5' phospholipid phosphatase SHIP-2 causes protein kinase B inactivation and cell cycle arrest in glioblastoma cells

Abstract

The tumor suppressor protein PTEN is mutated in glioblastoma multiform brain tumors, resulting in deregulated signaling through the phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB) pathway, which is critical for maintaining proliferation and survival. We have examined the relative roles of the two major phospholipid products of PI3K activity, phosphatidylinositol 3,4-biphosphate [PtdIns(3,4)P2] and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], in the regulation of PKB activity in glioblastoma cells containing high levels of both of these lipids due to defective PTEN expression. Reexpression of PTEN or treatment with the PI3K inhibitor LY294002 abolished the levels of both PtdIns(3, 4)P2 and PtdIns(3,4,5)P3, reduced phosphorylation of PKB on Thr308 and Ser473, and inhibited PKB activity. Overexpression of SHIP-2 abolished the levels of PtdIns(3,4,5)P3, whereas PtdIns(3,4)P2 levels remained high. However, PKB phosphorylation and activity were reduced to the same extent as they were with PTEN expression. PTEN and SHIP-2 also significantly decreased the amount of PKB associated with cell membranes. Reduction of SHIP-2 levels using antisense oligonucleotides increased PKB activity. SHIP-2 became tyrosine phosphorylated following stimulation by growth factors, but this did not significantly alter its phosphatase activity or ability to antagonize PKB activation. Finally we found that SHIP-2, like PTEN, caused a potent cell cycle arrest in G(1) in glioblastoma cells, which is associated with an increase in the stability of expression of the cell cycle inhibitor p27(KIP1). Our results suggest that SHIP-2 plays a negative role in regulating the PI3K-PKB pathway.

FIG. 1

Effect of SHIP-2 on PKB activity. (A) Schematic of the different SHIP-2 mutants used in this study. (B) U87-MG cells were transiently transfected with HA-tagged PKB plus empty vector, WT PTEN, or different SHIP-2 constructs (in duplicate). After 48 h, the cells were lysed and exogenous PKB was IPed using an anti-HA antibody. The immune complex was assayed for PKB activity towards the synthetic peptide substrate, Crosstide. Total levels of PKB, as well as phosphorylation of PKB on Thr308 and Ser473, were assessed by immunoblotting. This experiment was repeated at least three times with consistent results.

FIG. 2

PtdIns(3,4,5)P3 phosphatase activity of WT SHIP-2 and different SHIP-2 constructs. At 48 h posttransfection, U87-MG cells expressing Xpress-tagged WT SHIP-2, SH2-SHIP-2, NPXY-SHIP-2, or SHIP-2(D608A) or cells transfected with vector alone (pcDNA3) were lysed and precipitated with nickel-charged agarose resin. The IPs were incubated with [³²P]PtdIns(3,4,5)P3 (labeled at the 3′ position) for 45 min at 37°C. Samples were extracted in chloroform-methanol and separated by TLC. The phosphatase assays were repeated four to five times with similar results.

FIG. 3

Effect of SHIP-2, PTEN, and LY294002 on phospholipid levels in vivo. (A) Activity of endogenous PKB in U87-MG cells infected with control virus or PTEN- or SHIP-2-expressing adenovirus or treated with LY294002 (20 μM) for 1 h. The results are means of three experiments, calculated as the percentage of PKB activity in the uninfected cells. (B) U87-MG cells were left untreated (data not shown), infected with control (GFP-expressing or PTEN- or SHIP-2-expressing adenovirus) for 48 h, or treated with LY294002 for 1 h. Cells were labeled with 400 μCi of [³²P]phosphorus for 2 h, and the phospholipids were extracted, deacylated, and resolved by anion exchange with an increasing gradient of NaH₂PO₄, pH 3.8. Fractions were collected and counted for ³²P radioactivity. A sample of [³²P]PtdIns(3,4,5)P3 was also resolved in order to confirm the identity of the peak representing PtdIns(3,4,5)P3. The identity of the other peaks was determined based on the position of the PtdIns(3,4,5)P3 peak and previously published lipid profiles (7). (C) Same as panel B but showing the whole profile and the 24-h time point for SHIP-2 to demonstrate loss of PtdIns(4,5)P2. (D) At 48 h postinfection with control or SHIP-2 adenovirus, U87-MG cells were lysed and SHIP-2 was IPed using anti-Flag monoclonal antibody and protein G-Sepharose. The IPs were incubated with [³²P]PtdIns(3,4)P2 (labeled at the 3′ position) or [³²P]PtdIns(4,5)P2 (labeled at the 4′ position) for 45 min at 37°C. Samples were extracted in chloroform-methanol and separated by TLC. The percentage of PtdIns(4)P produced is indicated. The phosphatase assays were repeated three times with similar results.

