p87 and p101 subunits are distinct regulators determining class IB phosphoinositide 3-kinase (PI3K) specificity

Abstract

Class IB phosphoinositide 3-kinase γ (PI3Kγ) comprises a single catalytic p110γ subunit, which binds to two non-catalytic subunits, p87 or p101, and controls a plethora of fundamental cellular responses. The non-catalytic subunits are assumed to be redundant adaptors for Gβγ enabling G-protein-coupled receptor-mediated regulation of PI3Kγ. Growing experimental data provide contradictory evidence. To elucidate the roles of the non-catalytic subunits in determining the specificity of PI3Kγ, we tested the impact of p87 and p101 in heterodimeric p87-p110γ and p101-p110γ complexes on the modulation of PI3Kγ activity in vitro and in living cells. RT-PCR, biochemical, and imaging data provide four lines of evidence: (i) specific expression patterns of p87 and p101, (ii) up-regulation of p101, providing the basis to consider p87 as a protein forming a constitutively and p101 as a protein forming an inducibly expressed PI3Kγ, (iii) differences in basal and stimulated enzymatic activities, and (iv) differences in complex stability, all indicating apparent diversity within class IB PI3Kγ. In conclusion, expression and activities of PI3Kγ are modified differently by p87 and p101 in vitro and in living cells, arguing for specific regulatory roles of the non-catalytic subunits in the differentiation of PI3Kγ signaling pathways.

Keywords: G-proteins; Gβγ p101; Phosphatidylinositol 3-Kinase; Phosphatidylinositol Signaling; Phosphoinositide 3-Kinase γ (PI3Kγ); Phospholipid; Signal Transduction; p87.

FIGURE 1.

Expression of PI3Kγ regulatory subunits in human tissues. Human adult normal tissue total protein lysates obtained from BioChain were analyzed for expression of p87 and p101. A, representative blots showing expression of p87 and p101 in various human tissues. 50 μg of protein was loaded per lane for brain, lung, and rectum, and 20 μg of protein was loaded for thymus. B, p87 antibody raised against a human p87 peptide in rabbit was characterized by peptide blocking against human p87 overexpressed in HEK cells.

FIGURE 2.

Expression of PI3Kγ regulatory subunits upon serum stimulation in human peripheral blood mononuclear cells. The cells were isolated from human blood samples and cultured in the presence of fetal calf serum. At different time points the cells were harvested and analyzed by Western blotting. A, representative blots showing expression of p87, p101, and Hsp90. B, the histogram represents statistical evaluation of the expression of p87 and p101, normalized to Hsp90 (n = 12).

FIGURE 3.

Lipid kinase activities of phospholipid vesicle-associated class I_B PI3Ks. A, an experimental model of phospholipid vesicles recruiting equal amounts of PI3Kγ variants. To obtain equal recruitment of PI3Kγ variants, we designed phospholipid vesicles where p110γ and p87–p110γ were recruited via interaction with anionic phosphatidylserine (PS) and p101–p110γ via interaction with Gβ₁γ₂. B and C, lipid kinase activities of PI3Kγ variants equally associated with phospholipid vesicles in the presence and absence of 100 nm Gβ₁γ₂ were examined as described previously (35). To achieve equal association of PI3Kγ variants, phospholipid vesicles were prepared and incubated with 32 nm concentrations of enzyme as detailed under “Experimental Procedures.” The upper panels show representative immunoblots of sedimented phospholipid vesicles probed with specific anti-p110γ antiserum (top rows) and autoradiographs demonstrating formation of ³²P-labeled PtdIns(3,4,5)P₃ under identical experimental conditions (bottom rows). The histograms represent statistical evaluations (mean values ± S.E.) of four (A) or three (B) independent experiments. p101–p110γ displays higher basal and Gβ₁γ₂-induced lipid kinase activities as compared with p110γ and p87–p110γ, whereas the association of each PI3Kγ variants with phospholipid vesicles was comparable.

FIGURE 4.

Reconstitution of heterodimeric PI3Kγ. A, recombinant class I_A p85α subunit, individual subunits of PI3Kγ, and heterodimeric variants were expressed in and purified from Sf9 cells. Proteins were subjected to SDS/PAGE (10% acrylamide) and analyzed by Coomassie Brilliant Blue staining. Molecular mass is given in kDa. B, lipid kinase activity of monomeric p110γ subunit in the presence of 200 nm Gβ₁γ₂ was significantly increased after coincubation with either p87 or p101 and reached the level of intensity of coexpressed p87–p110γ or p101–p110γ, respectively. The assays were performed as described previously (35). For the reconstitution of heterodimeric PI3Kγ variants, p110γ was incubated with a 10-fold molar excess of p87 or p101. Coincubation of p110γ with class I_A p85α served as a negative control. The data shown here are mean values ± S.E. (n = 3). C, Gβ₁γ₂-mediated recruitment of p110γ, p101–p110γ, and p110γ coincubated with p101 to phospholipid vesicles. Pulldown assays were performed in the presence of 32 nm p110γ or p110γ/p101 and 32 nm p101 subunit as detailed previously (35). Sedimented phospholipid vesicles were subjected to SDS/PAGE (10% acrylamide) followed by immunoblotting using specific anti-p110γ antiserum. D and E, stimulation of lipid kinase activity and autophosphorylation of p110γ (○), p101–p110γ (●), and p110γ coincubated with p101 (▾) in response to increasing concentrations of Gβ₁γ₂ were studied. The assays were conducted as detailed previously (35) with some modifications. The lipid kinase assays (D) were performed in the presence of 1.6 nm p110γ or p101–p110γ and 16 nm p101, whereas autophosphorylation (E) was studied in the presence of 6.4 nm p110γ or p101–p110γ and 32 nm p101. Gβ₁γ₂-induced activation of PI3Kγ is illustrated as a percentage of the maximal stimulation of coexpressed heterodimeric p101–p110γ. The lipid kinase activity of monomeric p110γ subunit is known to be sensitive to Gβ₁γ₂-induced stimulation in vitro (26, 34, 35). The almost complete loss of p110γ sensitivity to Gβ₁γ₂ was due to the higher concentrations of non-ionic detergent, polyoxyethylene-10-lauryl ether, in the assay. The data shown here represent the averages of two independent experiments.

