SKIP negatively regulates insulin-induced GLUT4 translocation and membrane ruffle formation

Abstract

Skeletal muscle and kidney enriched inositol phosphatase (SKIP) is an inositol polyphosphate 5-phosphatase that hydrolyzes phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] to downregulate intracellular levels. In this study, we show that SKIP inhibits phosphoinositide 3-kinase signaling in insulin-stimulated CHO cells. Ectopic expression of SKIP did not inhibit insulin-induced PI(3,4,5)P3 generation but did rapidly decrease insulin-induced intracellular PI(3,4,5)P3 levels compared with those in control cells. Further, insulin-induced phosphorylation of some downstream targets such as Akt and p70 S6 kinase was markedly inhibited by the ectopic expression of SKIP, whereas phosphorylation of mitogen-activated protein kinase was not. In contrast, downregulation of intracellular SKIP levels by antisense oligonucleotides dramatically enhanced Akt (protein kinase B) phosphorylation in response to insulin, suggesting that endogenous SKIP downregulates insulin signaling. SKIP also markedly inhibited GLUT4 translocation and membrane ruffle formation. We conclude that SKIP preferentially regulates glucose transport and actin cytoskeletal rearrangement among a variety of PI(3,4,5)P3 downstream events.

FIG. 1.

Western blot analysis of CHO cells expressing GFP and SKIP. (A) Schematic representation of GFP (Ad-GFP), Myc-tagged wild-type SKIP protein (Ad-SKIP-WT), and phosphatase-negative mutant SKIP (Ad-SKIP-Δphos), which lacks the catalytic motifs of the wild type. (B and C) CHO cells were either left uninfected (0) or infected with increasing concentrations of recombinant adenovirus expressing GFP (B) or wild-type SKIP or its phosphatase-negative mutant (C) for 24 h. Cell lysates were prepared 48 h later as described in Materials and Methods and were immunoblotted with anti-GFP (B), anti-Myc (α-Myc) (C), or anti-SKIP (α-SKIP) (B and C) antibodies. m.o.i, multiplicity of infection.

FIG. 2.

Effect of SKIP on intracellular phosphoinositide levels. Control cells (A to D) or CHO cells infected with Ad-SKIP-WT (E to H) or Ad-SKIP-Δphos (I to L) were labeled with [³²P]orthophosphate for 2 h. Serum-deprived cells were either unstimulated (A, E, and I) or stimulated for 1 min (B, F, and J), 3 min (C, G, and K), or 5 min (D, H, and L) with insulin. Phospholipids were extracted, deacylated, and applied to a strong anion exchange column as described in Materials and Methods. The insets show the region of PI(3,4,5)P₃ elution from the column. Profiles were normalized to the total counts in each run. Values on the left of all panels indicate disintegrations per minute (10²).

FIG. 3.

Effect of SKIP on insulin-signaling proteins. (A) Insulin-stimulated tyrosine phosphorylation of IRS-1 and IRβ. CHO cells were infected with recombinant adenovirus and then stimulated with insulin (100 nM) for 10 min. Whole-cell lysates were immunoprecipitated with anti-phosphotyrosine antibodies and then blotted with anti-IRS-1 or anti-IRβ antibodies. (B) Effect of SKIP on insulin-stimulated PI 3-kinase activity in CHO cells. Adenovirus-infected cells were stimulated with insulin (100 nM) for 10 min. Cell lysates were immunoprecipitated with anti-IRS-1 antibodies and assayed for PI 3-kinase activity with PI as the substrate. The products were resolved by thin-layer chromatography and visualized by autoradiography.

FIG. 4.

SKIP involvement in insulin-induced Akt phosphorylation in CHO cells. (A) SKIP inhibition of Akt phosphorylation in CHO cells. CHO cells were infected with Ad-GFP, Ad-SKIP-WT, or Ad-SKIP-Δphos for 24 h. Control cells were uninfected. Serum-deprived cells were stimulated with insulin (100 nM) for up to 20 min. Lysates were immunoblotted with phospho-specific Akt antibodies (upper and middle panels) or anti-Akt antibody (lower panel). The phospho-specific antibody only detects Akt phosphorylated at Thr-308 or Ser-473. The Western blots shown are representative of three independent experiments. (B) Akt activation by SKIP inhibition. CHO cells were transfected with antisense, sense, or control oligonucleotides for 48 h. Serum-starved cells were stimulated with insulin (100 nM) for up to 30 min. Cells were lysed, and equal amounts of lysate were blotted with anti-PTEN antibody, anti-SKIP antibody, phospho-specific Akt antibodies, or anti-Akt antibody.

