11.1 Nuclear Phosphoinositide Signalling

Phosphorylation at the 3,4, or 5, position of the inositol head group of phosphatidylinositol generates seven different phosphoinositides that form the basis of a ubiquitous membrane signalling system. An array of tightly regulated phosphoinositide kinases and phosphatases, ultimately control the subcellular profile of phosphoinositides (Irvine 2005), which can regulate protein localisation, ion channel function and protein enzymatic activity and impact on cellular processes including vesicle transport, cytoskeletal dynamics, cell proliferation and survival, gene transcription, cell polarity and migration (McCrea and De Camilli 2009). Phosphoinositides are tethered tightly into the membrane and can recruit and localise proteins to specific subcellular membrane domains through specific phosphoinositide interacting domains (PID) (Lemmon 2003). Because the membrane can be considered more akin to a two dimensional system, membrane interaction is analogous to inducing protein/protein interactions and acts to concentrate upstream regulators and downstream targets together leading to enhanced downstream signalling and specificity. Phosphoinositide signalling occurs on many different intracellular membranes including the inner surface of the plasma-membrane, the Golgi, the endoplasmic reticulum and on membrane vesicles that move between these compartments and their deregulation has been implicated in an array of human diseases (McCrea and De Camilli 2009). Phosphoinositide metabolism also occurs within the nucleus. When isolated nuclei are incubated with radiolabeled 32P-ATP, radioactivity is incorporated into Phosphatidylinositol phosphate (PtdInsP), Phosphatidylinositol bisphosphate (PtdInsP 2) and Phosphatidic acid (PtdOH) (Smith and Wells 1983a, b, 1984a, b). As the nuclei were intact and not disrupted, phosphoinositides must be present in nuclei and the kinases that can make them are also present and located at the same sites.

11.1.1 The Location of Phosphoinositide Signalling in the Nucleus

Phosphoinositides are normally presented within the context of a membrane. Phosphoinositides contain two long hydrophobic fatty acyl tails linked to a glycerol group, which is coupled via a phosphodiester linkage to the phosphorylated inositol head group. This chemical structure is ideally suited to form the interface between the hydrophobic membrane, through insertion of the fatty acyl tails, and the cytosol. The nucleus is an organelle that is bounded by a double bilayer membrane, the outer part being contiguous with the endoplasmic reticulum. While one would imagine that inositide signalling in the nucleus might occur on the inner surface of this double bilayer, the first clues that this may not be the case came from studies in Murine erthythroleukemia (MEL) cells (Cocco et al. 1987). In this case nuclei were isolated from control MEL cells or from cells that had been differentiated down the erythroid pathway. The isolated nuclei were then incubated with radiolabelled ATP, which became incorporated into phosphoinositides. What was fascinating and previously undocumented, was that upon differentiation there were changes in the amount of radiolabelled phosphoinositide present in the nuclei. This suggested that phosphoinositides are dynamically regulated in response to extracellular signals and that phosphoinositides in the nucleus may constitute a signalling pathway that could specifically control nuclear functions. The physiochemical nature of phoshoinositides within the nucleus is still not clear. Experiments utilising detergents to remove the nuclear membrane prevented neither the radiolabelling of nuclear phosphoinositides in the control conditions nor the changes in phosphoinositide labelling observed upon differentiation (Cocco et al. 1987). In a more detailed analysis, we prepared nuclei from rat liver, which have a beautiful intact nuclear envelope after isolation, and used increasing concentrations of detergent to remove the envelope, which was analysed by electron microscopy. Radiolabelling of nuclear phosphoinositides and the mass of various phosphoinositides and phospholipids were also measured (Vann et al. 1997). The data clearly demonstrated that removal of the nuclear envelope correlated with loss of phospholipids such as phosphatidylcholine, but did not correlate with either the removal of phosphoinositides or phosphoinositide kinases. These data suggest that the nuclear phosphoinositide pools that are involved in regulating nuclear processes, are present within the nucleus rather than in the nuclear membrane envelope (Divecha et al. 1991; Banfic et al. 1993; Cocco et al. 1987, 1988; Payrastre et al. 1992). Using a specific PID or antibodies that interact specifically with the phosphoinositide phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P 2), it appears that PtdIns(4,5)P 2, and by inference, other phosphoinositides, are clustered in nuclear structures called interchromatin granules (Watt et al. 2002; Boronenkov et al. 1998; Mellman et al. 2008). These structures are also nuclear regions that are highly enriched in factors used for splicing mRNA. Although this would suggest a role for PtdIns(4,5)P 2 in splicing, it is not clear whether these regions are where splicing occurs or where splicing components are stored. However immunodepletion of PtdIns(4,5)P 2 from nuclear extracts attenuates in vitro splicing (Osborne et al. 2001). The exact chemical nature of how phosphoinositides are presented in these structures is far from clear but likely, phosphoinositides are sequestered by proteins that interact with and hide their hydrophobic tails but are able to present the inositol head group for further phosphorylation or phospholipase C mediated cleavage.

11.1.2 PtdIns(4,5)P2 Synthesis and Signalling in the Nucleus

Within the nucleus Phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P 2) is central to phosphoinositide signalling being a second messenger itself (van den Bout and Divecha 2009) and a substrate for both phosphatidylinositol-3-kinase (PtdIns-3-kinase) and phospholipase C (PLC).

For PtdIns(4,5)P 2 to function as a specific second messenger within the nucleus we expect that PtdIns(4,5)P 2 levels would be controlled by nuclear specific factors and in turn they would regulate nuclear specific downstream targets.

