Lipid second messenger regulation: the role of diacylglycerol kinases and their relevance to hypertension

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1 (2001) 15, Nature Publishing Group All rights reserved /01 $ REVIEW ARTICLE Lipid second messenger regulation: the role of diacylglycerol kinases and their relevance to hypertension University Department of Medicine, Manchester Royal Infirmary, Manchester, UK Extracellular stimuli elicit cellular responses through generation of intracellular second messengers. The lipid second messenger diacylglycerol is produced following activation of the phosphoinositide signalling system. Diacylglycerol is the physiological activator of protein kinase C but also interacts indirectly with other signalling molecules such as small G proteins. Diacylglycerol kinases convert diacylglycerol to phosphatidic acid so terminating signalling through diacylglycerol. However, phosphatidic acid itself has a lipid second messenger role, with targets distinct from those of its precursor diacylglycerol. Therefore, diacylglycerol kinases occupy a central position in signal transduction and regulation of their activity is crucial to cellular function. A family of nine mammalian diacylglycerol kinases have been identified. Their structural diversity and complex pattern of tissue expression suggests that they function in distinct cellular processes. In addition to the plasma membrane, diacylglycerol kinases are found at the nucleus and cytoskeleton and translocation between subcellular compartments occurs with agonist stimulation. In small arteries diacylglycerol kinase activity is increased by adrenergic stimulation implying a role in vascular smooth muscle responses. Due to their role as key regulators of protein kinase C activity diacylglycerol kinases may play a role in the cardiovascular changes that occur in hypertension and as such could represent novel therapeutic targets. (2001) 15, Keywords: signal transduction; lipid second messengers; diacylglycerol; phosphatidic acid; diacylglycerol kinase; vascular smooth muscle Introduction Second messengers are the essential intermediates linking extracellular stimuli via receptor activation, to the required intracellular response such as those seen in the nucleus, cytoskeleton or in the case of smooth muscle, the contractile apparatus. Early studies identified signalling pathways that proceeded through an apparently linear series of simple steps where an extracellular stimulus via its unique receptor, activated a phospholipase with the resultant hydrolysis of a membrane phospholipid and the production of a second messenger. In turn the second messenger activated an effector to induce a cellular response (Figure 1). However, it is now clear that signalling pathways do not act in isolation and the discovery of new components and connections has revealed that the transfer of information from extracellular stimulus to intracellular response occurs through complex networks rather than linear Correspondence: Dr J Ohanian, University Department of Medicine, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK. johanian man.ac.uk Received 9 June 2000; revised and accepted 13 August 2000 pathways. Indeed, the cellular response to any given stimulus will depend on the signalling molecules present, the regulation of their cellular levels as well as their intracellular locations. The phosphoinositide signalling system The phosphoinositide signalling system is one of the most common pathways used by growth factors and G protein coupled receptor agonists to initiate cellular responses. The primary event following receptor activation is hydrolysis of phosphoinositol 4,5- bisphosphate (PIP 2 ) by phosphoinositide-phospholipase C (PI-PLC) releasing two messengers, inositol- 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DG). DG functions as an allosteric activator of protein kinase C (PKC) 1 and the major role of IP 3 is to modulate intracellular calcium levels by controlling calcium channels at both the plasma membrane and endoplasmic reticulum, 2 a finding of particular relevance to smooth muscle contraction. 3 Signals transduced through PIP 2 hydrolysis are terminated by metabolism of the second messengers IP 3 and DG to inactive products. In addition to activating PKC, DG regulates other

