Evidence for functional redundancy of class IA PI3K isoforms in insulin signalling - PubMed (original) (raw)

Evidence for functional redundancy of class IA PI3K isoforms in insulin signalling

Claire Chaussade et al. Biochem J. 2007.

Abstract

Recent genetic knock-in and pharmacological approaches have suggested that, of class IA PI3Ks (phosphatidylinositol 3-kinases), it is the p110alpha isoform (PIK3CA) that plays the predominant role in insulin signalling. We have used isoform-selective inhibitors of class IA PI3K to dissect further the roles of individual p110 isoforms in insulin signalling. These include a p110alpha-specific inhibitor (PIK-75), a p110alpha-selective inhibitor (PI-103), a p110beta-specific inhibitor (TGX-221) and a p110delta-specific inhibitor (IC87114). Although we find that p110alpha is necessary for insulin-stimulated phosphorylation of PKB (protein kinase B) in several cell lines, we find that this is not the case in HepG2 hepatoma cells. Inhibition of p110beta or p110delta alone was also not sufficient to block insulin signalling to PKB in these cells, but, when added in combination with p110alpha inhibitors, they are able to significantly attenuate insulin signalling. Surprisingly, in J774.2 macrophage cells, insulin signalling to PKB was inhibited to a similar extent by inhibitors of p110alpha, p110beta or p110delta. These results provide evidence that p110beta and p110delta can play a role in insulin signalling and also provide the first evidence that there can be functional redundancy between p110 isoforms. Further, our results indicate that the degree of functional redundancy is linked to the relative levels of expression of each isoform in the target cells.

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Figures

Figure 1

Figure 1. Structures of the selected PI3K inhibitors

Figure 2

Figure 2. Effect of isoform-specific inhibitors on insulin-induced phosphorylation of PKB in CHO-IR cells

Overnight-starved CHO-IR cells were incubated for 5 min with the indicated PI3K inhibitors or DMSO and stimulated or not with insulin (1 nM, 10 min). Whole-cell lysates were then analysed by Western blotting using specific antibodies. (A) Effect of p110α-specific inhibitor (PIK-75, 100 nM) and p110β-specific inhibitor (TGX-221, 100 nM) on the insulin-induced stimulation of PKB phosphorylation on Ser473 (left) and Thr308 (right). A representative Western blot is shown in each of the upper panels and quantification is presented below. The relative stimulation of insulin over the basal level was taken as 100% and all values are calculated as a percentage of this. The results are means±S.E.M. for three independent experiments each performed in triplicate. (B) Dose–response effect of p110α-specific inhibitor (PIK-75) and p110β-specific inhibitor (TGX-221) on the insulin-induced stimulation of PKB (1 nM, 10 min). (C) Sigmoidal concentration–response curve of PKB phosphorylation obtained in the presence of increasing concentrations of PIK-75. (D) Dose–response effect of another structurally unrelated p110α-selective inhibitor (PI-103) on the insulin-induced stimulation of PKB phosphorylation (100 nM insulin, 10 min). (E) Effect of wortmannin (Wrt; 100 nM) and LY294002 (LY; 5 μM) on the insulin-stimulated phosphorylation of PKB (100 nM insulin, 10 min). These broadly active PI3K inhibitors were used as a control.

Figure 3

Figure 3. Effect of isoform-specific inhibitors on insulin signalling in 3T3-L1 fibroblasts

Overnight-starved 3T3-L1 fibroblasts were incubated for 5 min with the indicated PI3K inhibitors or DMSO and stimulated or not with insulin (100 nM, 10 min). Whole-cell lysates were then analysed by Western blotting using specific antibodies. (A) Effect of p110α-specific inhibitor (PIK-75, 100 nM) and p110β-specific inhibitor (TGX-221, 100 nM) on the insulin-induced stimulation of PKB phosphorylation. A representative Western blot is shown in each of the upper panels and quantification is presented below. The relative stimulation of insulin over the basal level was taken as 100% and all values are calculated as a percentage of this. The results are means±S.E.M. for three independent experiments each performed in triplicate. (B) Dose–response effect of another structurally unrelated p110α-selective inhibitor (PI-103) on the insulin-induced stimulation of PKB. (C) Effect of wortmannin (Wrt; 100 nM) and LY294002 (LY; 5 μM) on the phosphorylation of PKB. These broadly active PI3K inhibitors were used as a control.

