Serine phosphorylation proximal to its phosphotyrosine binding domain inhibits insulin receptor substrate 1 function and promotes insulin resistance

Abstract

Ser/Thr phosphorylation of insulin receptor substrate (IRS) proteins negatively modulates insulin signaling. Therefore, the identification of serine sites whose phosphorylation inhibit IRS protein functions is of physiological importance. Here we mutated seven Ser sites located proximal to the phosphotyrosine binding domain of insulin receptor substrate 1 (IRS-1) (S265, S302, S325, S336, S358, S407, and S408) into Ala. When overexpressed in rat hepatoma Fao or CHO cells, the mutated IRS-1 protein in which the seven Ser sites were mutated to Ala (IRS-1(7A)), unlike wild-type IRS-1 (IRS-1(WT)), maintained its Tyr-phosphorylated active conformation after prolonged insulin treatment or when the cells were challenged with inducers of insulin resistance prior to acute insulin treatment. This was due to the ability of IRS-1(7A) to remain complexed with the insulin receptor (IR), unlike IRS-1(WT), which underwent Ser phosphorylation, resulting in its dissociation from IR. Studies of truncated forms of IRS-1 revealed that the region between amino acids 365 to 430 is a main insulin-stimulated Ser phosphorylation domain. Indeed, IRS-1 mutated only at S408, which undergoes phosphorylation in vivo, partially maintained the properties of IRS-1(7A) and conferred protection against selected inducers of insulin resistance. These findings suggest that S408 and additional Ser sites among the seven mutated Ser sites are targets for IRS-1 kinases that play a key negative regulatory role in IRS-1 function and insulin action. These sites presumably serve as points of convergence, where physiological feedback control mechanisms, which are triggered by insulin-stimulated IRS kinases, overlap with IRS kinases triggered by inducers of insulin resistance to terminate insulin signaling.

FIG. 1.

Tyr phosphorylation of IRS-1^7A and its interactions with IR. (A) CHO-T cells stably overexpressing either WT IRS-1 (CHO-T^WT) or IRS-1^7A (CHO-T^7A) at 80% confluence were deprived of serum for 16 h prior to the experiment. The cells were then incubated with insulin for the indicated times at 37°C. Cell extracts (100 μg) were resolved by SDS-PAGE (7.5% polyacrylamide) and were immunoblotted with the indicated antibodies (anti-PY [α-PY] or anti-IRS-1 [α-IRS-1] antibodies). The results of one experiment, which were representative of six experiments, are shown. (B) IRS-1 was isolated from CHO-T^WT or CHO-T^7A cells. IRS-1^WT and IRS-1^7A were phosphorylated in vitro by IR as described in Materials and Methods. The reaction mixture was subjected to immunoprecipitation with anti-IRS-1 antibodies. Immunocomplexes were resolved by SDS-PAGE (7.5% polyacrylamide) and were immunoblotted with anti-pY or anti-IRS-1 antibodies. (C) CHO-T^WT and CHO-T^7A cells were deprived of serum for 16 h prior to the experiment. The cells were stimulated with 100 nM insulin for the indicated times at 37°C. Cell extracts were prepared, and samples (1 mg) were bound to immobilized IR. IR-IRS-1 complexes were resolved by SDS-PAGE (7.5% polyacrylamide) and immunoblotted with the indicated antibodies. In parallel, samples of total cell extracts (100 μg) were resolved by SDS-PAGE (7.5% polyacrylamide) and immunoblotted with anti-IRS-1 antibodies. The results of one experiment, which were representative of two experiments performed on duplicate samples, are shown.

FIG. 2.

Overexpression of Myc-IRS-1^WT and Myc-IRS-1^7A in Fao cells. Fao cells at 70 to 80% confluence were infected with Ad-Myc-IRS-1^WT or Ad-Myc-IRS-1^7A at the indicated PFU (A) or with an insert-free adenoviral construct at 7 × 10⁷ PFU/ml (B). After 48 h, cells were left untreated (A) or treated with insulin for the indicated times (B). Cell extracts were prepared, samples were resolved by SDS-PAGE (7.5% polyacrylamide) and immunoblotted with the indicated antibodies (anti-IRS-1 [α-IRS-1], anti-myc [α-myc], or anti-PY [α-PY] antibodies). The results of one experiment, which were representative of three experiments (B) or two experiments (A), are shown.

FIG. 3.

