Insulin-proliferative and metabolic actions in L6_Δ43 cells. (A) L6, L6_hIR, and L6_Δ43 myoblasts (clones C1, C2, and C8) were stimulated with 100 nM insulin for 10 min as indicated and then solubilized as described in Materials and Methods. Cell lysates (50 μg of protein/sample) were subjected to Western blotting (WB) with phospho-ERK (p-ERK1/2), followed by reblotting with ERK (ERK 1/2) antibodies. Filters were revealed by ECL and autoradiography. The autoradiographs shown are representative of four independent experiments. (B to D) The cells were incubated with 100 nM insulin for 12 h (B) or 10 min (C and D) and assayed for thymidine incorporation into nuclei, 2-deoxy-d-glucose uptake, or glycogen synthase activity as described in Materials and Methods. The bars represent the mean values plus standard deviations of data from three (B), five (C), and four (D) independent experiments, each in triplicate.

Insulin effect on PDK-1 tyrosine phosphorylation. (A and B) L6_hIR myoblasts (A) and L6 myoblasts expressing hemagglutinin (HA)-tagged wild-type PDK-1or the indicated PDK-1 mutants (B) were preincubated in the absence or the presence of LY294002 (100 μM for 15 min) or PP1 or PP2 (5 μM for 2 h). Insulin (100 nM) was added to the incubation medium for another 10 min. The cells were then solubilized, and equal amounts of proteins (200 μg) were precipitated (I.P.) with PDK-1 or HA antibodies, as indicated, and Western blotted (WB) with either phosphotyrosine (p-Tyr) or PDK-1 antibodies as described in Materials and Methods. The blots were revealed by ECL and autoradiography. The autoradiographs shown are representative of four (A) and three (B) independent experiments. (C and D) L6 myoblasts expressing either the wild type or the mutant PDK-1 or transfected with a specific PDK-1 siRNA were incubated with 100 nM insulin for 10 min and assayed for 2-deoxy-d-glucose uptake (left) and glycogen synthase activity (right) as described in Materials and Methods. The bars represent the mean values plus standard deviations of data from three independent experiments, each in triplicate. (E and F) L6_hIR myoblasts were stimulated with insulin as in panel A and solubilized, and cell proteins (200 μg) were precipitated with PDK-1 antibodies, followed by Western blotting with phosphotyrosine (p-Tyr) or PDK-1 antibodies (E) or with specific pTyr₉ or pTyr_373/376 antibodies (F), as indicated. The blots were revealed by ECL and autoradiography. The autoradiographs shown are representative of four (E) and three (F) independent experiments.

PDK-1 interaction with the insulin receptor in L6 cells and in vivo. (A) L6, L6_hIR, and L6_Δ43 myoblasts were preincubated with 100 μM LY294002 for 15 min; 100 nM insulin was added to the incubation medium for an additional 10 min, where indicated. The cells were then solubilized, and equal amounts of proteins (200 μg) were immunoprecipitated (I.P.) with IR α-subunit antibodies, followed by Western blotting (WB) with PDK-1 or IR α-subunit antibodies. (B) Male C57/BL6 mice fasted for 8 h and then were subjected to intraperitoneal fast-acting insulin injection (0.15 IU/g). After 5 min, the animals were sacrificed, followed by collection and processing of gastrocnemius muscles and perigonadal fat pads as described in Materials and Methods. The tissues were lysed, precipitated with insulin receptor antibodies, and blotted with either PDK-1 or phosphotyrosine (p-Tyr) antibodies. For control, aliquots of the lysates were also directly blotted with IR α-subunit or PDK-1 antibodies, as indicated. The blots were revealed by ECL and autoradiography. The autoradiograph shown is representative of four independent experiments. (C) (Left) wheat germ agglutinin-purified insulin receptor preparations (25 μg of proteins) were incubated with 100 nM insulin for 30 min as indicated and then blotted on nitrocellulose filters. Alternatively (right), immunoprecipitated insulin receptor α-subunits from insulin-stimulated L6_hIR and L6_Δ43 cells were used. Filters were probed with biotinylated recombinant PDK-1 and revealed by autoradiography. The autoradiographs shown are representative of four independent experiments. (D) L6_hIR myoblasts were transiently transfected with a cDNA encoding the p110 catalytic subunit of PI 3-K and incubated with 100 nM insulin for 10 min, as indicated. The cells were then solubilized and immunoprecipitated with insulin receptor antibodies, followed by blotting with either PDK-1 or insulin receptor antibodies (top and middle). For control, aliquots of the lysates were directly blotted with PI 3-K antibodies (bottom). The blots were revealed by ECL and autoradiography. The autoradiograph shown is representative of three independent experiments. (E) Immunoprecipitated human insulin receptors were blotted on nitrocellulose filters, and the blots were incubated for another 10 min with either the phosphorylated Δ43 or the Y→F peptide, as indicated. The blots were then probed with biotinylated recombinant PDK-1 and revealed by autoradiography. The autoradiograph shown is representative of three independent experiments.