PtdIns(4,5)P 2 can be synthesised by two different enzyme families that are highly related. Phosphatidylinositol-4-phosphate (PtdIns4P)-5-kinases (PIP5Ks) phosphorylate PtdIns4P (Loijens et al. 1996) on the 5-position while phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinases (PIP4Ks) phosphorylate PtdIns5P on the 4-position (van den Bout and Divecha 2009). So which family is responsible for the synthesis of PtdIns(4,5)P 2 in the nucleus? Isoforms of both families are present in the nucleus (Ciruela et al. 2000; Boronenkov et al. 1998; Mellman et al. 2008), however, the mass level of PtdIns4P is at least 20-fold higher than the level of PtdIns5P. This suggests that PtdIns(4,5)P 2 is synthesised primarily through the PIP5K pathway. In order to further analyse this, we incubated isolated nuclei with radiolabelled ATP for short time periods and then isolated the PtdIns(4,5)P 2 and determined on which position the label was incorporated. We found that the relative labelling ratio of the 5 to the 4 position was approximately 1.8 (Vann et al. 1997). There are two possible interpretations to these experimental data. The first is that approximately two times more PtdIns(4,5)P 2 is synthesised through the PIP5K than the PIP4K pathway (we cannot determine if the radiolabel was on the same molecule or on different molecules of PtdIns(4,5)P 2). The second is that the labelling of the 4-position occurs because of new synthesis of PtdIns4P from PtdIns phosphorylation, which is passed on to PIP5K for synthesis of PtdIns(4,5)P 2. In order to differentiate between these possibilities, we undertook a similar nuclear labelling experiment in the presence of inhibitors of PI4K that synthesise PtdIns4P. High concentrations of wortmannin inhibit the PI4KIII family of enzymes while adenosine is a specific inhibitor of the PI4KII family (Balla and Balla 2006). Interestingly, both inhibitors blocked PtdIns4P synthesis to about 50% each in isolated intact nuclei and to approximately 90% when incubated in combination. When we determined the ratio of radiolabelling of the 5 to the 4 position of PtdIns(4,5)P 2 labelled in the presence of the inhibitors we found that treatment with wortmannin increased the ratio to 10:1 while adenosine had no effect. Neither wortmannin nor adenosine had any effect on the in vitro activity of the PIP4K enzymes. These simple and elegant in vitro studies suggest that in nuclei there are two families of enzymes that synthesise PtdIns4P, but that only the wortmannin sensitive enzymes provide PtdIns4P that is used by the PIP5K to generate PtdIns(4,5)P 2. The adenosine sensitive pool of PtdIns4P may be involved in direct signalling. Furthermore it would appear that at least 90% of nuclear PtdIns(4,5)P 2 is derived from the PIP5K pathway with the PIP4K pathway possibly providing a small minority of the nuclear PtdIns(4,5)P 2. The data also suggest that the role of the PIP4K may not be related to their ability to generate PtdIns(4,5)P 2 but that they may have a more specialised function in the nucleus (see later).

11.1.3 Nuclear Specific Regulators of PtdIns(4,5)P2 Synthesis

It is still unclear which isoforms of PIP5K are present in the nucleus. This is in part a consequence of the lack of suitable antibodies and because when overexpressed in cells, PIP5K generally localise to the plasma-membrane. However, PIP5Ka (Mellman et al. 2008; Boronenkov et al. 1998) and two splice variants of PIP5Kg have been shown to be present in the nucleus (Schill and Anderson 2009). How their localisation is regulated is not clear. However, two nuclear specific regulators of PIP5K have been defined. We initially demonstrated that the Retinoblastoma protein (pRB) interacts with all isoforms of PIP5K (Divecha et al. 2002). pRB is a nuclear localised master regulator of differentiation, cell survival and progression through the cell cycle. Moreover the pRB pathway is deregulated in nearly all human tumours. pRB interacts with and stimulates the activity of PIP5K and pRB activity can control the synthesis of nuclear PtdIns(4,5)P 2. In fact we have shown that pRB acts as a scaffold protein for a number of different enzymes involved in phosphoinositide regulation including PIP4K and a Diacylglycerol kinase (DGK) (Los et al. 2006). Interestingly, DGKz, but not DGKa or DGKq, interacts with pRB in vitro and in vivo, and acts in vivo as a downstream effector of pRB to regulate nuclear levels of diacylglycerol and phosphatidic acid, and cell cycle progression in response to DNA damage induced by g-irradiation (Los et al. 2006). DGKz also localized mainly to the nucleus in C2C12 cells and its overexpression (but not of a kinase dead mutant or of a mutant that did not enter the nucleus), blocked C2C12 cells in the G1 phase of the cell cycle (Evangelisti et al. 2007). In contrast, the down-regulation of endogenous DGKz by siRNA increased the number of cells both in S and G2/M phases of the cell cycle. The cell cycle arrest of cells overexpressing wild-type DGKz was accompanied by decreased levels of pRB phosphorylated on Ser-807/811. pRB also interacts with the p55 regulatory subunit of PI-3-kinase (Xia et al. 2003) furthering the idea that pRB may act to scaffold nuclear inositide metabolising enzymes.

Another well characterised regulator of PIP5K has emerged from the Anderson laboratory. Using yeast two hybrid analysis, Star-PAP was identified as an interactor with PIP5Ka (Mellman et al. 2008). Interaction regulates the localisation of PIP5Ka to nuclear speckles, where PtdIns(4,5)P 2, presumably synthesised by PIP5Ka, regulates the activity of Star-PAP. Star-PAP is a poly(A) polymerase that regulates the length of the poly-A tail of a select set of mRNAs, some of which are involved in regulating responses to oxidative stress. RNAi mediated suppression of PIP5Ka leads to a decrease in a similar set of mRNAs that are also regulated by Star-PAP. In vitro, Star-PAP activity is dramatically stimulated by PtdIns(4,5)P 2, suggesting that it is also a downstream target for nuclear PtdIns(4,5)P 2 signalling. The data suggest that the Star-PAP complex acts as a hub for nuclear PtdIns(4,5)P 2 signalling to control the response to oxidative stress. pRB is also critical for responses to oxidative damage and thus may impinge on the Star-PAP pathway through regulation of PtdIns(4,5)P 2 synthesis. Whether Star-PAP is directly regulated by PtdIns(4,5)P 2 is not clear as star-PAP is also regulated by phosphorylation by Caesin kinase 1, an enzyme that is also regulated by PtdIns(4,5)P 2 (Gonzales et al. 2008).

Besides Star-PAP, few nuclear specific PtdIns(4,5)P 2 interactors have been identified. The BAF complex is a chromating regulating complex which is able to interact with PtdIns(4,5)P 2 (Zhao et al. 1998; Rando et al. 2002) although it is not clear which component of the complex interacts with this phosphoinostide nor what this interaction can do to the function of the BAF complex. PtdIns(4,5)P 2 also interacts with histone H1 and disrupts its ability to suppress basal transcription by RNA polymerase in vitro. PtdIns(4,5)P 2 interaction is abolished upon Protein Kinase C (PKC) mediated phosphorylation of the H1 (Yu et al. 1998). Furthermore the drosophila PIP5K homologue, skittles, interacts with ASH2, a component of a chromating remodelling complex and its functional complex modulates histone H1 hyperphosphorylation in vivo (Cheng and Shearn 2004). These data suggest that PtdIns(4,5)P 2 may play direct role in modulating chromatin assembly and regulating transcription. Using a number of different strategies to enrich and purify PtdIns(4,5)P 2 interacting proteins from the nuclei of MEL cells, we have identified an enrichment of proteins that are part of the pre-mRNA and mRNA splicing complexes and proteins involved in regulating DNA damage responses. While the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R-(Xn = 3-7)-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology (PH) or plant homeodomain (PHD) modules (Lewis et al. 2011). Proteins with no prior history of phosphoinositide interaction were also identified, some of which have functional roles in chromatin assembly. DNA topology was exemplar amongst these with the identification of topoisomerase IIa (TopoIIa). Biochemical assays validated our proteomic data supporting a direct interaction between PtdIns(4,5)P 2 and DNA TopoIIa. Furthermore, we also showed that in vitro, phosphoinositides could modulate TopoIIa decatenation activity (Lewis et al. 2011). Clearly further definition of PIP5K and PtdIns(4,5)P 2 interactors will be critical for understanding the complexity of PtdIns(4,5)P 2 synthesis in the nucleus. A snapshot of phosphoinositide signaling is shown in Fig. 11.1.

