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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S u m m a r y a n d
g e n e r a l d is c u s s io n
Lipid metabolism not only regulates the structure of biomembranes, but in addi- tion plays an essential role in signal transduction pathw ays. A n ex ample of such a signalling lipid ( second messenger) is diacylglycerol (D A G ) that is produced together w ith inositol-1 ,4 ,5 -trisphosphate (I P3) through receptor-stimulated hydro- lyses of phosphatidylinositol-4 ,5 -bisphosphate (PI( 4 ,5 )P2) by phospholipase C. D A G regulates a number of proteins including protein k inase C (PK C) ,Ras guanyl nucleotide-releasing protein (Ras-G RP) , chimaerins, and Munc-1 3 that are in- v olv ed in cell grow th, differentiation, apoptosis, cell morphology, and v esicle fusion. D A G lev els can be negativ ely regulated by phosphorylation to phospha- tidic acid (PA ) , catalysed by diacylglycerol k inases (D G Ks) . T here is ev idence that PA can also act as a second messenger. T hus, D G K s serv e to either attenuate D A G -mediated signalling or to stimulate PA -dependent signalling. T he importance of regulating D A G and PA lev els in cells is illustrated by the ex istence of as many as ten different D G K isoforms in mammals. T he aim of this study w as to inv esti- gate the properties and physiological functions of tw o of these isoforms, D G Ke and -c. Chapter 1 rev iew s the current k now ledge of the signalling functions of these tw o D G K isoz ymes.
I n Chapter 2 w e inv estigated w hich structural domains of D G Ke are impor- tant for D G K activ ity. D G Ke contains a conserv ed catalytic region containing an A T P-binding site, and three cysteine-rich domains (CRD s) that are thought to inter- act w ith D A G . I nterestingly, the third CRD is ex tended w ith a stretch of 1 5 amino acids that is conserv ed among all D G K isotypes. Point-mutation analysis rev ealed that this ex tension w as essential for D G K activ ity. A s the ex tension is specific for D G K s that not only bind to D A G but also phosphorylate D A G , w e proposed that this small domain is inv olv ed in presenting D A G to the catalytic region. W e further- more made many truncation mutants of D G K e and show ed that all conserv ed
Summary and general discussio
the proline-rich region, are req uired for full DGK activity.
Chapter 3 introduces the retinoblastoma family proteins, or pocket proteins, as we had the intriguing finding that a DGK activity was bound to the retinoblas- toma protein (pRB), an important regulator of the cell cycle, differentiation and apoptosis. In Chapter 4 , we identified DGKc as the DGK that physically binds to pRB.DGKc bound to pRB via its myristoylated alanine-rich C-kinase substrate (MARCKS ) phosphorylation site domain (PS D).DGKc also bound the pRB -related pocket proteins p10 7 and p130 . We found that all three pocket proteins stimulate DGK activity. We also found a physiological function for DGKc in pocket protein signalling: Mouse embryonic fibroblasts (ME Fs) derived from pRB knock-out mice are unable to arrest in G1 after a-irradiation, whereas wild-type ME Fs stop cycling to repair their DN A.DGKc overexpression in pRB knock-out ME Fs, however, recon- stituted a a-irradiation-induced cell cycle arrest in a DGK activity-dependent way.
AsDGKc did not affect radiation-induced cell cycle arrest in wild-type ME Fs, DGKc activity specifically stimulate a cell cycle arrest in a pRB-negative background.
In pRB-negative cells, increased active Ras levels are observed that is known to stimulate cell cycle progression. Therefore, DGKc may stimulate a a-irradiation- induced cell cycle arrest in pRB knock-out ME Fs by inhibiting DAG-mediated activation of Ras-GRP, thereby negatively regulating Ras signalling.
Chapter 5 describes our finding that PKC-mediated phosphorylation of the DGKc-MARCKS-PS D inhibits DGKc binding to pRB, suggesting that PKC can regu- late the interaction between pRB and DGKc. As pRB stimulates DGKc activity to phosphorylate DAG and so can attenuate DAG-mediated signalling, PKC-mediated disruption of the DGKc-pRB interaction is likely to lead to an increase in DAG lev- els and conseq uent stimulation of PKC activity or other DAG-regulated proteins.
To determine in which physiological process DGKc binding to pRB is involved, we investigated the role of DGKc in cell cycle regulation and differentiation.
Chapter 6 describes that overexpression of DGKc in different cell lines did not affect cell cycle profiles. Also under specific conditions that cause a pRB-medi- ated cell cycle arrest, the cell cycle profiles of DGKc overexpressing cells was unaffected. In addition, DGKc knock-out ME Fs showed comparable cell cycle profiles as wild-type ME Fs. These results suggest that DGKc overexpression or DGKc absence does not affect the cell cycle.
As pRB is known to regulate muscle differentiation, we studied the role of DGKc in this process (Chapter 7 ).DGKc was found to stimulate muscle differen- tiation in a DGK activity-dependent fashion and enhanced the expression of early- and late muscle-specific genes. Microarray analysis revealed that DGKc increased mRN A levels of diverse muscle genes during differentiation; especially targets of the myogenic transcription factor MyoD, suggesting that DGKc stimulates MyoD transcriptional activity. Indeed, DGKc stimulated promoter activity of the MyoD target genes, myogenin, muscle creatine kinase and of a synthetic promoter containing MyoD binding sites. To demonstrate the physiological role of DGKc in muscle differentiation, we showed that DGKc knock-out ME Fs were unable to differentiate or to express muscle-specific genes.