Expression of FLAG43 peptide in L6 cells. (A) L6_hIR cells were transiently transfected with 5 μg of the pcDNA3-FLAG expression vector encoding amino acids 1340 to 1382 of the insulin receptor COOH terminus (FLAG43) or with the empty vector (FLAG). The cells were stimulated with 100 nM insulin for 10 min as indicated and then solubilized, and equal amounts of proteins (50 μg) were blotted (WB) with FLAG antibodies. (B) Aliquots of the same lysates (200 μg of proteins) were immunoprecipitated (I.P.) with PDK-1 antibodies and blotted with FLAG antibodies. To ensure equal amounts of PDK-1 in each sample, filters were reblotted with PDK-1 antibodies. (C) Alternatively, the cell lysates were immunoprecipitated with IR α-subunit antibodies, followed by blotting with PDK-1 antibodies. The filters were further blotted with IR α-subunit antibodies to ensure equal amounts of insulin receptor in each sample. The filters were revealed by ECL and autoradiography. The autoradiographs shown are representative of four (A and C) and three (B) independent experiments.

Expression of hIR_Δ43 in L6 skeletal-muscle cells. (A) Untransfected L6 myoblasts (L6) and myoblasts expressing 3 × 10³ wild-type (Wt) (L6_hIR) (6) or carboxy-terminally truncated insulin receptors (L6_Δ43; clone A3, 4 × 10³ insulin receptors/cell; clones C1, C2, and C8, 3.1 × 10³ insulin receptors/cell) were solubilized, and equal amounts of proteins (50 μg/sample) were analyzed by Western blotting (WB) using IR α-subunit antibodies as described in Materials and Methods. The blots were revealed by ECL and autoradiography. (B) The cells were stimulated with 100 nM insulin for 5 min, as indicated, and lysates (200 μg of protein/sample) were immunoprecipitated with insulin receptor α-subunit antibodies, followed by blotting with phosphotyrosine antibodies (p-Tyr). (C) Alternatively, the cell lysates were immunoprecipitated with IRS-1 or IRS-2 antibodies, followed by blotting with phosphotyrosine, IRS-1, or IRS-2 antibodies, as indicated. The blots were revealed by ECL and autoradiography. The autoradiographs shown are representative of four (A) and three (B and C) independent experiments.

hIR_Δ43 signaling in L6 skeletal-muscle cells. (A and B) L6, L6_hIR, and L6_Δ43 myoblasts (C1, C2, and C8 clones) were stimulated with 100 nM insulin for 10 min (B) or the indicated times (A) and then assayed for PI 3-K or PDK-1 activities as described in Materials and Methods. The bars represent the mean values plus standard deviations of data from three (A) and four (B) independent experiments, in triplicate. (B, top) Alternatively, the cells were lysed and Western blotted (WB) with specific phospho-Ser241 (p-Ser₂₄₁) antibodies. The blots were revealed by ECL and autoradiography. The blot shown is representative of three independent experiments. (C) The cells were exposed to 100 nM insulin for 10 min and then solubilized as described in Materials and Methods. Cell lysates (50 μg of protein/sample) were blotted with specific phospho-threonine₃₀₈-PKB antibodies (p-Thr₃₀₈-PKB) and then reblotted with phospho-Ser₄₇₃-PKB (p-Ser₄₇₃-PKB) antibodies. To ensure equal Akt transfer, the filters were further blotted with PKB antibodies (PKB). The filters were revealed by ECL and autoradiography. The autoradiographs shown are representative of four independent experiments.

