(4,5)P₂ and other anionic lipids on DGKc activity is thought to be

a consequence of strong interactions with the positively charged DGKc-MARCKS- PSD that may tighten DGKc binding to membranes, like was shown for PI(4,5)P₂ binding to MARCKS (Wang et al., 2001). Our preliminary (unpublished) data from lipid blot assays suggest that the DGKc-^MARCKS-PSD indeed binds to the anionic phosphoinositides.

In an assay with large unilamellar vesicles that more closely resemble cellu- lar membranes, as they consist of a lipid bilayer, phosphatidylethanolamine (PE), which typically promotes negative curvature of the membrane, was shown to stimulate DGKc binding to the membrane and to increase DGKc activity (Fanani et al., 2004). Sphingomyelin inhibited DGKc activity. Cholesterol, another lipid that promotes negative curvature, enhanced membrane binding of DGKc but did not affect DGK activity. Cholesterol inhibited PE-mediated DGKc activation but reverted DGKc inhibition by sphingomyelin (Fanani et al., 2004), so that it seems to act as a moderating lipid. Since in biological membranes, sphingomyelin and cholesterol colocalise in caveolae/lipid raft microdomains, the above findings with artificial mixtures of these lipids could be of potential physiological relevance.

Category Cell type Factor Stimulatory/inhibitory Reference

Lipids in vitro PS,PI,PI(4,5)P2,PA + (Thirugnanam et al.,

2001)

PE + (Fanani et al., 2004)

SM -

Proteins PKC_ - (Luo et al., 2003b)

in vitro pRB, p107, p130 + (Los et al., 2006)

Growth

f actors gonadotrope cells GnRH + / translocation (Davidson et al., 2004)

Jurkat+muscarine

receptor carbachol translocation (Santos et al., 2002)

Category Cell type Factor Stimulatory/inhibitory Reference

Lipids in vitro PS + (Bregoli et al., 2001)

in vitro PS,PA (ddDGKA) + (Ostroski et al., 2005)

Proteins RhoA - (Houssa et al., 1999)

Growth

f actors ^A431 ATP/ bradykinin / thrombin translocation (van Baal et al., 2005) IIC9 fibroblasts _-thrombin + / translocation (Bregoli et al., 2001)

PC12 NGF + (Tabellini et al., 2004)

arteries Noradrenaline + / translocation (Walker et al., 2001)

Table 5 . Regulation of DGKc activity:

Table 6 . Regulation of DGKe activity:

However, no physical association of DGKc to such lipid domains has been reported to date.

Similar to DGKc,^DGKe is also stimulated by ^PS in mixed micelles assays, but other anionic lipids have not been tested (Bregoli et al., 2001). The Dictyostelium DGKe homologue, DGKA was shown to be activated by PS as well as PA (Ostroski et al., 2005).

4 . 2 . R e g u l a t i o n b y p r o t e i n s o r p r o t e i n m o d i f i c a t i o n s

PKC_ was shown to bind to DGKc and to phosphorylate the DGKc-MARCKS-PSD in HEK293 cells (Luo et al., 2003b). Phosphorylation of DGKc inhibits DGKc activity by 40%. In contrast, the retinoblastoma tumour suppressor protein (pRB) and its family members p107 and p130 also bind to DGKc and stimulate ^DGKc activity up to 5-fold (Los et al., 2006). These opposing effects of PKC_ and p^RB-type proteins allow tight regulation of DGKc activity.

DGKe binds to the effector loop of GTP-bound RhoA (Houssa et al., 1999).

Associated active RhoA completely inactivates DGKe, which may be explained by the binding of RhoA to the catalytic domain of DGKe (Los et al., 2004).

4 . 3 . Tr a n s l o c a t i o n a n d a c t i v a t i o n i n d u c e d b y g r o w t h f a c t o r s DGKc and DGKe are activated and/or translocated upon stimulation with certain growth factors. In HEK293 cells, endogenous DGK activity diminished after serum starvation and increased upon serum addition, which was also observed in cells that overexpress DGKc (Avila-Flores et al., 2005). This result indicated that ^DGKc is activated by growth factors. A growth factor that stimulates DGKc activity is gonadotrope releasing hormone (GnRH), which increased DGKc activity by 2.5 or 6-fold in, respectively, SCL60 and L`T2 gonadotrope cells (Davidson et al., 2004).