2 Diacylglycerol kinases in signal transduction 94 lipid biosynthesis. When cells are stimulated by a wide variety of agonists, DGK activity increases and drives the conversion of DG generated by PLC to PA. There will be three major consequences of this increased activity: (1) DG will be removed by conversion to PA so terminating the activation of PKC; (2) DG produced from PLC mediated PIP 2 hydrolysis will be recycled through PA to replenish the level of phosphoinositides; and (3) PA, a second messenger in its own right is formed. 5 8 DGKs therefore play a crucial role in maintaining normal cell function. The diacylglycerol kinase family Figure 1 Schema of the phosphoinositide signalling system. Receptor activation by vasoconstrictor hormones or growth factors stimulates PI-PLC mediated hydrolysis of PIP 2 and the production of two second messengers, IP 3 and DG. IP 3 releases calcium from intracellular stores and DG activates PKC. Activation of DGK will reduce DG levels so terminating PKC-mediated responses. However, PA the product of DGK activity may also act as a second messenger eliciting other responses, for instance through activation of mitogen activated protein kinases (MAPK). GPCR, G protein coupled receptor; TKR, tyrosine kinase receptor. signalling molecules such as the guanine nucleotide exchange factors vav and Ras-GRP. 4 This indicates an additional role for DG as a regulator of the small G proteins Ras and Rho which themselves are important for signalling to the cytoskeleton. DG is also an intermediate in glycerophospholipid synthesis and its conversion to phosphatidic acid (PA) is an essential step in the generation of phosphoinositol lipids. Clearly DG has diverse cellular roles and regulation of its levels is important for optimum cell function. Diacylglycerol kinases (DGK) phosphorylate DG to form PA (Figure 2). In resting cells, DGK activity is low so allowing DG to be used for glycero- Figure 2 Signalling molecules regulated by DG and PA. DGKs phosphorylate DG to form PA. Possible targets of these lipid second messengers are listed. Activation of DGKs will influence the lipid that predominates and hence the final cellular response. ERK, extracellular regulated kinase. To date, nine mammalian DGK isoforms have been identified which differ in their activators, substrate specificity, tissue expression and structural domains (Table 1) (reviewed in Topham and Prescott, 4 van Blitterswijk and Houssa, 9 Sakane and Kanoh 10 ). For example, while cysteine-rich domains (CRDs), putative DG binding sites, 11 are common to all known DGKs, only type I DGKs (, and ) possess EFhand motifs that bind to, and are activated by calcium. Type II DGKs ( and ) have N-terminal pleckstrin-homology (PH) domains (potential sites for protein-protein or protein-lipid interactions 12 ) but lack EF-hands. Type I, III ( ), IV ( ) and V ( ) DGKs are all activated by phosphatidylserine (PS) and only the type III isoform ( ) shows clear substrate specificity preference for arachidonate in the Sn2 position of the glycerol backbone. Furthermore, while DGK possesses a broad tissue distribution, the other isoforms do not (Table 1). DGK is structurally different from other characterised DGK isoforms and contains unique features such as three (instead of two) CRDs, a proline and glycine-rich region, and a ras-associating domain located within a PH domain. 13 Recently, active RhoA was shown to bind to and inactivate DGK 14 suggesting that agonists which signal through Rho (eg G protein-coupled receptor agonists) could regulate the activity of this isoform (see below). While very little is known about the role and regulation of DGK isoforms in signal transduction, their different properties and tissue distribution strongly suggest their involvement in a variety cellular functions. DGKs are also widely conserved having been identified in bacteria, 15 Drosophila melanogaster, Caenorhabditis elegans 4 and plants. 19 Little is known concerning the involvement of DGKs in human disease, although there is evidence for a role of certain isoforms in vision. Recently the gene responsible for retinal degeneration in the Drosophila visual mutant, rdga, was identified as a DGK, ddgk2. 18 Three mammalian DGK isoforms (DGK, and ) have been localised to the retina and DGK is the most closely related to ddgk2. 20 Several groups have demonstrated light-dependent hydrolysis of PIP 2 and PA generation in vertebrate retina and because PKC is implicated in phototransduction

3 Diacylglycerol kinases in signal transduction Table 1 Classification and characteristics of mammalian diacylglycerol kinases 95 Type Isoform MW Tissue distribution Substrate preference Domain structure I kda Thymus, spleen, kidney, brain None EF hands 2 CRDs 90 kda Brain, adrenal gland, intestine, None EF hands small arteries 2 CRDs 90 kda Retina None EF hands 2 CRDs II 130 kda Skeletal muscle, testis None PH, 2 CRDs, EPH 130 kda Testis, lung, spleen, brain, heart, None PH, 2 CRDs, EPH muscle, kidney, liver III 64 kda Testis, skeletal muscle, pancreas Arachidonate-containing 2CRDs IV 104 kda Thymus, brain, intestine, eye, None 2 CRDs, MARCKS, endothelial cells, heart, small ankyrin repeats arteries 130 kda Brain, retina None 2 CRDs, MARCKS, ankyrin repeats V 110 kda Brain, intestine, duodenum, None 3 CRDs, PH, RA liver, small arteries CRD, cysteine rich domains, putative DG binding sites; EF hands, calcium binding sites, EPH, EPH C-terminal homology domain; PH, pleckstrin homology domains, potential site for protein-protein or lipid-protein interaction; MARCKS, sequence homologous to the MARCKS phosphorylation domain, nuclear localisation sequence; ankyrin repeats, potential site for cytoskeletal interaction; RA, ras associating domain. For references see text and Topham and Prescott, 4 van Blitterswijk and Houssa. 