FIG. 4

Cellular localization of PKB in U87-MG cells overexpressing PTEN and SHIP-2. At 48 h postadenoviral infection or 1 h after addition of LY294002 (20 μM), 2.4 × 10⁴ cells were homogenized in hypotonic lysis buffer, then centrifuged at low speed to remove the nuclear fraction. After centrifugation at 100,000 × g for 30 min, the membrane fraction was resuspended in hypotonic lysis buffer containing 1% Triton X-100 and recentrifuged at 13,000 × g for 10 min to remove insoluble components. The cytosolic and membrane fractions were probed for the presence of PKB, phospho-Ser473 PKB, and PDK-1 by immunoblotting. The immunoblots are representative of five experiments. In addition, endogenous PKB was IPed from the cytosolic (c) and membrane (m) fractions and assayed for PKB activity. The PKB activity shown is from the same experiment used to derive the immunoblots of PKB and phospho-Ser473 PKB (the PDK-1 immunoblot was from a separate experiment) but is representative of all five experiments.

FIG. 5

Tyrosine phosphorylation of SHIP-2 in response to PDGF does not affect its ability to inhibit PKB activity. Primary astrocytes were infected with control, PTEN, or SHIP-2 adenoviruses for 48 h or treated with LY294002 (20 μM) for 1 h. The cells were serum-starved for 24 h, then incubated with PDGF (50 ng/ml; Boehringer Mannheim) for 15 min at 37°C prior to lysis. (A) SHIP-2 was IPed using anti-Flag antibody and protein G-Sepharose, and the IPs were probed for SHIP-2 and anti-phosphotyrosine (4G10). (B) Endogenous PKB was IPed and assayed for activity using Crosstide as the substrate. One representative experiment of three is shown.

FIG. 6

Tyrosine phosphorylation does not significantly alter SHIP-2 phosphatase activity. U87-MG cells were transiently transfected with vector alone (pcDNA3), WT SHIP-2 and pcDNA3 or WT SHIP-2 and v-Src. SHIP-2 was IPed using nickel-charged agarose resin, and assessed for 5′ inositol phosphatase activity as described for Fig. 2. In the case of the SHIP-2 plus v-Src samples, SHIP-2 was eluted from the nickel-charged agarose resin with 500 mM imidazole, and re-IPed with anti-phosphotyrosine (4G10) antibody and protein A-Sepharose before assessing phosphatase activity. Equal amounts of phosphorylated and nonphosphorylated SHIP-2, as assessed by immunoblotting, were used in the phosphatase assay. The percentage of PtdIns(3,4)P2 produced is indicated for each sample. This experiment was repeated three times with similar results.

FIG. 7

SHIP-2 causes cell cycle arrest in G₁ in U87-MG cells. (A and B) U87-MG cells were infected with control adenovirus (GFP-expressing) or SHIP-2-expressing adenovirus, or left uninfected. At 24 h after infection, either nocodazole (70 ng/ml) was added (B) or the cells were left untreated (A). After a further 18 h, the cells were fixed overnight in ethanol, stained with propidium iodide and analyzed by fluorescence-activated cell sorting. (C) U87-MG cells, infected with control adenovirus or PTEN- or SHIP-2-expressing adenovirus for 48 h, were treated with cycloheximide (200 μg/ml) for the indicated times. Equal amounts of cell lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by Western blotting for the presence of p27^KIP1. (D) Forty-eight hours after adenoviral infection, U87-MG cells, plated in 96-well plates at 10,000 cells/well, were pulsed with 3 μCi of [³H]thymidine per well for 16 h. Cells were harvested, and the incorporated radioactivity was assessed. Assays were performed in octuplicate and the results are the means ± the SD.

FIG. 7

SHIP-2 causes cell cycle arrest in G₁ in U87-MG cells. (A and B) U87-MG cells were infected with control adenovirus (GFP-expressing) or SHIP-2-expressing adenovirus, or left uninfected. At 24 h after infection, either nocodazole (70 ng/ml) was added (B) or the cells were left untreated (A). After a further 18 h, the cells were fixed overnight in ethanol, stained with propidium iodide and analyzed by fluorescence-activated cell sorting. (C) U87-MG cells, infected with control adenovirus or PTEN- or SHIP-2-expressing adenovirus for 48 h, were treated with cycloheximide (200 μg/ml) for the indicated times. Equal amounts of cell lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by Western blotting for the presence of p27^KIP1. (D) Forty-eight hours after adenoviral infection, U87-MG cells, plated in 96-well plates at 10,000 cells/well, were pulsed with 3 μCi of [³H]thymidine per well for 16 h. Cells were harvested, and the incorporated radioactivity was assessed. Assays were performed in octuplicate and the results are the means ± the SD.

FIG. 8

Reduction of endogenous SHIP-2 results in increased PKB activity. (A) U87-MG cells were transfected with oligonucleotide or Lipofectin reagent alone (control). Total RNA was extracted 24 h after transfection, and Northern blots were probed with SHIP-2 cDNA, stripped and reprobed for β-actin. 37, ISIS 30737; 38, ISIS 30738; 42, ISIS 30742; 44, ISIS 30744; 48, ISIS 30748; 50, ISIS 30750; 53, ISIS 30753; 80, ISIS 116280; 68, ISIS 116268. (B) HeLa cells were transfected with oligonucleotide ISIS 30737, ISIS 30738, or ISIS 30742 or Lipofectin alone (control). After 72 h, cell lysates, normalized for total protein, were assessed for SHIP-2 protein levels by immunoblotting. In addition, endogenous PKB was IPed and assayed for activity. These results are from one experiment representative of five.