Upon GnRH stimulation, DGKc was shown to bind to active c-Src, which is required for GnRH-mediated activation of DGKc and for translocation of cytosolic DGKc to the plasma membrane. In Jurkat T cells expressing a muscarinic type I receptor, stimulation of the receptor with carbachol causes a rapid translocation of GFP- tagged DGKc from the cytosol to the plasma membrane, which requires intact CRDs and phosphorylation of the MARCKS-PSD. Since CRDs are thought to bind to DAG, functional CRDs are probably required for DAG-mediated recruitment of DGKc to the plasma membrane (Santos et al., 2002).

DGKe also translocates upon stimulation with growth factors. DGKe trans- located from cytosol to plasma membrane upon stimulation with the vasocon trictor noradrenaline, but not angiotensin II, in rat small mesenteric arteries (Walker et al., 2001). Furthermore, noradrenaline increased DGKe activity in a PI 3-kinase dependent way. How PI 3-kinase regulates DGKe activity needs to be further inves- tigated, but Akt/PKB may be involved as increased DGK activity was physically associated with PKB/Akt upon noradrenaline stimulation. In A431 cells, extra- cellular ATP, bradykinin and thrombin that all signal via G-protein coupled recep- tors, induced DGKe translocation from cytosol to plasma membrane (van Baal et al., 2005). In contrast, stimulation of cells with epidermal growth factor (EGF), insulin or insulin-like growth factor that signals via tyrosine kinase receptors

failed to translocate DGKe.DGKe also translocated upon stimulation with high concentrations (100 µM) of short-chain DiC8, indicating that high amounts of DAG are sufficient for translocation. Similar to DGKc, mutation of residues in the DGKe-CRDs that are critical for DAG binding prevented growth factor and DiC8 induced translocation, suggesting that binding of DAG to CRDs causes DGKe translocation.

Nuclear translocations of DGKc and DGKe are also observed. Phosphoryla- tion of DGKc by activated PKC_ translocated DGKc from the nucleus to the cytosol (Topham et al., 1998). Stimulation of IIC9 fibroblasts with _-thrombin transiently increased nuclear DGKe protein and DGKe activity, suggesting that DGKe trans- located to the nucleus (Bregoli et al., 2001).

5. Signalling functions of

c and

e

From the previous sections, we can conclude that DGKc and DGKe have specif - ic characteristics, tissue distribution and subcellular localisation, and that their activities are regulated via enzyme translocation and regulation of catalytic activity. These specificities link them to different signalling pathways and physi- ological functions. In this review, we provide evidence that most signalling func- tions are based on the regulation of the DAG-binding proteins PKC,Ras-GRP, chimaerins and Munc-13. Others are based on signalling properties of the DGK product PA. The data are summarised in Table 7. The (possible) implications of DGKc activation for diverse signalling pathways are visualized in Figure 2.

5 . 1 .D G K

c

Accumulating evidence suggests that DGKs attenuate PKC signalling. In fact, a dynamic interplay exists between DGKs and PKC, in which the two proteins regu- late each other. Examples have been described especially for DGKc, but also for DGKe. The dynamic interplay between DGKs and PKC is best illustrated by four lines of evidence in which DGKc and ^PKC_ are shown to bind to each other and regulate activity and localisation of each other.

First, overexpression of DGKc in HEK293 cells has been shown to inhibit PKC_ activity, which was dependent on DGK activity (Luo et al., 2003a).