Insulin-induced Tyr phosphorylation of IRS-1^7A in Fao cells. (A) Fao cells were infected with Ad-Myc-IRS-1^WT or Ad-Myc-IRS-1^7A at 7 × 10⁷ PFU/ml. After 48 h, the cells were starved in serum-free RPMI medium for 16 h and then treated with insulin for the indicated times. Cell extracts were prepared, and samples (1 mg) were subjected to immunoprecipitation (IP) with anti-Myc (α-Myc) antibodies. Immunocomplexes were resolved by SDS-PAGE (7.5% polyacrylamide) and immunoblotted (IB) with anti-PY (α-PY) antibodies. In parallel, samples (100 μg) of total cell extracts were resolved by SDS-PAGE (7.5% polyacrylamide) and immunoblotted with anti-Myc antibodies. (B) The bands corresponding to anti-PY/total Myc-IRS-1 were quantitated by densitometry, and the results are shown in a bar graph. Results of a representative experiment performed with duplicate samples are shown.

FIG. 4.

Effects of insulin on activation of PKB, MAPK, and GSK-3β in Fao cells infected with IRS-1^WT or IRS-1^7A. Fao cells at 70% confluence were infected with Ad-Myc-IRS-1^WT or Ad-Myc-IRS-1^7A at 6 × 10⁷ PFU/ml. After 48 h, the cells were starved in serum-free medium for 16 h and incubated with 100 nM insulin for the indicated times (5 min [5′] to 60 min [60′]) at 37°C (−, not treated with insulin). Cytosolic extracts were prepared, and the samples (100 μg) were resolved by SDS-PAGE (7.5% polyacrylamide) and immunoblotted (IB) with the indicated antibodies. Antibodies directed against phosphorylated PKB (α-p-PKB), total PKB (α-total PKB), phosphorylated MAPK (α-p-MAPK), and total MAPK (α-total MAPK) were used. The results of four independent experiments were quantified and normalized relative to the total cellular content of the protein under study (A and C). Results of a representative experiment are shown in panel B.

FIG. 5.

Effects of TPA and anisomycin on insulin-induced Tyr phosphorylation of IRS-1^7A in Fao cells. Fao cells were infected with Ad-Myc-IRS-1^WT or Ad-Myc-IRS-1^7A at 7 × 10⁷ PFU/ml. After 48 h, the cells were deprived of serum for 16 h and treated (+) for 60 min with 200 nM TPA (A), 50 ng of anisomycin per ml (B), or 1 μg of anisomycin per ml (C) or not treated (−). This procedure was followed by treatment with 100 nM insulin for 2 min (A) or for the indicated times (2, 10, or 15 min [2′, 10′, or 15′], respectively) (B and C). Cell extracts were prepared, and samples (1 mg) were subjected to immunoprecipitation (IP) with anti-Myc (α-Myc) antibodies (A). Immunocomplexes were resolved by SDS-PAGE (7.5% polyacrylamide) and immunoblotted (IB) with the indicated antibodies (e.g., anti-myc antibody [α-myc], anti-phosphorylated Ser307 antibody [α-pSer307]). Alternatively, total cell extracts (B and C) were resolved by SDS-PAGE (7.5% polyacrylamide) and immunoblotted with the indicated antibodies. The results of one experiment, which were representative of four experiments, are shown. The results of two independent experiments performed in duplicate are quantified in the bar graph in panel A.

FIG. 6.

Effect of palmitic acid on insulin-induced Tyr phosphorylation of IRS-1 mutants in Fao cells. Fao cells were infected with Ad-Myc-IRS-1^WT or Ad-Myc-IRS-1^7A at 7 × 10⁷ PFU/ml. After 24 h, the cells were deprived of serum for 12 h and incubated for 12 h with (+) or without (−) the indicated concentration of palmitic acid in serum-free medium before being stimulated with 100 nM insulin for the indicated times (2 or 10 min [2′ or 10′], respectively). Cell extracts (100 μg) were resolved by SDS-PAGE (7.5% polyacrylamide) and immunoblotted with anti-PY and anti-Myc antibodies. The results of two independent experiments done in duplicate were quantified.

FIG. 7.