FIG. 5.

SKIP inhibition of p70 S6 kinase but not of MAP kinase. (A) Effect of SKIP on insulin-stimulated p70 S6 kinase activation. CHO cells that were uninfected (control) or infected with Ad-GFP, Ad-SKIP-WT, or Ad-SKIP-Δphos were incubated in the presence or absence of insulin for the indicated times. Cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with either phospho-specific p70 S6 kinase antibody, which detects p70 S6 kinase only when phosphorylated at Thr-389 (upper panel), or phospho-p70 S6 kinase antibody, which detects p70 S6 kinase phosphorylated at Thr-421 and Ser-424 (middle panel). The same lysates were blotted with anti-p70 S6 kinase antibody (lower panel). SKIP inhibited insulin-induced phosphorylation at Thr-389, whereas phosphorylation at Thr-421 and Ser-424 was only slightly inhibited. (B) Effect of SKIP on MAP kinase phosphorylation. CHO cells infected with adenovirus were either unstimulated or stimulated with insulin (100 nM) for 10 to 30 min. Cell lysates were immunoblotted with anti-phospho-p44 or anti-phospho-p42 MAP kinase (upper panel) or anti-MAP kinase antibody (lower panel). Neither wild-type SKIP nor its phosphatase-negative mutant inhibited MAP kinase activation after insulin stimulation for 30 min.

FIG. 6.

Effect of SKIP on insulin-induced calcium mobilization. CHO cells (A) and CHO cells expressing wild-type SKIP (B) or phosphatase-negative mutant SKIP (C) were loaded with fura-2 AM. Cells were stimulated with insulin (100 nM) 100 s into each experiment. The experiments were performed in triplicate, and the graph in each panel depicts a representative run.

FIG. 7.

Inhibition by SKIP of insulin-induced membrane ruffling in CHO cells. (A) Control CHO cells and those infected with adenovirus expressing GFP, wild-type SKIP, or phosphatase-negative mutant SKIP for 24 h were serum starved and stimulated with insulin (100 nM) for 10 min and stained with anti-Myc antibody (SKIP) or rhodamine-phalloidin (Actin). Membrane ruffle formation in SKIP-expressing cells was significantly inhibited. (B) CHO cells expressing GFP and SKIP were stimulated with insulin, and the numbers of cells exhibiting membrane ruffling were counted. Wild-type SKIP substantially inhibited insulin-induced membrane ruffling. Open bars represent the basal rate of membrane ruffle formation in the absence of insulin, whereas the solid bars represent values in the presence of insulin (100 nM). Results are means of five separate experiments.

FIG. 8.

Inhibition of GLUT4 translocation in SKIP-expressing cells. (A) CHO cells constitutively expressing GLUT4 were infected with adenovirus expressing wild-type SKIP or its phosphatase-negative mutant for 24 h. Cells were starved overnight and stimulated with insulin (100 nM) for 20 min. Cells were stained with anti-GLUT4 antibody and anti-SKIP antibody. The number of SKIP-positive cells exhibiting GLUT4 translocation was counted. SKIP expression inhibited insulin-induced GLUT4 translocation by approximately 75%. (B) CHO cells expressing GFP and SKIP were stimulated with insulin for 10 or 20 min, and the number of cells showing GLUT4 translocation was counted. Results are means of five separate experiments.

FIG. 9.

Inhibition by SKIP of insulin-induced glucose transport and glycogen synthesis in L6 myoblasts. (A) Insulin-induced glucose transport in adenovirus-infected cells. L6 myoblasts expressing GFP or SKIP were stimulated with insulin for 10 or 20 min. (B) Insulin-induced glucose incorporation into glycogen in transfected cells. Cells expressing GFP or SKIP were stimulated with insulin for 1 h. Results are means of three separate experiments.

FIG. 10.

Insulin-induced translocation of SKIP from intracellular compartments to the plasma membrane. CHO cells were serum starved for 16 h and then stimulated with insulin for 10 min. Cells were fixed and immunostained with anti-SKIP antibody (α-SKIP) (A and D) or anti-α-mannosidase (α-α-Mannosidase), a trans-Golgi marker (B and E). The combined images are also shown (C and F). Localization of SKIP to the trans-Golgi reticulum under resting conditions and translocation to the plasma membrane upon insulin stimulation can be observed.