11.2 Nuclear PLC and Cell Cycle Regulation

The study of nuclear inositide signalling has been fraught with difficulties, mainly due to the problems of obtaining intact nuclei, deprived of the outer membrane (that could carry endoplasmic reticulum (ER) contamination) combined with the complexity of isolating cells in a precise phase of the cell cycle.

Among the nuclear PI-metabolising enzymes, the inositide specific PLC has been one of the most extensively studied. The activation of nuclear PLC was first demonstrated in two “founder” reports, showing that insulin-like growth factor (IGF)-1 stimulation of Swiss 3T3 mouse fibroblasts produced a decrease in PtdIns4P and PtdIns(4,5)P 2 and a concomitant increase in diacylglycerol (DAG) levels in membrane-stripped nuclei (Divecha et al. 1991; Cocco et al. 1989). On the contrary, no changes in PtdIns4P, PtdIns(4,5)P 2, and DAG amount were detected in whole cell homogenates or in nuclei in which the envelope was maintained. Moreover, bombesin, which is another strong mitogen, stimulated inositide metabolism at the plasma membrane, but not in the nucleus, suggesting the existence of a nuclear polyphosphoinositide signalling system entirely distinct from the one at the plasma membrane (Divecha et al. 1991). It was also shown that PKC translocates from the cytoplasm to the nucleus in response to increased nuclear DAG levels (Divecha et al. 1991). PLCb1 was also shown to be present in nuclei of Swiss 3T3, and PLCb1 activity was up-regulated in response to IGF-1 stimulation (Martelli et al. 1992). The presence and activity of PLCb1 and PtdIns4P-5-Kinase was subsequently confirmed in rat liver nuclei (Divecha et al. 1993).

The regulation of nuclear PLCb1 has been investigated extensively. At the plasma membrane, PLCb1 is activated by both Gaq/a11 and Gbg subunits of heterotrimeric G-proteins. Activation of nuclear PLCb1 appears to involve an entirely different mechanism, involving mitogen-activated protein kinases (MAPK) phosphorylation (Fig. 11.2). Following IGF-1 stimulation of quiescent Swiss 3T3 mouse fibroblasts, activated p42/44 MAPK translocates to the nucleus where it phosphorylates Ser 982 in the C-terminal tail of PLCb1 (Xu et al. 2001). This phosphorylation is inhibited by the MAPK inhibitor PD098059. Phosphorylation of PLCb1 by MAPK also occurred in vitro using recombinant PLCb1 and MAPK proteins. However, phosphorylation of Ser 982 per se does not increase PLCb1 activity, as seen previously after IGF-1 stimulation (Martelli et al. 1992). Thus phosphorylation of Ser 982 in vivo might cause the recruitment of other components which stimulate PLCb1 activity. Nonetheless, Swiss 3T3 mouse fibroblasts, stably transfected with PLCb1 harboring a Ser 982 Gly mutation, showed a significant loss in mitogenesis in response to IGF-1 (Xu et al. 2001) similar to the one obtained through the down regulation of PLCb1 by anti-sense RNA (Manzoli et al. 1997). Recent data also suggest the involvement of the subunits Gq/11 in regulating PLCb1 in nuclei of striatal neurons (Kumar et al. 2008) and activation of nuclear PtdIns(4,5)P 2 hydrolysis in rat hepatocytes in response to insulin requires translocation of the insulin receptor to the nucleus (Rodrigues et al. 2008).

Fig. 11.1
figure 1

Nuclear phosphoinositides are metabolized by phosphatases, kinases and phospholipases (colored shapes next to arrows) to generate a profile of inositides and their derivatives within the nucleus. These nuclear signal transducers interact with and regulate the function of a set of nuclear effectors (dark blue shapes) in order to modulate nuclear specific pathways (green boxes). The modulation of these nuclear pathways eventually leads to the modulation of cell or organismal behavior (light blue boxes)

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Fig. 11.2
figure 2

Targets of PLCb1 in physiological and pathological conditions. Under physiological conditions PLCb1 activates a variety of downstream targets which ultimately regulate cell proliferation or differentiation. During MDS progression to AML, reduction in PLCb1 expression correlates with an increase in Akt which results in a worse prognosis. Cyclin D3 is a critical downstream transducer of PLCb1 common to both the physiological and pathological state

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In synchronized HL-60 cells two peaks of PLCb1 nuclear activity were observed 1 and 8,5 h after the release from a nocodazole block, that correlated with G2/M and late G1 phases of the cell cycle (Lukinovic-Skudar et al. 2005). The mechanism of PLCb1 activation involved an extracellular signal-regulated kinases (MEK) inhibitor sensitive to phosphorylation on a serine, similar to the one occurring in Swiss 3T3 cells (Lukinovic-Skudar et al. 2005). These studies were confirmed and extended in serum starved HL-60 cells progressing through the G1 phase by re-addition of serum (Lukinovic-Skudar et al. 2007). Also in this case two temporally distinct waves of nuclear PLCb1 activity occurred in cells mitogenically stimulated: one important for the G1/S and the other for the G2/M transition (Lukinovic-Skudar et al. 2007) and reviewed in (Visnjic and Banfic 2007). However, the mechanisms of interaction between PLCb1 products, DAG and inositol 1,4,5-triphosphate (Ins(1,4,5)P 3) with the cell cycle machinery are still unclear. Pioneering work by Fields and co-workers demonstrated that DAG levels rise to a peak in nuclei coincident with the G2/M transition, and this increase was sufficient to selectively stimulate PKCbII translocation to the nucleus, where it directly phosphorylates lamin B in the nuclear envelope, leading to nuclear lamina disassembly and mitosis progression (Hocevar and Fields 1991; Hocevar et al. 1993; Goss et al. 1994; Walker et al. 1995; Sun et al. 1997; Gokmen-Polar and Fields 1998). These authors also demonstrated that the generation of nuclear DAG at the G2/M transition was dependent on PLC activation. In MEL cells, PLCb1 is required for the activation of PKC and the phosphorylation of lamin B in G2/M (Fiume et al. 2009). MAPKs, in particular Jun N-terminal Kinase (JNK), can be activated by serum stimulation, and then it can translocate to the nucleus, where it mediates PLCb1 activation. These events can regulate PKCa-dependent phosphorylation of lamin B, nuclear envelope disassembly and thus cell cycle progression (Fiume et al. 2009). Nuclear PLCb1 also appears to be important in the resumption of meiosis in mouse oocyte (Avazeri et al. 2000). PLCb1 translocates to the nucleus, apparently to perichromatin and interchromatin granules, and this is followed by a later shift to the nucleoplasm, as demonstrated by immuno-electron microscopy analysis. Importantly, microinjection into the nucleus of an antibody to PLCb1 blocked germinal vesicle breakdown (Avazeri et al. 2000).