Figure 4

Figure 4. Effect of isoform-specific inhibitors on insulin signalling in 3T3-L1 adipocytes

Overnight-starved 3T3-L1 adipocytes were incubated for 5 min with the indicated PI3K inhibitors or DMSO and stimulated or not with insulin (100 nM, 10 min). Whole-cell lysates were then analysed by Western blotting using specific antibodies. (A) Effect of p110α-specific inhibitor (PIK-75, 100 nM) and p110β-specific inhibitor (TGX-221, 100 nM) on the insulin-induced stimulation of PKB phosphorylation. A representative Western blot is shown in each of the upper panels and quantification is presented below. The relative stimulation of insulin over the basal level was taken as 100% and all values are calculated as a percentage of this. The results are means±S.E.M. for three independent experiments each performed in triplicate. (B) Dose–response effect of two other structurally unrelated p110α-selective inhibitors (PI-103 and SN 30693) on the insulin-induced stimulation of PKB phosphorylation.

Figure 5

Figure 5. Effect of isoform-specific inhibitors on insulin signalling in HepG2 cells

Overnight-starved human heptoma cells (HepG2) were incubated for 5 min with the indicated PI3K inhibitors or DMSO and stimulated or not with insulin (100 nM, 10 min). Whole-cell lysates were then analysed by Western blotting using specific antibodies. (A) Dose–response effect of p110α-specific inhibitor (PIK-75) and p110β-specific inhibitor (TGX-221) on the insulin-induced stimulation of PKB phosphorylation. (B) Dose–response effect of two other structurally unrelated p110α-selective inhibitors (PI-103 and PI-SN 30693) on the insulin-induced stimulation of PKB phosphorylation. (C) Dose–response effect of p110δ-specific inhibitor (IC87114) and p110γ-specific inhibitor (AS 252424) on the insulin-induced stimulation of PKB phosphorylation. Wortmannin (Wrt) and LY294002 (Ly) are shown as controls. (D) Effect of inhibitors in combination on the insulin-induced stimulation of PKB phosphorylation. A representative Western blot is shown in the upper panel and quantification is presented below. The relative stimulation of insulin over the basal level was taken as 100% and all values are calculated as a percentage of this. The results are means±S.E.M. for three independent experiments each performed in triplicate. ***P<0.001; n.s, not significant.

Figure 6

Figure 6. Effect of isoform-specific inhibitors on insulin signalling in J774.2 cells

Overnight-starved J774.2 cells were incubated for 5 min with wortmannin (wrt; 100 nM), PIK-75 (50 nM), TGX-221 (50 nM), IC87114 (100 nM) or DMSO and stimulated or not with insulin (100 nM, 10 min). Whole-cell lysates were then analysed by Western blotting using antibodies recognizing phospho-Ser473 (pS473) of PKB. A representative Western blot is shown in the upper panel and quantification is presented below. The relative stimulation of insulin over the basal level was taken as 100% and all values are calculated as a percentage of this. Results are means±S.E.M. for three independent experiments each performed in duplicate. ***P<0.001.

Figure 7

Figure 7. Comparison of the relative levels of expression and activity of different class IA PI3K isoforms

(A) Western blotting using isoform-specific antibodies was used to compare the levels of expression of class IA p110 isoforms in different cell types: lane 1, J774.2; lane 2, 3T3-L1 fibroblasts; lane 3, 3T3-L1 adipocytes; lane 4, CHO-IR; lane 5, HepG2. For p110α detection, total class IA activity was immunoprecipitated from 200 μg of lysate using an antibody recognizing the p85α adapter subunit, and recombinant protein was loaded as a control (0.2 μg, lane marked +). For p110β and p110δ detection, 20 μg of total cell lysate was loaded in each case. (B–E) Total class IA activity was immunoprecipitated from 20 μg of lysate from the indicated cell type using an antibody recognizing the p85α adapter subunit. PI3K activity was then assayed in the presence of 100 nM of the indicated inhibitor. Cell types were J774.2 (B), 3T3-L1 fibroblasts (C), 3T3-L1 adipocytes (D), CHO-IR (E) and HepG2 (F). Results are expressed relative to the activity in the presence of DMSO and are means±S.E.M. for three independent determinations. WORT, wortmannin.

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