Posicionamiento en torno a la Interculturalidad

compounds.

Translocation of PKC isoforms is an important feature of their activation. However, Ca^+ regulated protein-lipid interactions are probably just one aspect of membrane targeting, as a class of proteins has been defined which are receptors for activated PKC, the so-called RACKs. These are not substrates for PKC but appear to be able to interact with PKCs, targeting them to specific membranes. In the case of RACKl, this interaction takes place via part of the C] domain in PKCp and peptides corresponding to this region block PKCp translocation to the plasma membrane (Ron et a i, 1995).

1 . 8 . 2 Regulation of PKC by PI pathways

The activation of PKC-Ç, PKC-e, PKC-T), and the PKC-related PRKl by the products of PI kinases has already been discussed (Section 1.5.4.a). Although the activation of these PKC isoforms has been demonstrated in vitro, it is unclear whether direct interaction of Pis with PKC is responsible for the PI 3-kinase-dependent activation of PKC isoforms observed in vivo (Moriya et a i, 1996; Toker et al., 1995). Recently a mechanism whereby PI 3-kinase may activate PKCs was suggested by the demonstration that PDKl can phosphorylate key regulatory sites in the activation loop of PKCÔ and PKCÇ in vitro (Le Good et al., 1998; Chou et a i, 1998). This phosphorylation was shown to be dependent on PI 3-kinase activity when PDKl and PKCô or PKCÇ were co-expressed in human embryonic kidney (HEK) 293 cells. Furthermore, ectopically expressed PKCÔ in HEK 293 cells becomes dephosphorylated upon serum starvation and is rephosphorylated in a PI 3-kinase-dependent manner when cells are restimulated with serum (Le Good et al.,

1998). It is not yet clear whether binding of Ptdlns(3,4)?2 or Ptdlns(3,4,5 ) ? 3 to the PH

domain of PDKl is required for these phosphorylation events, nevertheless, the concept that PDKl acts as a link between PI 3-kinase signals and the activation of protein kinases suggests an interesting research direction.

1.9

Nuclear PI signalling

Evidence for PI signalling in the nucleus has been reviewed elsewhere (Divecha et al., 1993) and therefore will not be discussed in detail here.

When isolated rat liver or Friend cell nuclei are incubated with [y-32p]ATP, radioactivity becomes incorporated into PtdlnsP, Ptdlns?2, and PtdOH (Cocco et al., 1987; Smith and

Wells, 1984) suggesting the presence of an autonomous PI cycle in nuclei. In support of this, stimulation of quiescent Swiss 3T3 fibroblasts with lGF-1 leads to a specific increase in nuclear DAG and a decrease in nuclear Ptdlns? 2 (Divecha, 1991). Furthermore, a

number of enzymes in the PI cycle have been found associated with nuclear fractions including Pl-kinases, DAG-kinase, PKC, P L C pl, PLCÔ4, and PtdlnsTP (Liu et al..

1996; Payrastre et a l, 1992; Snoek et a l, 1993). However, it should be noted that it is difficult to assess the purity of nuclear preparations used in these studies and it remains a possibility that they may be contaminated with perinuclear or even plasma membranes, both known to contain Pl-kinases and PtdlnsTPs. In some cases detergents have been used to remove the nuclear membrane but the question of the presence of detergent insoluble membrane rafts (Section 1.2.4) in nuclei has not be specifically addressed.

1.10 Phosphoinositides and Cytoskeietal

Reorganisation

PtdIns(4,5)P2 binds to a variety of actin regulating proteins including profllin, cofilin, gelsolin, CapZ and gCap39, suppressing the function of these proteins (Janmey and Stossel, 1987; Lassing and Lindberg, 1985; Stossel, 1993; Yonezawa et a l, 1991; Yu et a l, 1990), for review). Ptdlns(4,5)?2 also binds to the actin crosslinking protein a - actinin and promotes actin filament bundling (Fukami et a l, 1994; Fukami et a l, 1992). It has long been suggested that PtdIns(4,5)P2 controls actin polymerisation and depolymerisation through its interaction with actin regulatory proteins. This hypothesis was strengthened by the observation that complexes of actin and profilin, an actin monomer-sequestering protein, could be dissociated by PtdIns(4)P and PtdIns(4,5)P2 (Janmey and Stossel, 1987; Lassing and Lindberg, 1988), and also that the amount of PtdIns(4,5)P2 bound to a-actinin and vinculin decreases on stimulation of fibroblasts with

PDGF and correlates with actin depolymerisation (Fukami et a l, 1994).

Despite the many reported effects of Pis on actin organisation, convincing evidence for the regulation of the actin cytoskeleton by phosphoinositides has been scarce, mainly due to the fact that studies on actin de/polymerisation have relied on in vitro methods and because of the potential for artefacts in protein-phospholipid interactions. However, the hypothesis that PI signalling is directly linked to reorganisation of the actin cytoskeleton is becoming more compelling. Firstly, there is the finding that PtdlnsPKs are regulated by small GTPases Rac and Rho which are known to perform key functions in controlling the actin cytoskeleton (see below). Secondly, actin polymerisation induced by thrombin in platelets has been found to occur through a Rac pathway and actin uncapping and polymerisation was PtdIns(4,5)P2-dependent (Hartwig et a l, 1995). Thirdly, LPA-

stimulated stress fibre formation in PAE cells is sensitive to inhibitors of PLD activity (Cross et a l, 1996). The product of PLD activity, PtdOH, was able to stimulate stress fibre formation alone when added to cells. PLD is known to be activated by PtdIns(4,5)P2

which suggests that there is potential for the regulation of PLD activity via a PtdlnsPK. It is notable that type I PtdlnsPKs are known to affect actin structures in vivo (see below) and are activated by PtdOH in vitro (Section 1.5.2.b). However, it appears that in PAE

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