Nuclear phospholipid metabolism is also particularly important during G1/S transition and S phase. Nuclear specific phosphatidylinositol (PtdIns) lipid breakdown occurs during S phase, releasing nuclear inositol phosphates, including inositol(1,4)-bisphosphate (Ins(1,4)P 2), which may function to stimulate DNA polymerase activity (York and Majerus 1994; Sylvia et al. 1988, 1989). In the G1/S transition PLCd1 accumulates in the nucleus by binding to PtdIns(4,5)P 2 (Stallings et al. 2005), and the suppression of PLCd1 alters S phase progression and inhibits cell proliferation, possibly through a block in S-phase exit. PLCd1 suppression also increased cyclin E level (Stallings et al. 2008) which has also been shown to attenuate S-phase exit. PLCb1 overexpression in MEL cell nuclei (Faenza et al. 2000), leads to an upregulation of cyclin D3, along with its kinase (cdk4), even in cells that are serum-starved. As a consequence of increased cyclin D3 levels, retinoblastoma protein (pRB) is phosphorylated and this leads to the activation of the E2F-1 transcription factor (Faenza et al. 2000) (Fig. 11.2).

The contribution of PtdIns(4,5)P 2 in S phase entry has been linked also to cyclin A2. In Swiss 3T3 cells nuclear PtdIns(4,5)P 2 down-regulation may cause a delay in phorbol ester-induced S phase entry and this was at least in part channeled through cyclin A2 at the transcriptional level, thus identifying cyclin A2 as a downstream effector of the nuclear PtdIns(4,5)P 2 signalling network (Nelson et al. 2008).

All in all, the above mentioned studies clearly demonstrate that nuclear inositide signalling is able to integrate cellular signals to control important master regulators of the cell cycle in order to impinge on progression through the cell cycle.

11.3 NuclearPLCb1 During Cell Differentiation

PLCb1 has been implicated in the control of differentiation. For instance, nuclear PI metabolism changes during dimethyl sulfoxide (DMSO)-induced erythroid differentiation of MEL cells (Cocco et al. 1987). We have also demonstrated that the DMSO-induced differentiation of these cells is accompanied by an accumulation of nuclear PtdIns(4,5)P 2 (Manzoli et al. 1989), concomitant with a decrease of PLCb1 in the nucleus (Martelli et al. 1994) and a decrease in nuclear DAG level (Divecha et al. 1995). The nuclear localization of PLCb1 was shown to be crucial for the differentiation of MEL cells and targets a reduction in the expression of the transcription factor p45/NF-E2 which is required for the expression of the b-globin gene (Matteucci et al. 1998) (Fig. 11.2). p45/NF-E2 is a highly specific target for PLCb1 signalling as other transcription factors involved in erythroid differentiation of MEL cells, such as members of the GATA family, are not regulated (Faenza et al. 2002). A proteomic approach also identified SRp20, a member of the highly conserved serine/arginine-rich splicing factor (SR) family of splicing regulators as a target of nuclear PLCb1 in MEL cells. In addition, by immunoprecipitation and subcellular fractioning, it has been shown that endogenous PLCb1 and SRp20 physically interact in the nucleus (Bavelloni et al. 2006). Nuclear PLCb1 also up-regulates the expression of CD24 in MEL cells. CD24 is an antigen involved in differentiation and haematopoiesis, is overexpressed in a number of leukemias and is considered as a critical molecule in the metastasizing ability of solid tumors. When PLCb1 expression is reduced by RNAi, CD24 expression is also down-regulated. The regulation of PLCb1 on CD24 is mediated at the transcriptional level at least in part, since PLCb1 affects the promoter activity of CD24 (Fiume et al. 2005).

In general nuclear PLCb1 correlates with increased replicative capacity of the cell. However in some models of differentiation nuclear PLCb1 activity appear to be required for differentiation. Nuclear PLCb1 is increased upon insulin induced differentiation of C2C12 mouse myoblasts (Faenza et al. 2003). Skeletal muscle differentiation is characterized by terminal withdrawal from the cell cycle, the activation of muscle-specific gene expression, and morphological changes including myoblast alignment, elongation, and fusion of mononucleated myotubes. These events are coordinated by a family of four muscle-specific basic helix-loop-helix transcription factors: MyoD1, Myf5, myogenin, and Mrf4, termed the muscle regulatory factors (MRFs) (Lassar et al. 1994). An imbalance of nuclear and cytoplasmic PLCb1 suppresses myogenesis as the overexpression of a cytoplasmic PLCb1 mutant that lacks a nuclear localization sequence suppresses the differentiation of C2C12 myoblasts (Faenza et al. 2003), while the expression of the wild type PLC1b1 or PLCg1 induces C2C12 differentiation. Upon differentiation PLCb1 becomes highly concentrated in the nuclei, while PLCg1 increases in the cytosol, suggesting that they may target a common pathway but in an independent manner (Faenza et al. 2004). In fact both PLCg1 and b1 activate transcription of cyclin D3, however they appear to do this in different ways. PLCb1 targets the activation of the AP1/Jun pathway while the regulation of cylin D3 transcription by PLCγ1 is not clear (Ramazzotti et al. 2008). Increased cyclin D3 levels are known to play an important role in regulating myocyte differentiation (Kiess et al. 1995; Cenciarelli et al. 1999; Chu and Lim 2000).