9 (reviewed in Topham and Prescott 4 ), a role for DGK activity in mammalian vision is probable. Regulation of DGK activity DGKs are found within the cytoplasm, plasma membrane, cytoskeleton and nucleus, 9 and studies have reported their redistribution, towards the plasma membrane and/or nucleus upon stimulation with agonists such as chemotactic factors, 21 adrenergic agonists, 22 phorbol esters 23 and diacylglycerol. 24 These observations suggest that an important step in the regulation of DGK activity is presentation of the kinase at the site of receptor stimulated production of its substrate DG. However, the trigger for DGK translocation remains unclear. For Type I DGKs it is tempting to implicate calcium as these isoforms possess calcium-binding sites 25 and IP 3 induced increases in intracellular calcium occur during receptor stimulated PI-PLC mediated hydrolysis of PIP 2. However, there is no firm evidence linking rises in intracellular calcium to DGK translocation or activation. Indeed, DGK, a Type I isoform translocates to the nucleus following cytokine stimulation in the absence of any change in calcium levels. 26 Another possibility is that receptor-mediated PLC activation and the generation of DG would induce DGK translocation and activation particularly if the cysteine-rich domains (CRD) of DGK perform a similar function to those of PKC. 11 There are a number of reports demonstrating that treatment of cells with exogenous PLC or cell permeable diglycerides to raise membrane DG levels increases membrane-associated DGK activity. 21,23,24,27 How- ever, interpretation of these studies is complicated by the fact that such treatments would be expected to activate PKC also, which may in turn modulate DGK activity. Indeed, there is evidence that PKCs phosphorylate DGKs 28 and in bradykinin stimulated fibroblasts 29 or carbachol stimulated taenia coli tissue, 30 PKC is involved in DGK activation. In addition, PKC-mediated phosphorylation is an important regulator of DGK activity in the nucleus (see below). There are also a number of reports that do not support a role for increased DG levels as a regulator of DGK localisation or activity Certainly in rat mesenteric small arteries angiotensin II stimulation increases cellular DG but does not activate membrane-associated DGK 22 and cell permeable DGs do not increase DGK activity per se. 33 Therefore, whether DG itself directly induces DGK translocation or activation remains unresolved. DGKs and the cytoskeleton PA, the product of DGK activity, is implicated in regulation of the actin cytoskeleton by inducing actin polymerisation and formation of stress fibres. 34,35 Additionally, it can interact directly with actin-binding proteins 36 and activate phosphoinositide 5-kinase (PI5-K) 37 increasing the formation of PIP 2 which is itself an important regulator of the cytoskeleton. 36 The major pathway for PA production is PLD mediated hydrolysis of phosphatidylcholine 38 and it is unclear whether PA formed by DGKs acts as a second messenger. 7 However, there is evidence to support a role for DGKs in cytoskeletal reorganisation. For instance, agonist-induced trans-

4 96 Diacylglycerol kinases in signal transduction location of DGK to the cytoskeleton has been observed 39,40 (Ohanian, unpublished data). In addition, DGKs are known to associate with the Rho family of G proteins (Rac, Rho and CDC42) best known for their ability to regulate cytoskeletal remodelling in response to extracellular signals, leading to changes in cell morphology, adhesion and motility. 41 Tolias et al 37 found DGK activity associated in a complex with Rac1, Rho-GDI (guanine nucleotide dissociation inhibitor) and P15-K. They proposed that following cell stimulation, DGK-generated PA would activate P15-K producing PIP 2 which in turn would activate Rac1 thereby inducing cytoskeletal changes. The DGK isoform(s) present in such a complex has not been identified. Houssa et al 14 have shown that DGK binds activated RhoA and that when associated with active RhoA its kinase activity is inhibited. As RhoA is known to translocate to the membrane following agonist stimulation, 42 this may be a mechanism by which DGK is targeted to the plasma membrane. However, the mechanism of the subsequent dissociation necessary for DGK to become active is not known. DGKs in the nucleus An emerging body of evidence suggests that nuclear lipid signalling partly regulates the cell cycle, and several groups have shown that there is a nuclear phosphoinositide cycle distinct from its plasma membrane counterpart. 43 Intriguingly, four DGK isoforms, DGK,, and have been found in the nucleus. 9 DGK translocated to the perinuclear region in T-lymphocytes treated with interleukin-2 and inhibition of its activity prevented cell progression to S phase of the cell cycle. 