Insulin induces binding of 14-3-3 to endogenous IRS-2 and phosphorylates Ser-573 on IRS-2.

<p><i>A.</i> Fao cells were starved for serum overnight and incubated for 30 min with either 10 nM insulin or 50 ng/ml IGF-1. 250 µg protein was immunoprecipitated with IRS-2 antibody and separated on a 5–15% gradient gel. Overlay assay followed stripping and reprobing with IRS-2 antibody as loading control. Successful stimulation is shown as phosphorylation of p-Thr-308 and corresponding Akt/PKB reblot. <i>B.</i> Fao cells were starved for serum overnight and stimulated with 10 nM insulin for the indicated time points. 100 µg of protein was separated on a 7.5% gel and membranes were probed with specific antibodies against p-Ser-573 of IRS-2 and p-Thr-308 of Akt/PKB. For loading control membranes were stripped and reprobed with protein antibody.</p

Co-immunoprecipitation and overlay assays indicate interaction of 14-3-3 and IRS-2 upon IGF-1/insulin stimulation.

<p><i>A.</i> HEK293 cells were transiently transfected with GFP or GFP-IRS2 and after serum starvation cells were incubated with 50 ng/ml IGF-1 for 30 min or after preincubation with 100 nM wortmannin for 30 min. 400 µg total protein was used for immunoprecipitation with 14-3-3 antibody (C-17) and samples were separated on 5–15% gradient gel. Upper membrane was incubated with IRS-2 antibody, lower membrane with 14-3-3 antibody (K-19). <i>B.</i> HEK293 cells were transfected with GFP-IRS2 and stimulation was carried out after starvation for serum overnight with 50 ng/ml IGF-1 for 30 min or subsequently after preincubation with 1 µM PI-103 for 30 min. 250 µg of total protein was pulled down using GFP-Trap®. SDS-PAGE followed transfer onto nitrocellulose membranes. Overlay assay followed stripping of the membrane and reprobing with GFP antibody as loading control. <i>C.</i> Extent of interaction was quantified by scanning densitometry of blots and normalization for GFP-IRS2 serum starved condition (mean ± SEM; n = 4; *p<0.05 serum starved vs. IGF-1 or IGF-1 vs. PI-103/IGF-1). <i>D</i>. Male C57Bl/6 mice were fasted overnight and injected intravenously with 2 IU (international units) insulin. After 10 min liver was taken and 500 µg of total protein was immunoprecipitated with IRS-2 antibody. After performing overlay assay membrane was stripped and reprobed with IRS-2 antibody as loading control. Two mice of each group are shown. <i>E</i>. Densitometric analyses of 14-3-3 interaction with IRS-2. Overlay signal was normalized for total IRS-2 protein content (mean ± SEM; n = 4; *p<0.05 fasted vs. insulin). <i>F</i>. Male C57Bl/6 mice were fasted overnight, refed for 4 hours or injected intraperitoneally with insulin for 30 min. Procedure as in <i>G</i>. Four mice of each group are shown. <i>G</i>. 14-3-3 interaction with IRS-2 was quantified by scanning densitometry of immunoblots and normalization for IRS-2 protein (mean ± SEM; n = 4; *p<0.05 fasted vs. refed and insulin stimulation).</p

Ser-573 of IRS-2 is an IGF-1-dependent 14-3-3 binding site.

<p><i>A.</i> HEK293 cells were transiently transfected with GFP-IRS2, GFP-IRS2-S303A, GFP-IRS2-T401A, GFP-IRS-2-T517A, GFP-IRS2-S556A or GFP-IRS2-S573A and stimulated with 50 ng/ml IGF-1 for 30 min or after preincubation with 1 µM PI-103 30 min prior IGF-1 stimulation. 250 µg of total protein were used for GFP pulldown and overlay assay was performed. Stripping followed reprobing of the membrane with GFP antibody as loading and expression control. <i>B.</i> Transiently with GFP-IRS2 or GFP-IRS2-S573A transfected HEK293 cells were stimulated with 50 ng/ml IGF-1 alone for 30 min or after preincubation with 100 nM wortmannin for 30 min. 100 µg of total protein was used for GFP pulldown, separated on 5–15% SDS gels and overlay assay was performed. Corresponding GFP reblot as expression and loading control is shown <i>C</i>. Flp-In HEK293 cells stably expressing GFP-IRS2 or GFP-IRS2-S573A were stimulated as in <i>A</i> and cell lysis was followed by pull down of 400 µg total protein. Samples were divided into two equal volumes and separated on SDS-PAGE. One membrane was used for overlay assay, the other one was directly incubated with GFP antibody. <i>D</i>. Interaction of 14-3-3 with IRS-2 from <i>B</i> was quantified scanning densitometry of Western blots (mean ± SEM; n = 3; *p<0.05 IRS-2 wild type IGF-1 vs. IRS-2 wild type wortmannin/IGF-1).</p

With mass spectrometry identified residues with surrounding amino acids that correspond to a 14-3-3 binding motif.

<p>Shown are the identified residues indicated in bold with pS or pT and the surrounding 6 amino acids before and after the phosphorylated residue.</p

Sequence alignments and characterization of a polyclonal antibody raised in sheep against position Ser-573 on IRS-2.