Second, PKC_ regulates DGKc localisation by phosphorylation of serines within the MARCKS-PSD of DGKc (Topham et al., 1998). PKC_ and PKCa stimulated nuclear export of DGKc in COS-7 cells. Nuclear export was regulated by phosphor- ylation of serines in the MARCKS-PSD of DGKc as S/D-DGKc mutants, in which these serines are replaced by aspartates that mimic phosphorylation, localised in the cytoplasm, whereas S/N-DGKc mutants that cannot be phosphorylated local- ised in the nucleus. However, these studies were performed by confocal scanning laser microscopy and the percentage of cells with the specific localisation has not been described. Inhibiting nuclear export of DGKc by PKC downregulation inhibited EGF-induced increases in nuclear DAG levels in A172 neuroblastoma

Introduction to the signalling functions of diacylglycerol kinase-c and -e isoforms

cells, suggesting that DGKc regulates nuclear DAG levels and, with that, nuclear PKC activity. Therefore, activated nuclear PKC_ enhanced its own activity by stim- ulating DGKc export. In contrast, in certain cell lines ^PKC-mediated phosphoryla- tion translocated DGKc to the plasma membrane, suggesting a negative feedback loop in which PKC facilitates DAG removal by DGKc. For example, in T cells, PKC phosphorylation of the DGKc-MARCKS-PSD is required (but not sufficient) for DGKc translocation to the plasma membrane upon receptor activation, as demon- strated by PKC downregulation and DGKc-MARCKS-PSD mutant studies (Santos et al., 2002). Furthermore, in C2C12 myoblasts, S/D-DGKc showed increased plasma membrane localisation that was independent of receptor stimulation, suggesting that PKC-mediated phosphorylation is sufficient for membrane localisation in these cells (Hogan et al., 2001). However, PKC-regulated localisation of DGKc in the latter study was only based on the S/D-DGKc substitution mutant and no direct evidence involving PKC was shown. Thus, depending on the cell system, PKC stimulated DGKc export from the nucleus or DGKc translocation to the plasma membrane by phosphorylating the DGKc-MARCKS-PSD.

Third, PKC regulates DGKc activity by phosphorylation. Part of the catalytic domain of DGKc binds to PKC_ and this interaction is inhibited upon TPA or PDGF- mediated activation of PKC_ that phosphorylates serines of the DGKc-MARCKS- PSD (Luo et al., 2003a; Luo et al., 2003b). The S/D-DGKc mutant in which PKC phosphorylation is mimicked was unable to bind PKC_, had reduced DGK activity, and was unable to inhibit PKC_ activity. In contrast, the ^S/N-DGKc mutant that cannot be phosphorylated, was insensitive for PKC_ regulation. As expected, S/D-DGKc-transfected HEK293 cells had higher DAG levels compared to S/N-DGKc- transfected cells (Luo et al., 2003b), but whether DAG levels were increased com- pared to wild-type DGKc was not shown. These results comply with a model in which DGKc inhibits PKC_ activity by keeping DAG levels low. Upon activation of PKC_,DGKc is inhibited by phosphorylation which prolongs PKC_ activation.

However, this model is based on overexpression studies, as a direct relationship between PKC activity, DAG levels and DGKc activity has not been performed in cells that express only endogenous DGKc and PKC_. In addition, these experiments were performed in whole cells rather than in nuclei, while PKC_ was shown to regulate nuclear localisation of DGKc, which may have an effect on ^DAG levels in whole cells. Therefore, it would be interesting to do these experiments also in isolated nuclei.

Fourth, we found an additional level of complexity by which PKC_ regulates DGKc activity. DGKc was shown to bind to the retinoblastoma protein (pRB) via its DGKc-MARCKS-PSD, which stimulated DGK activity (Los et al., 2006). Phos- phorylation of the DGKc-MARCKS-PSD by PKC_ inhibited the interaction between DGKc and pRB (manuscript accepted). Therefore, in addition to the above men- tioned model, nuclear DGKc bound to pRB may keep DAG levels low, but upon activation of PKC_ the interaction is disrupted and ^DGKc stimulation by p^RB is replaced by PKC_-mediated inhibition of ^DGKc activity and nuclear export of DGKc.

Three examples have been described in which DGKc inhibits PKC signalling.