A common theme of the role of PLCb1 in differentiation of MEL cells and C2C12 cells is the activation of cyclin D3. Nuclear PLCb1 activates cyclin D3 in both systems. Cyclin D3, however, has opposite effect in the two cell types, stimulating the progression through G1 phase of the cell cycle in the case of MEL cells (Faenza et al. 2005; Cocco et al. 2009) and promoting the differentiation of myoblasts to myotubes in the case of C2C12 cells. Also during 3T3-L1 adipocyte differentiation nuclear PLCb1 regulates the expression of cyclin D3. During 3T3-L1 adipocyte differentiation there are two phases of PLCb1 activity; the first occurs within 5 min of treatment with differentiation medium, does not require translocation of PLCb1 to the nucleus but is regulated by ERK and PKCa. The second phase occurs from day 2 of differentiation, requires translocation of PLCb1 to the nucleus and is independent of regulation by ERK and PKCa. Over-expression of PLC mutants, which either lack the ERK phosphorylation site or the nuclear localization sequence, revealed that both phases of PLCb1 activity are required for terminal differentiation to occur. Inhibition of PLCb1 activity prevents the upregulation of cyclin D3 and cdk4 protein, suggesting that PLCb1 plays a role in the control of the cell cycle during differentiation (O’Carroll et al. 2009). How PLCb1 controls differentiation is not clear, however, using a combination of proteomics, immunocytochemistry and molecular biology, we identified a functional signaling cascade elicited by PLCb1 in the nucleus during C2C12 myogenic differentiation. DAG generation from PLC-mediated PtdIns(4,5)P 2 hydrolysis results in the activation of nuclear PKCbI and the subsequent phosphorylation of the eukaryotic elongation factor 1A (eEF1A) on Ser 53 (Piazzi et al. 2010). PLCb1 also co-localizes and interacts with DGKz, in nuclear speckles in C2C12 cells. Like PLCb1, nuclear DGKz also increases during myoblast differentiation, and impairment of DGKz upregulation markedly inhibits differentiation (Evangelisti et al. 2006). Furthermore over expression of DGKz facilitates differentiation, although this appears to be through the inhibition of cyclin D1 transcription. These data would suggest that there may also be a role for nuclear phosphatidic acid in the regulation of myogenesis.

Nuclear PLC signalling therefore targets the activity of numerous proteins involved in cell cycle machinery in order to regulate cell specific fate such as proliferation or differentiation.

11.4 PLC and the Regulation of Nuclear Inositol Phosphates

Phospholipase C mediated cleavage of PtdIns(4,5)P 2 also generates DAG and inositol(1,4,5)trisphosphate (Ins(1,4,5)P 3). DAG is a potent activator of PKC (Nishizuka 1984), which translocates to the nucleus in response to IGF-1 stimulation (Divecha et al. 1991; Banfic et al. 1993; Martelli et al. 1991). There are many nuclear substrates of PKC, however, if any of them regulate proliferation in response to IGF-1 stimulation is not clear. The other second messenger, Ins(1,4,5)P 3 can regulate a number of pathways. Ins(1,4,5)P 3 receptors that regulate calcium flux have been found on the inner nuclear envelope (Malviya et al. 1990; Humbert et al. 1996) and recent studies have suggested that nuclear Ins(1,4,5)P 3 may specifically mediate increases in nuclear calcium (Rodrigues et al. 2007, 2008, 2009; Gomes et al. 2008). Increased nuclear calcium could potentially regulate an array of transcriptional regulators to modulate cell behaviour (Bading et al. 1997; Hardingham et al. 1997). Ins(1,4,5)P 3 can also be further phosphorylated in the nucleus to generate a number of highly phosphorylated inositols. Indeed the inositol polyphosphate multikinase (IPMK), which phosphorylates Ins(1,4,5)P 3 to generate higher phosphorylated inositols, localizes in the nucleus and regulates transcription (Resnick et al. 2005; Resnick and Saiardi 2008). The recent characterization of IPMK knockout mice demonstrates critical roles for IPMK in embryogenesis and central nervous system development (Frederick et al. 2005). Highly phosphorylated inositols are water soluble second messengers, which have been implicated in the control of chromatin remodelling, mRNA export and telomere function (Tsui and York 2010). In a similar manner to phosphoinositides, inositol phosphates also regulate protein function by specifically interacting with protein domains. In this case they are unlikely to regulate localisation, but are more likely to modulate protein conformation which in turn regulates their activity and function.

11.4.1 Class I PI-3-Kinase

PtdIns(4,5)P 2 can also be converted to PtdIns(3,4,5)P 3 by a nuclear PI-3-kinase. While PI-3-kinase has been shown to be present in the nucleus (Bacqueville et al. 2001; Deleris et al. 2006; Metjian et al. 1999), is not clear whether PtdIns(3,4,5)P 3 is actually synthesized in the nucleus. The most convincing data showing the presence of PtdIns(3,4,5)P 3 in nuclei has come from Lindsay et al. (2006). In this study the authors used the PH domain from GRP1, which shows high specificity towards PtdIns(3,4,5)P 3. This phosphoinositide probe was used as an affinity probe on electron microscopy sections of control cells and of those stimulated with PDGF. PDGF stimulated approximately a twofold increase in the PtdIns(3,4,5)P 3 signal seen in nuclei. Interestingly the increase in PtdIns(3,4,5)P 3 signal was sensitive to pretreatment with the PI-3-kinase inhibitor wortmannin, however, it was not sensitive to expression of a nuclear targeted PTEN (a PtdIns(3,4,5)P 3 phosphatase), although the increase in PtdIns(3,4,5)P 3 seen in the plasma membrane was sensitive to both. This may suggest that the microenvironment of PtdIns(3,4,5)P 3 within the nucleus is not conducive to PTEN mediated dephosphorylation. Interestingly a FRET probe developed to visualise PtdIns(3,4,5)P 3 in vivo was also unable to detect a significant pool of nuclear PIP 3 although the probe could detect hormone activated PtdIns(3,4,5)P 3 signalling at the plasmamembrane (Ananthanarayanan et al. 2005). There are two class I PI-3kinases, that convert PtdIns(4,5)P 2 into PtdIns(3,4,5)P 3 and both have been found in the nucleus. Class IA PI-3-kinase can be regulated by PIKE (PI-3-kinase enhancer), which localizes in the nucleus and can bind GTP. PLCg1 interacts directly with PIKE and stimulates binding of GTP and the ability of PIKE to interact with and activate PI-3-kinase (Ahn et al. 2004; Ahn and Ye 2005; Ye 2005).

