filosófica Nuevo poder espiritual Construcción Religiosa
2.3. La crítica de la escuela francesa postestructuralista a los derechos humanos
The dose-response curve for bryostatin 1-mediated activation of PKD was also investigated by treating quiescent cultures of Swiss 3T3 cells with increasing concentrations (0.3-100 nM) of bryostatin 1 before PKD immunoprécipitation and in vitro kinase assays. Stimulation of intact cells with bryostatin 1 was found to induce a striking dose-dependent increase in PKD activity (Fig.3.6a), with half-maximal and maximal stimulation achieved at -1 nM and 5-10 nM, respectively. Maximal PKD activation induced by bryostatin 1 was comparable to that induced by the phorbol ester PDBu (Fig. 3.6a). Strikingly, higher concentrations of bryostatin 1 (>10 nM) activated PKD to a lesser extent. The biphasic pattern of PKD activity was not due to the induction of PKD degradation by high concentrations of bryostatin 1 since western blot analysis demonstrated equivalent PKD protein levels at all bryostatin 1 concentrations used (Fig. 3.6a).
Bryostatin 1-mediated activation of PKD was also demonstrated when PKD activity was measured by the phosphorylation of an exogenous substrate peptide, Syntide-2, a previously described in vitro substrate for PKD (Valverde et al., 1994; Van Lint et ai.,
Br-1 (nM) 0.3 I 3 10 30 100 < IV K a-PKD
II
ï î
100 80 60 40 20 0 C ) - PDBu l 10 100 1 10 100 1000 B r - 1 ( i i M ) Br-1 2 10 20 too 200 500 1000 — - 0.5 2 5 20 50 200 500 P D B u ( n M ) T P A ( n M )Fig. 3.6. B ryostatin 1, unlike phorbol esters, induces biphasic PK D activation in Swiss 3T3 fibroblasts.
(a) Confluent and quiescent cultures of Swiss 3T3 fibroblasts were incubated with increasing concentrations
o f bryostatin 1 (U-IUU n M ) for 3U min or with 2UU n M PD B u for lU min and lysed. P K D was
immunoprecipitated from the lysates using the PA-1 antiserum and subjected lo in vitro kinase assays, SDS-
P A G E and autoradiography (lA K ) Alternatively, lysates were incubated at 37"C for 3U min (to active
endogenous protein phosphatases) before P K D was immunoprecipitated and analysed by S D S -P A G E and western blotting using a specific P K D antibody (sc-935). One representative experiment in shown (upper panel) and the mean + s.e. increase in P K D autophosphorylation from 3 independent experiments (as quantified by scanning densitometry) are shown (low er panel), expressed as a percentage of the maximum response induced by bryostatin I.
(b) Conlluent and quiescent cultures o f Swiss 3T 3 fibroblasts were incubated in the presence of either
increasing concentrations of bryostatin 1 for 3Ü min (solid bars), 2ÜU nM PDBu for lU min (hatched bar) or were left unstimulated (open bar). The cells were lysed and P K D immunoprecipitated with the PA-1 anti serum. P K D activity was measured in a syntide-2 phosphorylation assay as described in Chapter 2. Results shown are expressed as the fold increase in syntide-2 phosphorylation over that induced by P K D isolated from resting cells and are the mean + s.e. o f two independent experiments, each performed in duplicate.
(c) Conlluent and quiescent cultures of Swiss 3T3 fibroblasts were stimulated with increasing concentrations
of either PDBu or T P A for lU min, lysed and P K D immunoprecipitated with the P A - 1 antibody. P K D activity
(as assessed by autophosphorylation) was analysed by in v itro kinase assays, S D S -P A G E and
Br-1 (n M ) 0.3 1 3 10 30 100 < 100 1 10 100 Br-1 (nM ) 1000
B)
200 + + + PS 10 50 100 Br-1 (nM )Fig. 3.7. B ryostatin 1 induces biphasic PKD activation in mouse em bryo fibroblasts but not d urin g d irect in vitrostim ulation.
(a) ConHuent and quiescent cultures o f MEFs were treated w ith increasing concentrations o f bryostatin 1 (Ü-1ÜÜ nM) for 30 min or with 200 iiM PDBu for 10 min as indicated, lysed and PKD immunoprecipitated w ith PA-1 antisernm. PKD activity, as shown by autophosphorylation, was determined by in vitro kinase assays follow ed by SDS-PAGE, autoradiography and scanning densitometry. Results shown are the mean ± s.e. o f three independent experiments (lower panel). A representative autoradiogram is shown (upper panel).
(b) COS-7 cells were transiently transfected w ith pcDNA3-PKD and after 72 h viable cells were lysed and PKD immunoprecipitated using the PA-1 antiserum. PKD was then eluted from immunocomplexes and activated in vitro by incubation w ith [y-^^PJ-ATP plus increasing concentrations o f bryostatin 1 (0-100 nM) in the presence o f 125 //g ml phosphatidyl-L-serine (PS), as indicated for 10 min at 30°C, followed by SDS-PAGE and autoradiography . Results shown are representative o f two independent experiments.
1995). As shown in Figure 3.6b, a biphasic pattern of syntide-2 phosphorylation w as detected in PKD immunoprecipitates (prepared from cells stimulated with increasing concentrations of bryostatin 1), comparable to the biphasic pattern of PKD autophosphorylation shown in Fig. 3.6a.
In contrast, phorbol esters did not induce a biphasic activation of PKD (Fig. 3.6c). The maximal effects of either PDBu orTPA on PKD activity (achieved at 100 nM and 20 nM, respectively) were not effected by increasing the concentrations of these agents by 10- and 25-fold respectively. Thus, bryostatin 1, unlike phorbol esters, induces a biphasic activation of PKD in intact Swiss 3T3 cells.
To confirm that the biphasic pattern of PKD activity induced by bryostatin 1 w as not restricted to the Swiss 3T3 fibroblast cell line, experiments were performed using murine embryonic fibroblasts (MEF). As shown in Fig. 3.7a, a dose-dependent biphasic activation of PKD induced by bryostatin 1 was observed in quiescent cultures of secondary MEF, with a maximum response occurring at -10 nM. Thus, the biphasic activation of PKD by bryostatin 1 was not restricted to immortalised cell lines. Strikingly however the dose-response curve for the direct activation of PKD by bryostatin 1 and phosphatidyl-L-serine in vitro was not biphasic (Fig. 3.7b), indicating that the biphasic regulation of PKD by bryostatin 1 is not due to a direct effect on PKD.