In serum-starved HEK293 cells in which 60% of endogenous DGKc is downregulat ed by DGKc si^RNA, different PKC isoforms translocated to the plasma membrane and become active. In contrast, control cells expressing normal DGKc levels do not contain active PKC, suggesting that DGKc keeps DAG levels low to prevent PKC activation under serum-starved conditions (Avila-Flores et al., 2005). Further- more, in gonadotrope cells, DGKc is recruited and activated to inhibit PKC signal- ling. Activation of gonadotrope releasing hormone (GnRH) receptors continuously activates PLC`1 to produce Ca²⁺ and DAG. To regulate DAG levels and PKC signal- ling, GnRH receptors activate c-Src, which recruits DGKc to the plasma membrane and stimulate its activity (Davidson et al., 2004). Thereby, GnRH receptors also in-duce an off-switch for DAG production to limit PKC-mediated gonadotropin release to a short time interval. In addition, a physiological role of DGKc in atten- uating PKC signalling was found in cardiac hypertrophy, in which cardiomyocytes increase in size as a result of hypertrophic signals or increased functional demands.

Several PKC isoforms, including PKC¡, are involved in cardiomyocyte hypertrophy and heart failure and therefore PKC activity need to be regulated. In cardiomyo- cytes overexpressing DGKc or in hearts of transgenic mice with cardiac-specific DGKc expression, hypertrophic agonist-mediated PKC¡ translocation to the plas- ma membrane is inhibited (Takahashi et al., 2005; Arimoto et al., 2006). Further- more, DGKc suppressed transcription of the hypertrophic gene atrial natriuretic factor, protein synthesis, enlargement of cardiomyocytes and increase in heart weight; characteristics for cardiomyocyte hypertrophy. Since hypertrophic ago- nists increase myocardial DAG levels in hearts from wild-type mice, which was completely suppressed in hearts from transgenic DGKc mice, DGKc probably inhibits cardiomyo-cyte hypertrophy by removing DAG and, with that, by attenu- ating PKC signalling. However, no direct evidence is provided that DGKc regu - lates PKC activity and therefore a role for other DAG-binding proteins on cardiac hypertrophy cannot be excluded.

5 . 2 .D G K

e

DGKe also has been shown to be regulated by ^PKC and, conversely, may negatively regulate PKC signalling. In A431 cells, activation of PKC¡ by its pseudo-¡^RACK peptide resulted in translocation of PKC¡ to the plasma membrane within 3 minutes, followed by DGKe translocation within 6 minutes of PKC¡ stimulation (van Baal et al., 2005). Consequently, PKC¡ activation is restricted to a short time interval. How PKC¡ facilitates DGKe translocation is not known, but since PKC¡, and alsoPKCd, bound to and phosphorylated DGKe, it possibly increases the affinity of DGKe-CRDs for DAG, induces DGKe binding to the plasma membrane via another domain, or stimulates binding of proteins that guide DGKe to the plasma membrane.

5 . 3 . R o l e o f D G K

c

Many receptors signal via the Ras-Mitogen-activated protein kinase (MAPK) signalling pathway that is implicated in the regulation of a variety of responses, including proliferation, senescence, differentiation and apoptosis (Dent et al.,

Introduction to the signalling functions of diacylglycerol kinase-c and -e isoforms

2003). This signalling cascade is stimulated upon activation of the small G-protein Ras that leads to the sequential activation of Raf1,MEK and MAPK, also known as extracellular signal-regulated kinase (ERK). Activated ERK, in turn, regulates downstream kinases or transcription factors (Sebolt-Leopold and Herrera, 2004).

Two DAG-dependent proteins regulate Ras activation: PKC and Ras guanyl nucleotide-releasing protein (Ras-GRP). It is not known how PKC stimulates Ras activity. The involvement of PKC in Ras-ERK signalling is based on PKC inhibitors and prolonged TPA treatment, which inhibit, in some cases, growth factor-mediated ERK activation (Chiloeches et al., 1999; Bonfil et al., 2004; Clerk et al., 2006). Ras- GRP is an exchange factor that activates Ras by releasing GDP and thus facilitat- ing the binding of GTP (Kazanietz, 2000). DAG binds to the C1 domain of Ras-GRP, which is required for Ras-GRP activation (Ebinu et al., 1998; Tognon et al., 1998).