There are few targets that have been well characterized as acting downstream of nuclear PI-3-kinase signalling. Nucleophosmin interacts with PtdIns(3,4,5)P 3 and with CAD (caspase activated DNase) and the trimeric complex appears to be important in inhibiting DNA fragmentation (Ahn and Ye 2005). Akt/PKB is a well characterized PtdIns(3,4,5)P 3-regulated target at the plasma membrane, which also plays a role in the nucleus. However, whether PKB is activated by nuclear PIP 3 or is activated at the plasmamembrane and then translocated into the nucleus is not clear. There are many potential targets for nuclear Akt activity however few of them have been well characterized. Using a proteomic screening procedure for novel substrates of Akt in C2C12 myoblasts, lamin A/C was found to be a bone fide nuclear substrate of Akt (Cenni et al. 2008). Endogenous lamin A/C and Akt proteins interact, and lamin A/C is phosphorylated by Akt not only in vitro but also in vivo in response to insulin stimulation. By mass spectrometry and mutagenesis, Akt was shown to phosphorylate lamin A at Ser404, in the evolutionary conserved RSRGRASSH Akt motif. Since arginine at - 3 is a prerequisite for Akt phosphorylation, these data suggest why lamin A/C mutated at Arg401, which is found in primary EDMD-2 (Emery-Dreifuss muscular dystrophy-2) cells, is not phosphorylated in vitro by recombinant Akt. Moreover, in primary myoblasts transfected with lamin S404A, the presence of misshapen nuclei and nuclear abnormalities, such as nuclear envelope breaches, blebs, and honeycomb lamina structures together with concentrated foci of lamin A in the nucleoplasm was observed, which are hallmarks of the EDMD-2 phenotype (Cenni et al. 2008; Marmiroli et al. 2009). Also Akt2 localizes in the nucleus of the differentiated myoblasts and plays a specific role in the commitment of myoblasts to differentiation (Vandromme et al. 2001). HL-60 cells differentiate into monocyte-like cells following exposure to interferon-Q (IFN-Q) and vitamin D3. All-trans-retinoic acid (ATRA) and DMSO induce maturation along neutrophilic pathways and phorbol 12-myristate 13-acetate (PMA) causes the cells to differentiate into a macrophage- like phenotype (Collins 1987). PI3K activity also progressively increases in the nuclei of ATRA-treated HL-60 cells, and wortmannin, a PI3K inhibitor, prevented ATRA mediated antiproliferative and differentiative effects (Bertagnolo et al. 2004). Moreover, the level of active nuclear Akt increases in both HL-60 and NB4 (Matkovic et al. 2006) cells after 96 h of ATRA-treatment.

11.4.2 Class II PI-3-Kinase

Several studies demonstrated the presence of Class II PI-3-kinase in the nucleus. Class II enzymes predominantly convert PtdIns to PtdIns3P and PtdIns3P has been shown to be present in the nuclei. Phosphatidylinositol 3-kinase C2alpha (PI3K-C2a) was found to be present in nuclei of HeLa cells, where it localised to nuclear speckles. Inhibition of RNA polymerase II activity led to phosphorylation of PI3K-C2a suggesting that PI3K-C2a may play a role in transcription or splicing (Didichenko and Thelen 2001). Human promyelocytic leukemia HL-60 cells have been extensively studied as an experimental model for leukemic and myelocytic differentiation. The activity of nuclear phosphoinositide 3-kinase C2b (PI3K-C2b) was investigated in HL-60 cells induced to differentiate along granulocytic or monocytic lineages. Visnjic et al. demonstrated a significant increase in the activity of PI3K-C2b immunoprecipitated from both the nuclei and the nuclear envelopes of ATRA differentiated HL-60 cells. They also showed an increased level of tyrosine phosphorylation of the enzyme and a parallel increase in the level of nuclear PtdIns3P, suggesting that the enzyme may be activated by tyrosine phosphorylation (Visnjic et al. 2002; Visnjic and Banfic 2007). An increase in nuclear PI3K-C2b was also demonstrated during cell cycle progression in HL-60 cells (Visnjic et al. 2003) and in vivo during compensatory hepatic growth after partial hepatectomy (Sindic et al. 2006). How increased nuclear PI3K-C2 activity regulates nuclear functions and which cellular pathways are regulated are not clear.

11.4.3 PtdIns5P Signalling in the Nucleus

Using specific assays we showed that PtdIns5P is present in the nucleus and its levels are regulated in response to cellular stressors such as oxidative imbalance or UV irradiation through the activation of the p38 stress activated kinase (Jones et al. 2006). So how are PtdIns5P levels regulated in response to stress activation? We demonstrated that in C.Elegans and in Drosophila, knockout of the single PIP4K enzyme leads to increased levels of PtdIns5P, without significant changes in the levels of PtdIns(4,5)P 2. These data suggest that in vivo the role of PIP4K is to regulate PtdIns5P levels. There are three isoforms of PIP4K, a, b and g and we showed that the a isoform was predominantly cytosolic, while the b isoform was cytosolic and nuclear (Ciruela et al. 2000) and the γ isoform was found on intracellular membranes. We showed that in response to UV-irradiation PIP4Kb was directly phosphorylated by p38 at serine 326 and that this phosphorylation led to a decrease in PIP4K activity associated with PIP4Kb. To demonstrate that PIP4Kb controls nuclear PtdIns5P levels, we showed that overexpression of PIP4Kb decreased, while RNAi mediated suppression of PIP4Kb increased nuclear PtdIns5P (Jones et al. 2006). Interestingly, detailed analysis of the difference in the activities of the three isoforms of PIP4K showed that PIP4Kb has 2000 times less PIP4K activity compared to PIP4Ka (Bultsma et al. 2010; Wang et al. 2010). Therefore, how can PIP4Kb, which has very little PIP4K activity, regulate nuclear PtdIns5P? To begin to understand this we immunoprecipitated PIP4Kb from cells and identified associated proteins by using mass spectrometry. Interestingly, PIP4Kb associates with PIP4Ka. We then carried out a series of experiments to demonstrate that in vivo the majority of PIP4K activity in a PIP4Kb immunoprecipitate was actually derived from its association with PIP4Ka (Bultsma et al. 2010). Our previous data demonstrated that PIP4Ka was actually a cytosolic enzyme while PIP4Kb was a nuclear enzyme. So how does PIP4Kb regulate nuclear PtdIns5P levels? We found that when co-overexpressed PIP4Kb was able to target the activity of PIP4Ka to the nucleus (Bultsma et al. 2010), where it can presumably regulate the levels of nuclear PtdIns5P. How phosphorylation by the p38 pathway regulates PIP4K activity associated with PIP4Kb is not clear, but it may regulate the association between PIP4Ka and PIP4Kb.

While PIP4Ks are able to regulate PtdIns5P levels by phosphorylating it to PtdIns(4,5)P 2 what is really unclear is how PtdIns5P is synthesised. There are other enzymatic activities, present in the nucleus, which could synthesise PtdIns5P. The PIP5K family can synthesise PtdIns5P from PtdIns, albeit very inefficiently. PtdIns5P can also be generated by dephosphorylation of PtdIns(4,5)P 2 and a PtdIns(4,5)P 2-4-phosphatase has been characterised in mammalian cells that translocates to the nucleus upon stress induction (Ungewickell et al. 2005; Zou et al. 2007). Finally myotubularins can dephosphorylate PtdIns(3,5)P 2 to generate PtdIns5P (Coronas et al. 2008; Walker et al. 2001) although PtdIns(3,5)P 2 has not been demonstrated in the nucleus. Alternatively, and perhaps more interesting, there may be a novel enzymatic activity that synthesises nuclear PtdIns5P.

11.4.4 Targets for Nuclear PtdIns5P Signalling

The level of nuclear PtdIns5P is increased in response to oxidative stress and UV treatment and this occurs downstream of the activation of the stress activated p38 pathway (Jones et al. 2006). So what are the consequences of increased PtdIns5P in the nucleus? A seminal paper from Gozani et al (Gozani et al. 2003) demonstrated that the PHD finger of the growth inhibitory protein 2 (ING2) was able to interact with phosphoinositides and in particular PtdIns5P. Of interest, PHD fingers are generally only found in nuclear proteins many of which are involved in regulating gene transcription through the modulation of chromatin structure. ING2 also regulates the level of acetylation of the tumour suppressor p53 and increases in PtdIns5P induced acetylation and activation of p53 in a stress dependent manner. P53 is a master regulator of cell proliferation and is highly mutated and inactivated in human tumours. These data therefore link stress-activated modulation of nuclear PtdIns5P to the function of an important human tumour suppressor gene. To determine how common PHD interaction with phosphoinositides is, we have cloned over 30 of them and have assessed their interaction with phosphoinositides. We have found that among these, 10 PHD fingers interact strongly with phospholipids. Some PHD fingers also interact with trimethylated lysine 4 of histone H3 (H3K4me3) suggesting that they can also translate the histone code (Shi et al. 2006; Wysocka et al. 2006). For example TAF3 is a component of the basal transcription complex and interaction between the PHD finger of TAF3 and H3K4me3 stimulates transcription (Vermeulen et al. 2007). However, of the 30 PHD fingers that we have cloned only four showed interaction with peptides containing modified or unmodified sequences of the histone H3 tail. A subset of PHD fingers interact both with phosphoinositides and with modified histones, however it is not clear if phosphoinositide interaction can modulate or compete with histone interaction.

11.4.5 PtdIns5P Levels Can Regulate Histone Modification

In vivo, DNA is wrapped around histone octamers to form nucleosomes, which can be further packaged to form dense arrays of nucleosomes. Regulating the packing of these arrays and the actual positioning of the histone octamers are important in the regulation of transcription and gene expression. In all species, histones are highly conserved and residues within their N-terminal tails are targets for a number of different post-translational modifications, such as methylation, acetylation, ubiquitination and phosphorylation. Post translational modifications of histone proteins control gene expression by recruiting protein complexes that are able to modulate nucleosomal packing and positioning and modulate transcription directly (Ruthenburg et al. 2007). Interestingly, many of the proteins that contain PHD fingers are also present in these complexes (Musselman and Kutateladze 2009). The PHD finger of ATX1, a plant homologue of the mammalian trithorax proteins, does not interact with H3K4me3but shows exquisite preference for interaction with PtdIns5P (Alvarez-Venegas et al. 2006). ATX1 is a master controller of plant development and flowering (Alvarez-Venegas et al. 2003; Pien et al. 2008) and also plays a role in the regulation of gene transcription in response to environmental stress. ATX1 contains a SET domain that can trimethylate lysine 4 of histone H3, a histone tail modification that has been shown to be present at the promoter of genes that are being actively transcribed. Using expression arrays the WRKY70 gene was identified as a gene that was regulated both by ATX1 and by increased levels of drought stress. The expression of WRKY70 and the level of H3K4me3 on nucleosomes around its promoter was used to study how changing PtdIns5P modulated ATX1 activity in vivo. We showed that drought stress induced an increase in total levels of cellular PtdIns5P, that was mediated by the plant homologue of myotubularin, a PtdIns(3,5)P 2 3-phosphatase. The increase in PtdIns5P led to a decrease in the presence of H3K4me3 at the promoter of WRKY70. Using CHIP analysis we found that increased PtdIns5P also led to a decrease in the levels of ATX1 associated with promoters. In fact using immunofluorescence microscopy we showed that increased cellular PtdIns5P led to a change in the localisation of ATX1 from the nucleus to the cytoplasm which was dependent on the integrity of the PHD finger. Thus cellular PtdIns5P levels directly impinge on the activity of an important SET-domain containing protein to regulate the levels of H3K4me3 at specific promoters (Ndamukong et al. 2010) (Fig. 11.3). Other PHD fingers also interact with a different subset of histone marks including acetylated histones (Zeng et al. 2010; Matsuyama et al. 2010) and again according to our own data some of these are also able to interact with phosphoinositides. The data suggest that nuclear PtdIns5P levels may have an important role in modulating where and to what extend PHD finger containing proteins are activated and how they then impinge on chromatin structure and gene expression.

Fig. 11.3
figure 3

ATX-1 is a plant trithorax homologue that tri-methylates lysine 4 of histone H3, which is often present at actively transcribed promoters. H3K4me3 can recruit the TAF complex protein TAF3 to increase transcription of genes such as WRKY70. Upon drought stress, PtdIns5P levels increase as a consequence of the activation of myotubularin. Increased PtdIns5P leads to decrease in the localisation of ATX-1 and H3K4me3 at promoters. This may lead to a decrease in TAF3 at the promoter and hence a decrease in WRKY70 transcription

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11.4.6 Nuclear Phosphoinositides and Human Disease

As nuclear PLCb1 and nuclear inositides are involved in key steps of cell growth and differentiation, it is likely that they also play a role in disease development. Myelodysplastic syndrome (MDS) are a heterogeneous group of bone marrow disorders characterized by an impaired stem cell differentiation leading to progressive cytopenia and an increased, although variable, risk of evolution to acute myeloid leukemia (AML) transformation (Scott and Deeg 2010). The MDS diagnosis is currently based on morphological evaluations, according to either the French-American-British (FAB) (Bennett et al. 1982) or World Health Organization (WHO) (Vardiman et al. 2009) classification, as well as following two more complex systems, based on the percentage of marrow blasts, number of cytopenias, and bone marrow cytogenetic findings, useful for the assessment of the risk of evolution into AML, i.e. the International Prognostic Scoring System (IPSS) (Greenberg et al. 1997) and/or the WHO classification-based Scoring System (WPSS) (Malcovati et al. 2007). The identification of factors that define the risk of progression of MDS to AML or those that identify population that are beneficially effected by certain therapeutic regimes is essential in dictating the best overall therapeutic approach for individual patients.