<p><i>A.</i> Sequence alignment of the amino acids adjacent to the 14-3-3 binding site Ser-573 of IRS-2 in different species. <i>B</i>. Shown is the amino acid sequence surrounding Ser-573 in mouse IRS-2 and the sequence surrounding the homologue position Ser-522 in mouse/rat IRS-1. <i>C</i>. Flp-In HEK293 cells stably expressing GFP-IRS2 or GFP-IRS2-S573A were starved for serum followed by stimulation for 30 min with 50 ng/ml IGF-1 or after preincubation with 100 nM wortmannin for 30 min. 200 µg of lysate was separated on a 7.5% SDS gel, membrane was probed with p-Ser-573 antibody. Expression was checked by stripping the membrane and reprobing with GFP antibody. <i>D</i>. HEK293 cells were co-transfected transiently with mouse IRS-2/IR (insulin receptor), human IRS-2/IR and rat IRS-1/IR and stimulated with 10 nM insulin for 30 min. Membranes were incubated with p-Ser-573 antibody and reprobed with the corresponding protein antibodies. IR expression was also checked.</p

The area spanning amino acids 301–600 on IRS-2 is the 14-3-3 binding region.

<p><i>A.</i> Schematic illustration of truncated IRS-2 constructs to identify the 14-3-3 binding region. <i>B.</i> 20 µg of protein was separated on a 5–15% gradient gel and membrane was probed with GFP antibody to check expression and molecular weight of truncated IRS-2 versions. The arrow indicates a longer exposure time. <i>C.</i> HEK293 cells were transfected with either GFP-IRS2 or truncated versions of IRS-2 (GFP-IRS2-1-300, GFP-IRS2-1-600, GFP-IRS2-301-1321, GFP-IRS2-601-1321, GFP-IRS2-301-600), starved for serum overnight and stimulated for 30 min with 50 ng/ml IGF-1 or subsequently after preincubation with 1 µM PI-103 for 30 min. With GFP-Trap® 250 µg protein was pulled down and samples were subjected to overlay assay. For loading and expression control membranes were stripped and reprobed for GFP.</p

Inhibition of Akt/PKB reduces IGF-1-induced phosphorylation of serine 573 and 14-3-3 binding.

<p><i>A</i>. Flp-In HEK293 cells stably expressing GFP-IRS2 were starved for serum and incubated for 30 min with 50 ng/ml IGF-1 or 1 µM Akti-1/2 alone, or Akti-1/2 preincubation followed IGF-1 stimulation. 100 µg of total protein was separated on 7.5% SDS gels and membranes were probed with p-Ser-573 and p-Thr-308 of Akt/PKB. Membranes were stripped and reprobed with respective antibodies for detection of protein levels. <i>B</i>. Effect of Akt/PKB inhibition on serine 573 phosphorylation was assessed by scanning densitometry of blots and normalization for protein (mean ± SEM; n = 3; *p<0.05 IGF-1 vs. Akti/IGF-1). <i>C</i>. GFP pulldown from Flp-In HEK293 cells stably expressing GFP-IRS2, stimulated as in <i>A</i>. 200 µg protein was pulled down and overlay assay followed reprobing with IRS-2 antibody. 100 µg of total protein was separated on 7.5% SDS gel and membrane was checked for p-Thr-308 phosphorylation and reprobed with Akt/PKB protein antibody.</p

GST pulldown of transiently transfected IRS-2 and mutants.

<p><i>A</i> and <i>B</i>. GFP-IRS2, GFP-IRS2-S573A and GFP-IRS2-S556A/S573A were expressed transiently in HEK293 cells and cells were stimulated with 50 ng/ml IGF-1 for 30 min or after preincubation with 100 nM wortmannin for 30 min. Lysates were incubated with 2 µg GST-14-3-3β or ε for 2 hours. Samples were separated on 7.5% SDS gels and membranes were probed with GFP and GST antibodies.</p

Ser-573 influences phosphorylation of Akt/PKB.

<p><i>A</i>. HEK293 cells transiently expressing GFP-IRS2 or GFP-IRS2-S573A were stimulated with 50 ng/ml IGF-1 for the indicated time points. 40 µg of total protein was separated on 7.5% SDS gels and membranes were incubated with GFP antibody to ensure equal expression levels and with p-Thr-308 and p-Ser-473 antibody respectively. Corresponding Akt/PKB reblots are shown. <i>B</i>. Densitometric analyses of Akt/PKB phosphorylation. Black diamonds represent IRS-2 wild type, white squares IRS2-S573A mutant. Phosphorylation was normalized against total protein and IRS-2 wild type stimulated with IGF-1 for 5 min was set as 1 (mean ± SEM; n = 3; *p<0.05 IRS-2 120 min vs. S573A 120 min; IRS-2 240 min vs. S573A 240 min).</p

Sequence alignments of IRS-1 and -2 species.

<p>Protein sequences from mouse IRS-2 (NP_001074681.1), human IRS-2 (NP_003740.2), mouse IRS-1 (NP_034700.2) and human IRS-1 (NP_005535.1) were aligned. The sequences were searched for homologue residues of mouse IRS-2 that were identified by mass spectrometry and marked in bold red. Shown are only parts of the sequences that contain an identified phosphorylated serine/threonine residue. The term “numbering” indicates the amino acid position of the first amino acid in every line.</p