26 In contrast, PKC-mediated phosphorylation of the nuclear targeting domain of DGK causes this isoform to relocate to the cytoplasm allowing nuclear DG levels to rise and progression through the cell cycle to occur. 44 Taken together, these studies suggest that nuclear DGKs play an important role in cell growth through regulation of nuclear levels of DG and PA. DGKs in vascular smooth muscle In vascular smooth muscle vasoconstrictor hormones stimulate contraction through activation of the phosphoinositide signalling system. The resultant increase in IP 3 releases calcium from intracellular stores initiating a contractile response. 3 However, the role of DG is less clear. Although PKC has been implicated in agonist-induced smooth muscle contraction its precise role remains controversial (reviewed in Ohanian et al, 8 Lee and Severson, 45 Horowitz et al 46 ). Furthermore, the assumption that DG levels are increased in smooth muscle following vasoconstrictor hormone stimulation may not be correct. 47,48 For instance, we have shown no accumulation of inositol lipid-derived DG species in intact rat small arteries stimulated with vasopressin or noradrenaline. Although angiotensin II did increase DG levels. In contrast, PA levels were increased in response to noradrenaline and vasopressin but not angiotensin II suggesting agonist specific activation of DGK. 48 Indeed, our further studies have shown that noradrenaline activates membraneassociated DGK(s) whereas angiotensin II does not. 22 DGKs have also been implicated in the response to carbachol in taenia coli 30 and PDGF in rat aortic smooth muscle cells. 49 Little is known concerning the role of individual DGK isoforms in smooth muscle. Northern blot analysis has shown the presence of Type II (DGK ) and Type IV (DGK ) in heart and Type V (DGK ) in small intestine. 13,50,51 In rat mesenteric small arteries we have demonstrated the presence of messenger RNA for Type I DGK, and by immunodetection expression of Type IV DGK and Type V DGK (unpublished observations). The presence of multiple DGK isoforms in smooth muscle suggests that they will play specific roles in different cellular responses. The interaction between DGKs and the Rho family of small G proteins is of potential interest for smooth muscle in light of the involvement of Rho and Rho-kinase in contraction. 52,53 Diacylglycerol kinases in hypertension Essential hypertension is characterised by structural changes in the vasculature, such that the walls of resistance arteries are thickened and cardiac hypertrophy is present. 54 In addition, there may be exaggerated contractile responses in some vascular beds. 55 The molecular mechanisms of these changes are not certain, although there is evidence that vasoconstrictors such as angiotensin II, endothelin and noradrenaline play a role in their development. 56 There have been no systematic studies of DGK activity in hypertension. However, three lines of evidence suggest that DGKs may have an important regulatory role in the cardiovascular system: (1) vascular smooth muscle and cardiac myocytes express multiple DGK isoforms, (2) DGKs are crucial regulators of PKC activity, and (3) vasoconstrictor hormones differentially regulate their activity in vascular smooth muscle. Certainly transformed cells have elevated DG levels and reduced DGK activity 27 demonstrating the consequences of deregulation of this signalling system. Moreover, recent evidence involving DGKs in control of the cell cycle 26,44 further supports the notion that DGKs are important regulators of cell growth. Taken together, these observations suggest that DGKs may play a role in changes in the vasculature and myocardium that occur in hypertension. Furthermore as our understanding of the roles played by individual isoforms increases, potential targets for therapeutic intervention in cardiovascular disease may emerge.

5 Concluding remarks Although we are only in the early stages of understanding the functions of DGKs, it is clear that their role extends far beyond that of a housekeeping enzyme. Similar to other signalling molecules, eg PLC and PKC, DGKs exist as a family of structurally diverse enzymes with complex patterns of tissue expression suggesting they will be crucial in differing cellular processes. By removing DG, DGKs will act as an off switch for PKC signalling. However, the product of that same reaction, ie PA may act simultaneously to switch on other signalling pathways. The consequence of these two effects will undoubtedly reflect the isotypes expressed, their regulation and intracellular localisation. Acknowledgements This work was funded by the British Heart Foundation. References 1 Newton AC. Regulation of protein kinase C. Curr Opin Cell Biol 1997; 9: Berridge MJ. Inositol trisphosphate and diacylglycerol: two interacting second messengers. 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