The p85 Regulatory Subunit of PI3K Mediates cAMP-PKA and Insulin Biological Effects on MCF-7 Cell Growth and Motility
Figure 1
Phosphorylation of S83 modulates the IRS-1/p85 interaction. Total protein extract obtained from MCF-7 cells untreated and treated with 10 nM insulin for 10 min in the pretreated or not with 10 μg/mL PKAi for 15 min was immunoprecipitated with anti-p85α antibody and probed with anti-phospho (Ser/Thr) PKA substrate antibody (P-S/T) (a). MCF-7 cells transfected with p85WT or p85A or p85D were serum starved for 6 h and treated with 10 nM insulin for 10 min. Cell lysates were immunoprecipitated with anti-p85 antibody and then analysed by Western blot with anti-IRS-1, anti-p85, and anti-FLAG antibodies (b). , IRS-1, and β-tubulin expression levels in input lysates were shown (c). The upper bands in the insert of IRS-1 are not displaced by the control peptide and are considered nonspecific signals (IRS1-specific bands are indicated by the arrow). The histogram (d) represents the quantitation of IRS-1/ signals ratio obtained from different co-IP experiments. To minimize differences in transfection and/or antibody efficiency, the data shown in the panel (d) were obtained by the comparison of different experiments performed with anti-p85α (three experiments), anti-IRS-1 (three experiments), and anti-FLAG (three experiments; see Figure S2). The histograms show the fold variation relative to the untreated control cells of the ratio IRS-1/p85. Values are expressed as (). Differences between treatments were tested for statistical significance using Student’s matched pairs -test: versus 0.5% FCS treated cells; versus nontransfected cells; versus wild-type or p85D expressing cells or control cells after insulin treatment.