Insulin effect on PDK-1 and Akt plasma membrane translocation in L6_Δ43 cells. L6, L6_hIR, and L6_Δ43 myoblasts were incubated with 100 nM insulin for the indicated times, followed by cell subfractionation as described in Materials and Methods. Equal amounts of proteins (35 μg) from the purified plasma membrane (Mem) or the cytosolic (Cyt) fractions were then assayed by Western blotting (WB) with PDK-1 (A) or PKB (C) antibodies. The blots were revealed by ECL and autoradiography and subjected to densitometric analysis. The bars represent the mean values plus standard deviations of data from four (B) and three (D) independent experiments. Representative autoradiographs are also shown.

In vitro phosphorylation of PDK-1 by insulin receptor. (A) L6_hIR and L6_Δ43 myoblasts were solubilized, and insulin receptors were partially purified by wheat germ agglutinin chromatography. Equal amounts of purified receptors were then stimulated with 100 nM insulin for 10 min, as indicated, and incubated with immunoprecipitated PDK-1 preparations from untransfected L6 myoblasts as described in Materials and Methods. The proteins were blotted with phosphotyrosine antibodies (P-Tyr), and the identities of IR and PDK-1 bands were assessed by M_r and confirmed by reblotting the filters with IR and PDK-1 antibodies, as also shown on the bottom, to ensure equal amounts of PDK-1 in each assay. The blots were revealed by ECL and autoradiography. The autoradiographs shown are representative of four independent experiments. WB, Western blotting; Wt, wild type. (B) WGA-purified hIRs were stimulated with 100 nM insulin and incubated with recombinant PDK-1 in the presence of the indicated concentrations of either the Δ43 or the Y→F peptide, as indicated. The proteins were blotted with phosphotyrosine antibodies and revealed by ECL and autoradiography. Quantitation of the IR beta subunit (open symbols) and PDK-1 bands (filled symbols) was achieved by laser densitometry. The data points are the means ± standard deviations of four independent experiments. Mut, mutant; WT, wild type.

Effect of FLAG43 peptide on PDK-1 activity and membrane translocation. (A) L6 and L6_hIR cells were transiently transfected with 5 μg of the pcDNA3-FLAG expression vector encoding amino acids 1340 to 1382 of the insulin receptor COOH terminus (FLAG43) or with the empty vector (FLAG). The cells were stimulated with 100 nM insulin for 10 min as indicated and then solubilized, and equal amounts of proteins (200 μg) were immunoprecipitated (I.P.) with PDK-1 antibodies and blotted (WB) with phosphotyrosine antibodies (p-Tyr). To ensure equal amounts of PDK-1 in each sample, filters were reblotted with PDK-1 antibodies. (B) Alternatively, aliquots of the cell lysates (50 μg of proteins) were directly blotted with specific antibodies to either the Ser₄₃₇ or the Thr₃₀₈ residue of PKB (p-Ser₄₃₇- and p-Thr₃₀₈-PKB). For controls, the lysates were also reblotted with PKB antibodies. Filters were revealed by ECL and autoradiography. The autoradiographs shown are representative of three (A) and four (B) independent experiments. (C) FLAG43- and FLAG-transfected cells were incubated with 100 nM insulin for 10 min and assayed for 2-deoxy-d-glucose uptake as described in Materials and Methods. The bars represent the mean values plus standard deviations (SD) of data from three independent experiments, each in triplicate. (D and E) Cells transfected with FLAG43 or the empty vector were stimulated with 100 nM insulin for the indicated times, followed by cell subfractionation as described in Materials and Methods. Equal amounts of protein from the membrane (35 μg) or the cytosolic (35 μg) fraction were then assayed by Western blotting with PDK-1 antibodies. The blots were revealed by ECL and autoradiography and quantitated by densitometric analysis. The autoradiographs shown in panel D are from a representative experiment. The bars (E) represent the means ± SD of four independent experiments.