DGKc was found to inhibit ^Ras-GRP activity and downstream signalling via the Ras-ERK pathway (Topham and Prescott, 2001). In HEK293 cells, co-expression of Ras and Ras-GRP with DGKc, but not kinase-inactive DGKc, decreased Ras-GTP levels, suggesting that removal of DAG prevents Ras-GRP activation. Since DGKc is part of a signalling complex containing Ras-GRP and H-Ras, it may spatially regulate DAG levels to locally regulate Ras activation after receptor stimulation.

Indeed, in several cell systems, DGKc negatively regulates Ras-ERK signalling upon G-protein coupled receptor stimulation. Whether either PKC or Ras-GRP is involved in DAG-dependent Ras activation in these systems is not entirely clear.

Overexpression of DGKc in ^L`^T2 gonadotrope cells or cardiomyocytes shortened ERK activation upon stimulation with, respectively, GnRH or endothelin (Davidson et al., 2004; Takahashi et al., 2005). In T cells in which DGKc is overexpressed, ERK activation was decreased upon T cell receptor ligation, whereas overexpression of kinase-inactive DGKc prolonged ERK activation (Zhong et al., 2002; Topham and Prescott, 2001). Physiological evidence for DGKc in regulating Ras-ERK signal- ling was obtained in T cells isolated from DGKc knock-out mice. These DGKc- deficient T cells have increased active Ras and have enhanced and prolonged MEK and ERK activation upon T cell receptor stimulation (Zhong et al., 2003).

Since the Ras-ERK pathway regulates large numbers of protein kinases and transcription factors, the functional consequence of DGKc-mediated inhibition is dependent on the cell system. In L`<sup>T</sup>2 gonadotrope cells, DGKc prevents <sup>ERK</sup>- mediated activation of p90 ribosomal S6 kinase (Davidson et al., 2004), which regulates transcription factors and is involved in protein synthesis. Since gonado- trope releasing hormone stimulates transcription of the `-subunits of luteinizing hormone and follicle stimulating hormone in an ERK-dependent way (Kanasaki et al., 2005), DGKc may regulate transcription of these gonadotropins. In other cell systems, DGKc inhibits ERK-mediated activation of the transcription factor AP1, which stimulates in cardiomyocytes the expression of the hypertrophic gene atrial natriuretic factor, and, in T cells, the activation marker CD69 (Takahashi et al., 2005; Zhong et al., 2002). Thus, although the functional outcome is completely different, DGKc regulates signalling in these cells in a similar way.

Conversely, DGKc was found to be a substrate for ERK. In Cos-7 and HEK293 cells stimulated with serum, DGKc is phosphorylated on serine residues near

the ankyrin repeats that conform to the minimal MAPK phosphorylation consensus sequence (Abramovici et al., 2003; Avila-Flores et al., 2005). Activated ERK indeed phosphorylated DGKc in ^COS-7 cells. This phosphorylation was found to block DGKc association to the cytoskeleton (Abramovici et al., 2003). The functional conse- quence is not known. It would be interesting to investigate if phosphorylation of DGKc by ERK reflects a feedback mechanism that allows tight regulation of the Ras-ERK pathway.

5 . 4 . R o l e o f D G K

c

DGKc is involved in actin rearrangement by a mechanism that is not entirely clear.

At least, DGKc associates with the cytoskeleton in different cell systems and is involved in Rac signalling, as will be discussed in more detail.

DGKc binds to the cytoskeleton, which is subject to regulation. Phosphor- ylation of DGKc by ERK inhibited binding to the cytoskeleton in C2C12 myoblasts, whereas GnRH stimulation of gonadotrope L`T2 cells increased DGK activity (prob- ably DGKc) bound to the cytoskeleton (Abramovici et al., 2003; Davidson et al., 2004). In skeletal muscle and neurons, DGKc binds indirectly to the actin cytoskel- eton via syntrophins (Abramovici et al., 2003). Syntrophin is a component of the dystrophin glycoprotein complex that provides a strong mechanical link between the actin cytoskeleton and the extracellular matrix, which protects muscle cells for contraction-induced membrane damage (Lapidos et al., 2004). DGKc binds in a complex to syntrophin and dystrophin, which localises DGKc at the sarcolemma (Hogan et al., 2001; Abramovici et al., 2003). Since DGKc is present at sites were active actin remodelling occurs, it is thought to regulate actin rearrangement (see below). Interestingly, in mdx (X-chromosome-linked muscular dystrophy) mice that contain a point-mutation in dystrophin causing a Duchenne muscular dystrophy- like phenotype, DGKc was mislocalised to the nucleus, which may play a role in abnormal cytoskeletal changes that contribute to the pathogenesis.