FIGURE 5.

Different stabilities of heterodimeric PI3Kγ complexes. A, heterodimeric p87–p110γ and p101–p110γ complexes were incubated with increasing concentrations of individually purified p87 or p101 as indicated above for 30 min at 4 °C, and the lipid kinase activities were estimated in the presence of 200 nm Gβ₁γ₂ as described previously (35). The assays are represented as the percentage of p101–p110γ stimulation by 200 nm Gβ₁γ₂. Coincubation of PI3Kγ with class I_A p85α subunit served as a negative control. The data shown here are the averages of three independent experiments. B, purified heterodimeric p87–p110γ and p101–p110γ complexes were immunoprecipitated (IP) using specific anti-p110γ antibody as detailed under “Experimental Procedures.” Interaction of the antibody with catalytic p110γ subunit leads to significant release of p87 from the heterodimeric p87–p110γ complex, whereas the heterodimeric state of p101–p110γ is unaltered. C, interaction of p87 and p101 with recombinant purified PI3Kγ variants. Incubation of proteins and copurification using Ni²⁺-Sepharose^TM 6 Fast Flow beads (GE Healthcare) was performed as detailed under “Experimental Procedures.” Aliquots of the eluates were separated by SDS/PAGE (10% acrylamide) and analyzed by immunoblotting using antibodies raised against p87, p101, and p110γ. p101 associated with p110γ in heterodimeric p87–p110γ complexes. D, p87–p110γ releasing the non-catalytic p87 subunit. Incubation of PI3Kγ variants with non-catalytic subunits and filtration through Amicon® Ultra-4 MWCO 100 kDa (Millipore) centrifugal filtration devices was done as detailed under “Experimental Procedures.” Aliquots of inputs (5 μl) and filtrates (15 μl) were subjected to SDS/PAGE (10% acrylamide) followed by immunoblotting using specific antibodies against p87 and p101. p87 was observed in the filtrate of the centrifugal filtration device indicating dissociation of the heterodimeric p87–p110γ complex.

FIGURE 6.

Lipid kinase activities of constitutively membrane-associated class I_B PI3Ks in living cells. A–C, HEK 293 cells were transfected with plasmids encoding PI3Kγ (p110γ_CAAX, p110γ_CAAX with CFP-p85α, p87–p110γ_CAAX, and p101–p110γ_CAAX) and GFP-Grp1_PH. After starvation for 18 h the cells were imaged (confocal laser-scanning microscope slices of 1 μm) and then lysed. A, cellular distribution of GFP-Grp1_PH in PI3K-expressing cells. Shown are representative cells (confocal laser-scanning microscope slices of 1 μm) from three independent experiments (scale bar, 10 μm). B, protein expression in HEK 293 cells evaluated by immunoblotting using anti-p110γ, anti-p87, anti-p101, and anti-GFP (CFP) antibodies. Anti-GFP (CFP) was used to detect CFP-p85α and GFP-Grp1_PH. C, the scatter plot represents the statistical evaluation of the membrane translocation of the PtdIns(3,4,5)P₃ sensor, GFP-Grp1_PH, in the corresponding experiments. The data shown here are mean values ± S.E. of three independent experiments comprising a total of 15–18 cells per condition. D, generation of PtdIns(3,4,5)P₃ requires catalytic activity of the constitutively membrane-associated p110γ_CAAX. HEK 293 cells were transfected with plasmids encoding kinase-defective YFP-p110γ(K833R)_CAAX mutant and GFP-Grp1_PH. Shown here are representative images of cells starved for 18 h (confocal laser-scanning microscope slices of 1 μm) from three independent experiments (scale bar, 10 μm). Although YFP-p110γ(K833R)_CAAX is localized at the plasma membrane, loss of its catalytic activity impairs PtdIns(3,4,5)P₃ synthesis and, hence, translocation of its sensor, GFP-Grp1_PH, to the plasma membrane. E and F, activity of constitutively membrane-associated p110γ_CAAX is not affected by endogenous Gβ₁γ₂ or Ras. E, HEK 293 cells were transfected with plasmids encoding PI3Kγ (p110γ_CAAX, p87–p110γ_CAAX and p101–p110γ_CAAX), GFP-Grp1_PH, β-adrenergic receptor kinase (βArk)-CFP and FLAG-neurofibromin 1 (NF1). After starvation for 18 h the cells were imaged (confocal laser-scanning microscope slices of 1 μm) and then lysed. The cell lysates were analyzed by immunoblotting for the expression of the plasmids using anti-p110γ, anti-FLAG, and anti-GFP (CFP) antibodies. F, the histogram represents statistical evaluation of the membrane translocation of GFP-Grp1_PH. The data represent the mean values ± S.E. of three independent experiments comprising a total of 15–18 cells per condition. Although p87 displays some stimulation of membrane-associated p110γ_CAAX, this effect was not statistically significant. The activity of p110γ_CAAX was significantly enhanced by p101.