In vivo phosphorylation of PH/PTB-L in response to insulin. (A) CHO-T cells were transiently transfected with pcDNA3-Myc-PH/PTB (nt 1 to 1290) or with an insert-free plasmid (control). Twenty-four hours posttransfection, the cells were deprived of serum for 16 h and treated with insulin for the indicated time periods. Cell extracts (100 μg) were resolved by SDS-PAGE (10% polyacrylamide) and immunoblotted with anti-PY (α-PY) or anti-Myc (α-Myc) antibodies. (B) In parallel, samples (100 μg, 40 μl) were incubated (+) with 3 U of calf intestine phosphatase (CIP) at 37°C for 1 h. The samples were resolved by SDS-PAGE (10% polyacrylamide) and immunoblotted with anti-Myc antibodies. The results of one experiment, which were representative of two experiments, are shown. (C) CHO^PH/PTB-L cells were starved in serum-free F12 medium for 16 h. The cells were treated for 30 min with the indicated inhibitors or not treated with an inhibitor (−) before being treated with 100 nM insulin for 30 min. Cell extracts were prepared, and samples were resolved by SDS-PAGE (10% polyacrylamide) and immunoblotted with anti-Myc antibodies. The results of one experiment, which were representative of three experiments, are shown.

FIG. 8.

Effects of insulin on serine phosphorylation of Myc-PH/PTB-L^7A. CHO-T cells were transiently transfected with constructs encoding Myc-PH/PTB-L (nt 1 to 1290), either wild type (WT) or the 7A mutant (A). Alternatively, the cells were transfected with the constructs encoding Myc-PH/PTB-L (nt 1 to 1290; aa 1 to 430) or Myc-PH/PTB-S (nt 1 to 1095; aa 1 to 365) (B). After 24 h, the cells were incubated in serum-free F12 medium for 16 h before being treated with insulin for the indicated times. Cell extracts were resolved by SDS-PAGE (10% polyacrylamide) and immunoblotted with anti-Myc (α-Myc) antibodies. The results of one experiment, which were representative of three experiments, are shown.

FIG. 9.

Effects of insulin and calyculin A on Ser phosphorylation of PH/PTB-L mutants. (A) CHO-T cells were transiently transfected with constructs expressing either Myc-PH/PTB^WT or the following PH/PTB mutants: S336A, S407A, S408A, S407A/S408A (S407/8A), and S336A/S407A/S408A (S3A). After 24 h, the cells were deprived of serum for 16 h and treated with insulin for the indicated times. Cell extracts were resolved by SDS-PAGE (10% polyacrylamide) and immunoblotted with anti-Myc (α-myc) antibodies. The results of one experiment, which were representative of two or three experiments, are shown. (B) CHO-T cells were transiently transfected with pcDNA3-myc-PH/PTB^WT or pcDNA3-myc-PH/PTB^S408A. After 24 h, the cells were starved for 16 h and treated either with 100 nM insulin for 30 min or with 25 nM calyculin A for 60 min or left alone (−). Cell extracts were resolved by SDS-PAGE (10% polyacrylamide) and immunoblotted with anti-Myc antibodies. The results of one experiment, which were representative of two experiments, are shown.

FIG. 10.

Phosphorylation of S408 of IRS-1 in cultured Fao cells. (A) Fao cells were infected with Adeno-myc-IRS-1 (wild type [WT]), the 7A mutant, or the S408A mutant at a titer of 4 × 10⁷ to 6 × 10⁷ PFU/ml in order to obtain comparable expression levels of the proteins under study (compare with Fig. 11). After 48 h, the cells were starved for 16 h. The medium was removed, and the cells were incubated with 100 nM insulin or without insulin (−) for the indicated times (5, 15, or 60 min [5′, 15′, or 60′], respectively). (B) Alternatively, the cells were incubated with insulin for 2 or 60 min or incubated with sphingomyelinase (SMase) or calyculin A as indicated (+). Cytosolic extracts (100 μg) were resolved by SDS-PAGE (7.5% polyacrylamide) and immunoblotted (IB) with anti-phospho-Ser408 (α-p-Ser408) or anti-PY (α-PY) antibodies as indicated.

FIG. 11.

Effects of TPA and anisomycin on insulin-stimulated Tyr phosphorylation of IRS-1^WT, IRS-1^7A, and IRS-1^408A. Fao cells were infected with Adeno-myc-IRS-1 (wild type [WT]), the 7A mutant, or the S408A mutant at a titer of 4 × 10⁷ to 6 × 10⁷ PFU/ml. After 48 h, the cells were deprived of serum for 16 h and treated for 60 min (60′) with 200 nM TPA (A) or 50 ng of anisomycin per ml (B), followed by treatment with 100 nM insulin for the indicated times (2 min [2′] or 60 min [60′]). Cell extracts were prepared, and samples (1 mg) were subjected to immunoprecipitation (IP) with anti-Myc antibodies. Immunocomplexes were resolved by SDS-PAGE (7.5% polyacrylamide) and immunoblotted (IB) with anti-PY (α-PY) or anti-IRS-1 (α-IRS-1) antibodies. (C) Quantitation of the results of two independent experiments done in duplicate is presented.