G eneral discussion
AsDGKs regulate the levels of two second messengers, DAG and PA, their activity needs to be tightly regulated. In this thesis we provided evidence that DGK activity is highly regulated. First, our finding that all conserved structural domains of DGKe are required for DGK activity suggests that either the folding of the protein is very critical or that protein-protein and/ or protein-lipid interactions via the dif- ferent domains are very important. Second, DGKc activity is stimulated by pRB- type pocket proteins, suggesting that these pocket proteins can regulate DAG and PA levels via DGKc. Third, PKC inhibits the interaction between DGKc and pRB, thereby attenuating pRB-mediated DGKc activation. PKC was also shown to inhibit DGKc activity, suggesting that PKC coordinates the negative regulation of DGKc.
In this way PKC stimulates its own activity and that of other DAG binding proteins via a positive feed-back mechanism.
Such a positive feed-back mechanism may be very important in regulating the balance between DAG and PA. We surmise that this balance is spatially and temporally regulated and can not be easily disturbed: O verexpression of DGKc in different cell lines did not have any effect on the cell cycle or on pRB-mediated G1-arrest, possibly because sufficient DGK activity is present at specific sites, so that overexpressed DGKc does not have an additive effect on cell cycle regula- tion. Also DGKc knock-out MEFs showed similar cell cycle profiles compared to wild-type MEFs, suggesting that the DAG-PA balance is not dramatically disturbed, possibly because of compensation by one of the nine other DGKs. H owever, in pRB knock-out MEFs, overexpression of DGKc (but not of kinase-inactive DGKc) could reconstitute a a-irradiation-induced cell cycle arrest, suggesting that active DGKc may restore an imbalance between DAG and PA after radiation. DGKc did not affect an irradiation-induced G1-arrest in wild-type MEFs, suggesting that the bal- ance between DAG and PA may be regulated by endogenous DGKc.
O verexpression in myoblasts of active but not inactive DGKc stimulated mus- cle differentiation in a DGK activity-dependent way, indicating that changes in the cellular levels of either DAG and/ or PA can regulate the differentiation proc- ess. Furthermore, DGKc knock-out MEFs were unable to differentiate. These data suggest that DGKc appears to have an essential function during myoblast differ- entiation that cannot be compensated by other DGKs. Therefore it is likely that a shift in the DAG-PA balance is required for the differentiation process to proceed.
DAG inhibits differentiation by stimulating PKC that phosphorylates myogenic transcription factors, including MyoD, thereby inhibiting muscle specific gene expression. In addition, DAG stimulates Ras-GRP that activates Ras, which inhib- its muscle differentiation through the activation of the MAPK pathway. O n the other hand, PA stimulates muscle differentiation by activating mTO R, although the exact mechanism is poorly understood. In addition, PA may be involved in transcription regulation of muscle-specific genes, as PA was shown to regulate a transcription factor in yeast.
Summary and general discussio
To determine the mechanism by which DGKc regulates the above-mentioned physiological processes, it seems important to measure the cellular DAG and PA levels. However, as the changes in the ratio between DAG and PA are likely to be very small and/or local, a biochemical assay will probably not be sensitive enough and, therefore, new methods need to be developed. In addition, it would be inter- esting to determine what DAG- and/or PA-binding proteins are involved in mediat- ing the physiological effects of DGKc, for example in irradiation-induced cell cycle arrest of pRB knock-out MEFs and in differentiation.
In order to study the role of DGKc in pRB signalling it may be interesting to generate pRB mutants that are unable to interact with DGKc but still able to inter- act with other pRB targets. These could then be put back into tumour, differen- tiation and cell cycle assays. Furthermore, the reconstitution of muscle differen- tiation of DGKc knock-out MEFs with mutants of DGKc unable to interact with pRB would be interesting.
Studies in DGKc knock-out mice may also give more insight in the function of DGKc. Although these mice have no obvious phenotype, more careful examination of these mice under more extreme conditions might yet unveil a possible pheno- type. For example, as DGKc regulates muscle differentiation, DGKc knock-out mice may have a muscle defect that is not directly visible, but becomes evident when the mice have to exercise intensively or when controlled movements are required.
Because of the existence of ten different DGK isoforms in mammals, it is dif- ficult to find specific functions for each DGK. Therefore, it would help to generate a knock-out mouse or cell line with reduced amounts of DGK isoforms, thereby making it easier to investigate isoform-specific functions. In addition, phenotypes of double-knock-out mice may help to understand in which physiological proc- esses specific DGK isoforms are involved.
All together, future research is required to understand more about isoform- specific functions. Each DGK isoform may be functionally non-redundant under very specific conditions. In this thesis, we provided evidence that DGKc is required for such specific functions, including irradiation-induced cell cycle arrest in pRB-negative cells and in muscle differentiation. Future experiments will define new physiological functions of DGK isoforms.
