EL SENADO EN LA CONSTITUCIÓN DE 1837 1 Contexto histórico

cells to study their subcellular localisation. Both isoforms were also expressed in A431 and MDCK cells using plasmid transfection and microinjection (Figure 6.6). The fact that both Ptdlns 4Kp isoforms lack membrane targeting sequences and yet are not exclusively cytosolic, raises the question of the mechanism for membrane localisation. Ptdlns 4Kp and Ptdlns 4KpI showed identical immunofluorescence staining, suggesting that the insert region was unlikely to direct subcellular localisation under the conditions used in this study. It is possible that interaction with membranes, whether direct or indirect, is mediated by the LKH3 region or by posttranslational modification, although this remains to be determined. It is also not known whether a fraction is constitutively associated with membranes or if Ptdlns 4KP membrane-association is regulated. Furthermore, it must be considered that the use of overexpression may cause the accumulation of a delocalised pool of Ptdlns 4Kp in the cytosol, whereas under normal conditions a smaller amount of Ptdlns 4Kp might be exclusively membrane localised.

The finding that Ptdlns 4Kp is sensitive to PI 3-kinase inhibitors is important because it calls the specificity of such reagents into question. Wortmannin has been a valuable tool in dissecting PI 3-kinase function because of the potency of this fungal metabolite and its apparent specificity (Arcaro and Wymann, 1993; Ui et aL, 1995). Recently enzymes other that PI 3-kinases, some with important roles in signalling, have been found to be inhibited at concentrations in the nanomolar range, for example the phospholipase PLA% (Cross et aL, 1995), and protein kinases such as myosin light chain kinase (Nakanishi et aL, 1992). The mammalian type III Ptdlns 4K has also been found to be sensitive to wortmannin (Balia et aL, 1997) and a structural analogue of wortmannin, demethoxy viridin, has been found to inhibit a S. pombe membrane Ptdlns 4K activity (Woscholski et aL, 1994). This is likely to be an STT4-like activity since STT4p is inhibited by wortmannin (Cutler et aL, 1997). Given the structural relationship between the catalytic domains of PI 3-kinases and Ptdlns 4Ks and the invariance of the wortmannin-binding lysine residue (Wymann et aL, 1996) within the catalytic domains of this family, it is hardly surprising that wortmannin should have multiple targets amongst the PI3/4-kinases. Such findings advocate caution when interpreting inhibition data, in particular with whole cell experiments which frequently use elevated inhibitor concentrations in order to overcome the problem of instability. Also, because wortmannin inhibits by irreversible covalent binding to its target, the pre-incubation time used in different experiments is a critical parameter. The existence of multiple wortmannin targets which may lie in the same pathways, and the fact that some PI 3- kinases such as the class II and class III proteins display reduced sensitivity to wortmannin (Domin et aL, 1997), may explain the wide range of effects of PI 3-kinase inhibitors on whole cells and complex preparations.

There is mounting evidence for the existence of a wortmannin-sensitive cellular pool of PtdIns(4)P and Ptdlns(4,5)f2- However, it was initially reported that wortmannin

inhibited angiotensin II-stimulated inositol phosphate and calcium signalling in adrenal glomerulosa cells (Nakanishi et aL, 1995). It was subsequently shown that incubation of cultured adrenal glomerulosa cells or human Jurkat cells with wortmannin lead to a decrease in total PtdIns(4)P and PtdIns(4,5)P2 levels in unstimulated cells. Furthermore, upon stimulation with angiotensin II, PDGF, or the TCR agonist OKT-3, the initial increase in PtdIns(4,5)P2, InsPg and calcium mobilisation were not substantially affected. However, production of these key messengers during the sustained phase from 2-10 min post stimulation was significantly impaired (Nakanishi et aL, 1995). In these studies wortmannin was used at a concentration of 10 |iM, a concentration known to completely inhibit Ptdlns 4K|J in vitro, however this concentration of wortmannin would also inhibit the type III enzyme whose yeast homologue may lie upstream of PKC (Yoshida et aL, 1994). Alternatively, wortmannin sensitivity may arise if a Ptdlns 4K activity is regulated by the product of a PI 3-kinase as proposed for PLCy regulation (Bae et aL, 1998; Gratacap et aL, 1998; Rameh et aL, 1998). It is interesting to note that a previous study of PI turnover in T-cells postulated that the sustained phase of PtdIns(4,5)P2 synthesis may involve the activation of a distinct Ptdlns(4)f-generating pathway (Inokuchi and Imboden, 1990); this pathway may correspond to the wortmannin-sensitive PI pool. It will be crucial to test whether overexpression of, or reconstitution with exogenous Ptdlns 4Kp can overcome inhibition of PtdIns(4,5)P2 signalling by PI kinase inhibitors.

During the cloning of Ptdlns 4Kp a splice variant was isolated which contained a short sequence with a large proportion of acidic and serine residues. Analysis of the sequence suggested that it contained a casein kinase II phosphorylation site. Also, because of its close juxtaposition to LKH3, a novel Ptdlns 4K-specific sequence of unknown function, it was hypothesised that CKII phosphorylation may have role in regulating the function of LKH3. In vitro kinase assays with recombinant human CKII showed that Ptdlns 4KP could act as a substrate for CKII and that Ptdlns 4KpI was phosphorylated to a much greater degree. Using mass spectrometry the phosphorylation site was mapped to the insert region of Ptdlns 4KpI indicating that alternative splicing generates a protein with potential for regulation by CKII. The effect of CKII phosphorylation on Ptdlns 4K activity was measured and it was found that CKII phosphorylation of Ptdlns 4Kp and -pi led to only modest increases in activity (25 ±5% and 46 ±11%, respectively, data not shown). Estimates of the stoichiometry of CKII phosphorylation, assuming a single phosphorylation site, were in the order of 1.5 x 10’^ mol phosphate/mol for Ptdlns 4Kpi and two orders of magnitude lower for Ptdlns 4Kp. Consequently, the figures for activation could be underestimates. It is also possible that other factors are required to activate CKII towards Ptdlns 4KpI in vivo.

I next looked at the ability of cell lysates to phosphorylate Ptdlns 4Kp isoforms with the aim of characterising cellular protein kinases. Incubation of both Ptdlns 4K isoforms with Triton X-100 extracts from Jurkat cells led to a massive phosphorylation of Ptdlns

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