Actin cytoskeletal remodelling is important for changes in cell morphology, adhesion and motility. Several signalling pathways regulate actin remodelling in which the Rho family of small GTPases, including RhoA,Rac and Cdc42, play a central role. In Swiss 3T3 cells, RhoA induces the formation of stress fibers and the assembly of focal adhesions, Rac regulates the formation of lamellipodia and membrane ruffles, whereas Cdc42 controls the formation of filopodia and focal adhesions (Etienne-Manneville and Hall, 2002). DGKc binds to Rac1, both in its active (Rac1^V12) and inactive (Rac1^N17) form (Yakubchyk et al., 2005). Although the Rac1 binding region of DGKc is localised in or near the cysteine-rich domains, point-mutations that mimic (PKC-mediated) phosphorylation of the DGKc-MARCKS- PSD (S/D-DGKc) prevented the binding to Rac1^V12 as well as to Rac1^N17, suggesting that PKC regulates DGKc binding to Rac. Indeed, TPA stimulation inhibited DGKc binding to Rac1, which was abolished by a PKC inhibitor (Yakubchyk et al., 2005).

In several cell systems, DGKc and ^Rac1 colocalised, which provides further evidence that the two proteins associate: In C2C12 myoblasts and in N1E-115 neu- roblastoma cells, DGKc colocalised with Rac and F-actin in membrane ruffles and at the leading edge of lamellipodia (Abramovici et al., 2003; Yakubchyk et al., 2005).

Introduction to the signalling functions of diacylglycerol kinase-c and -e isoforms

Signalling

pathw ay/effect DG K function DG K isoform ( possible) physiological

function References

PKC inhibiting PKC (DAG?) DGKc /DGKe Downregulating PKC signalling

(Luo et al., 2003a; van Baal et al., 2005; Avila- Flores et al., 2005)

DGKc prevention of cardiac

hypertrophy (Arimoto et al., 2006)

DGKc inhibition of

gonadotropin release? (Davidson et al., 2004)

stimulating PKC (PAB)

(Jose Lopez-Andreo et al., 2003; Limatola et al., 1994)

Ras /ERK inhibiting Ras-GRP/PKC

(DAG?) ^DGKc Inhibiting Ras-ERK

signalling

(Topham and Prescott, 2001; Zhong et al., 2003; Zhong et al., 2002; Takahashi et al., 2005; Davidson et al., 2004)

DGKc Inhibition of

transcription?

(Davidson et al., 2004;

Takahashi et al., 2005)

DGKc inhibition of T cell

activation (Zhong et al., 2003) stimulating Raf1

translocation or a Ras-GTPase inhibitor, or inhibiting Ras-GAP by PA?

(Rizzo et al., 2000; Tsai et al., 1990; Tsai et al., 1989)

Actin

rearrangement small GTPase signalling DGKc

neurite formation / actin rearrangement in lamellipodia /

acetylcholine clustering at NMJ?

(Abramovici et al., 2003; Yakubchyk et al., 2005)

small GTPase signalling DGKe (Houssa et al., 1999;

McMullan et al., 2006) inhibiting chimaerins

(DAG?)?

stimulating PIP5K (PAB) DGKc (Luo et al., 2004a)

stimulating PAK1 (PAB) (Bokoch et al., 1998)

Neuronal functioning

inhibiting Munc f amily proteins (DAG?)

ceDGK1 DGKc?

DGKe?

regulating synaptic plasticity / involved in learning and memory?

(Nurrish et al., 1999) (Chase et al., 2004) (Matsuki et al., 2006) (McMullan et al., 2006) (Hozumi et al., 2003)

stimulating NSF (PAB) (Manif ava et al., 2001)

Table 7 . Signalling functions of DGKc and DGKe: