Clues for the Nuclear Localization of the Inositol Lipid Signalling System

A remarkable feature of the inositol lipid cycle present in the nucleus with respect to that found in the cytoplasm is the fact that it is located mainly outside of membrane bilayers. The second and possibly related distinctive feature is its operational distinctiveness from the cycle present at the plasma membrane. This implies that many agonists that stimulate the membrane cycle do not activate the nuclear system and vice versa, or that, when they are both respondent, they do so in a temporally distinct manner, or through different regulation mechanisms (Irvine, 2003).

The identification of the nuclear localization sites of inositol lipids and of the enzymes involved in their metabolism was a crucial issue for the interpretation of the functional significance of a signal transduction system located at the nuclear interior. In fact, compartmentalization is a potential solution to account for the different responses given by signal transduction pathways that share their molecular constituents.

The enzymes involved in inositol lipid metabolism were found not associated with the nuclear membrane but with intranuclear components. Cell fractionation methods did not ensure an absolute exclusion of contaminating membrane-derived lipids in the preparation of nuclei. Therefore, the presence of a given lipid substrate or of a related enzyme within the nucleus has to be demonstrated also by morphological techniques in whole cells not subjected to fractionation (Maraldi et al., 1992). Moreover, the identification of the intranuclear sites where the inositol lipid signalling systems are located could clarify which nuclear domain mediates the transduction of signals to the genome (Fig. 2).

The availability of a reliable antibody allowed the identification of the nuclear sites of localization of PI(4,5)P2 (Mazzotti et al., 1995). The sites of PI(4,5)P2

Fig. 2 Phosphoinositide signalling at the nucleus. PI(4,5)P2 is recruited at the cell nucleus through the PI-binding consensus sequence -K(X) KHKK- present in many chromatin and nuclear matrix proteins. Phospholipases and inositol kinases are recruited at the same sites by a lipid substrate-dependent compartmentalization. Agonists at the cell membrane can trigger the nuclear inositide signalling system either through translocation of the lipid kinase PI 3-kinase, or of protein kinases, such as PKB/Akt or PKCa, which activates nuclear PLCP1. This activation requires the translocation of MAP kinase, mediated by the cytoskeleton (bent arrow)

Fig. 2 Phosphoinositide signalling at the nucleus. PI(4,5)P2 is recruited at the cell nucleus through the PI-binding consensus sequence -K(X) KHKK- present in many chromatin and nuclear matrix proteins. Phospholipases and inositol kinases are recruited at the same sites by a lipid substrate-dependent compartmentalization. Agonists at the cell membrane can trigger the nuclear inositide signalling system either through translocation of the lipid kinase PI 3-kinase, or of protein kinases, such as PKB/Akt or PKCa, which activates nuclear PLCP1. This activation requires the translocation of MAP kinase, mediated by the cytoskeleton (bent arrow)

localization are multiple, due to the presence of a PI-binding consensus sequence in several nuclear proteins. The stability of PI(4,5)P2 association with nuclear proteins is more or less strong; in fact, part of the PI(4,5)P2 pool associated with heterochromatin is lost during nuclei isolation in mild ionic conditions (Mazzotti et al., 1995; Maraldi et al., 1995). The other part of PI(4,5)P2 pool is localized at the perichromatin fibrils and at the interchromatin granule clusters (Maraldi et al., 1995); these interactions are more stable and are maintained also in chromatin-depleted nuclear matrix preparations (Mazzotti et al., 1995). This suggests that inositol lipids form lipoprotein complexes in which the lipids are not oriented as in membranes (Maraldi et al., 1992).

PI(4,5)P2 is a minor phospholipid (0.3% of total phospholipids); nevertheless, the amount of the nuclear PI(4,5)P2, estimated about 0.5 mmol/mg nuclear protein, represents more than 20% of the PI(4,5)P2 detectable in the cell (Watt et al., 2002). This finding is particularly significant because it has been obtained with a method completely different with respect to that used in the first demonstration of a presence of PI(4,5)P2 within the nucleus (Mazzotti et al., 1995). In fact, instead of using a post-embedding immunocytochemical detection of the anti-PI(4,5)P2 antibody, the method utilizes the PH domain of PLCS in thawed cryosectioned material (Watt et al., 2002). In any case, the quantitative evaluations of the nuclear PI(4,5)P2 amount are quite similar, as well as the sites where the lipid is located within the nucleus (electron-dense patches of heterochromatin). A similar localization has been obtained at lower resolution by confocal microscopy, using PI(4,5)P2 fluorescently tagged with a green NBD fluorophore, using Texas Red-labelled histone H1 as carrier (Ozaki et al., 2000).

To address the problem of the functional role of nuclear polyphosphoinositides, the detection and quantitative evaluation of the enzymes involved in their synthesis, phosphorylation and hydrolysis has been widely investigated. An inverse proportion has been reported between the PI(4,5)P2 amount detectable within the nucleus by immunogold electron microscopy and the nuclear amount and activity of nuclear PI-phospholipase PLC|1 (Maraldi et al., 1995).

PI(4,5)P2 is also present at other intranuclear sites, in association with the other elements of the inositide signalling system, such as PIP kinases, PI 3-kinase (Boronenkov et al., 1998), PLC isoforms, and PKC isoforms (Maraldi et al., 1999). The speckles detectable by fluorescence microscopy correspond to clusters of interchromatin granules that represent sites of accumulation of splicing factors and hnRNPs (Boronenkov et al., 1998). The members of the inositide signalling system have been localized at the nuclear speckles by fluorescence microscopy, and at the interchromatin granule clusters, as well as at the perichromatin fibrils associated with the periphery of heterochromatin (Maraldi et al., 1999). The inositol signalling elements, found at these sites, can be essential for the activation of protein kinases and phosphatases to induce the re-location of splicing factors (Misteli and Spector, 1997).

Hydrolysis of nuclear PI(4,5)P2 by PLC might provide a localized release of IP3 that, in addition to the regulation of Ca2+ from cell stores, could serve as precursor of inositol polyphosphates, which are essential for RNA transport (Odom et al., 2000; Osborne et al., 2001).

Finally, PI(4,5)P2 mediated actin-dependent mechanisms have been demonstrated to be involved in chromatin remodelling (Zhao et al., 1998). PI(4,5)P2 but not PIP enhances the binding of the BAF complex to the nuclear matrix (Zhao et al., 1998); PI(4,5)P2 binds to BAF at one molecule per complex, which, in turn, increases actin polymerisation in a PI(4,5)P2-sensitive manner, suggesting that PI(4,5)P2 can induce the uncapping of actin, leading to nucleation or filament assembly (Rando, 2002).

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