Aberrations in DNA methylation, often affecting critical cancer-related signalling pathways, can lead to gene inactivation and contribute to tumorigenesis. However, DNA hypermethylation can be reversed by demethylating treatments, and indeed the current therapeutic approach for MDS patients is based on azacitidine, a DNA methyltransferase (DNMT) inhibitor which increases the overall survival and delays the MDS progression towards AML (Fenaux et al. 2009). However, the molecular mechanisms underlying this therapy are not completely understood, although they do lead to increased expression of genes involved in the control of cell cycle, such as p15/INK4B, p21WAF/Cip1 and p73 (Daskalakis et al. 2002; Raj et al. 2007). Furthermore, it is not clear why some patients benefit from azacitidine treatment, while others do not.

Among the enzymes of the nuclear PI cycle, PLCb1 appears to play a fundamental role in MDS as it is deregulated at both a genetic and an epigenetic level. Fluorescence in situ hybridization (FISH) analyses showed that the PLCb1 gene mapped to chromosome 20p12 (Peruzzi et al. 2000). Recent reports demonstrated that MDS patients can show a specific, cryptic and interstitial mono-allelic gene deletion of PLCb1. In fact, in a study involving 80 MDS cases belonging to all of the IPSS risk groups, about 30% of all of the MDS patients showed the PLCb1 gene deletion (Follo et al. 2009a). Interestingly, MDS patients bearing this mono-allelic deletion rapidly evolved to AML, suggesting that the alteration of the PLCb1 signalling can be linked to a higher risk of MDS progression. Furthermore, PLCb1 is aberrantly expressed in high-risk MDS, as compared to healthy donors (Follo et al. 2006). Interestingly, cells from MDS patients always expressed higher levels of the PLCb1b splicing variant, which is localized predominantly in the nucleus compared to PLCb1a, localised both in the nucleus and in the cytoplasm, hinting that an imbalance in nuclear versus cytoplasmatic PLCb1 signalling could be important in the MDS phenotype. PLCb1 promoter methylation and gene expression were quantified in high-risk MDS patients during azacitidine administration and compared to the expression of patients treated with only best supportive care as well as healthy subjects (Follo et al. 2009b). Strikingly, PLCb1 promoter methylation was decreased upon azacitidine treatment and this correlated with an increase in PLCb1 gene expression. Importantly, changes in PLCb1 expression induced by azacitidine treatment correlated with and anticipated the clinical outcome. The variation in PLCb1 expression, increase or decrease, in response to azacitidine were detectable much earlier compared to the clinical improvement or worsening, respectively, suggesting that monitoring PLCb1 levels could lead therapeutic decisions (Follo et al. 2009b). Moreover, the combination of azacitidine (AZA) and valproic acid (VPA) treatment, in high-risk myelodysplastic patients, leads to a synergistic increase in PLCb1 and cyclin D3 expression suggesting a potential for the combined activity of AZA and VPA in inducing PLCb1 signalling and positively affecting clinical outcome (Follo et al. 2011). Our recent demonstration that there is an inverse correlation between PLCb1 expression and Akt activation (Follo et al. 2008, 2009b) and that MDS patients showing a positive response had reduced levels of activated Akt (Follo et al. 2008), may underlie the importance of PLCb1 expression levels in determining clinical outcome (Fig. 11.2). All in all, these data hint at a possible role for nuclear PLCb1 in the pathogenesis of myeloid malignancies, and offer new tools for both diagnosis and prognosis of human MDS.

Many PHD finger containing proteins have also been implicated in the development of human immunodeficiency disease, neuronal dysfunction and cancer; moreover disease associated mutations often target the integrity of the PHD finger. In some cases, the disease associated mutations are linked to the ability of the PHD finger to associate with histone tails (both modified and unmodified), however, a number of mutations also lie outside of the PHD finger and in many cases the function of the PHD finger is unknown. For example somatic mutations in inhibitor of growth 1 (ING1) are linked to the development of breast cancer, melanoma, squamous cell carcinoma, head and neck cancer, and many of the mutations target the interaction of the PHD finger with H3K4me3. However there is a disease associated mutation in ING1 (260stop), which inserts a stop codon leaving an intact PHD finger. The stop codon however, truncates the final polybasic region and we presume from studies on ING2 (a close isoform of ING1), that this will attenuate its interaction with phosphoinositides. In the case of Borjeson-Forssman-Lehmann syndrome, disease associated mutations occur within the PHD fingers of PHF6, however no function has been associated with the PHD finger. Interestingly, we have found that one of the PHD fingers from PHF6 interacts with phosphoinositides. Ours and others studies (Shi et al. 2006) have shown that it is possible to generate mutants that dissociate the interaction of PHD fingers with histones from their ability to interact with phosphoinositides. These mutants will enable us to discern the role of the interaction between PHD fingers and various ligands in the development of human diseases.

11.5 Summary

The existence and function of inositide signalling in the nucleus is well documented and now has been linked to the regulation of nuclear specific functions to control proliferation, differentiation and cellular responses to environmental stressors. Although we are now beginning to understand some of the mechanisms that control nuclear phosphoinositide metabolism, more regulators and downstream targets need to be identified in order to exploit this independent signalling pathway for therapeutic purposes. We and others have focused on PLCb1 which is the most extensively investigated PLC isoform in the nuclear compartment. PLCb1 is a key player in the regulation of nuclear inositol lipid signalling, however it is neither clear how nuclear PLCb1 activity is transduced into changes in nuclear function nor whether other isoforms of PLC are also important in regulating nuclear inositide metabolism. It is possible and highly likely that phoshorylation of Ins(1,4,5)P 3 to generate higher phosphorylated derivatives such as IP 6 and IP 7 will be important in regulating nuclear function. In yeast, IP 6 and IP 7 have been implicated in the regulation of mRNA export (York et al. 1999) and chromatin regulation (Odom et al. 2000; Lee et al. 2007) and it is clear from knockout studies that they have important function in mammalian physiology (Tsui and York 2010). Phosphoinositides themselves through their interaction with specific chromatin remodelling complexes can also regulate chromatin conformation and gene transcription. The evidence, in a number of patients with myelodysplastic syndromes, that the mono-allelic deletion of PLCb1 is associated with an increased risk of developing acute myeloid leukemia paves the way for an entirely new field of investigation. Indeed the genetic defect evidenced, in addition to being a useful prognostic tool, also suggests that altered expression of this enzyme could have a role in the pathogenesis of this disease by causing an imbalance between proliferation and apoptosis. Targeting the enzymes that control nuclear phosphoinositides will likely yield novel therapeutic targets.