Oncogenic transformation induced by the p110beta, -gamma, and -delta isoforms of class I phosphoinositide 3-kinase

Abstract

Class I phosphoinositide 3-kinase contains four isoforms of the catalytic subunit, p110alpha, -beta, -gamma, and -delta. At physiological levels of expression, the wild-type p110alpha isoform lacks oncogenic potential, but gain-of-function mutations and overexpression of p110alpha are correlated with oncogenicity. The p110beta, -gamma, and -delta isoforms induce transformation of cultured cells as wild-type proteins. This oncogenic potential requires kinase activity and can be suppressed by the target of rapamycin inhibitor rapamycin. The p110delta isoform constitutively activates the Akt signaling pathway; p110gamma activates Akt only in the presence of serum. The isoforms differ in their requirements for upstream signaling. The transforming activity of the p110gamma isoform depends on rat sarcoma viral oncogene homolog (Ras) binding; preliminary data suggest the same for p110beta and indicate Ras-independent oncogenic potential of p110delta. The surprising oncogenic potential of the wild-type non-alpha isoforms of class I phosphoinositide 3-kinase may explain the dearth of cancer-specific mutations in these proteins, because these non-alpha isoforms could contribute to the oncogenic phenotype of the cell by differential expression.

Fig. 1.

Cellular transformation induced by class I p110 isoforms in the absence or presence of a myristylation signal. CEF were transfected with RCAS vectors encoding the wild-type or myristylated p110α (chicken), p110β (human), p110δ (human), and p110γ (human) isoforms by using various amounts of DNA. Each six-well plate was transfected with 500 (Upper Left), 200 (Upper Middle), 100 (Upper Right), 50 (Lower Left), 20 (Lower Middle), or 0 ng(Lower Right) of DNA. The cultures were overlaid with nutrient agar and fixed and stained with crystal violet on day 10.

Fig. 2.

Kinase activity is required for transformation induced by the H1047R mutant of p110α and by wild-type p110δ or p110γ. CEF on 10-cm plates were transfected with RCAS vectors encoding wild-type, lipid, and protein kinase-negative mutants, and mutants that are lipid kinase negative only. The p110γ-R1076H mutant, which carries the histidine residue corresponding to H1047 in the nononcogenic wild-type p110α, is included as additional control. The R1076H mutation does not affect the native oncogenic potential of p110γ.

Fig. 3.

Activation of Akt by p110δ and p110γ. (A) Serum-starved conditions reveal constitutive activation of Akt by p110δ. CEF that were transfected with empty RCAS vector, wild-type p110α (human), the H1047R mutant of p110α, or wild-type p110β, p110δ, p110γ, or Vps34p (class III PI3K) were serum starved for 42 h. Cells were then lysed, and the proteins were separated on a 3–8% gradient SDS/polyacrylamide gel. The transferred blot was probed with antibodies as indicated. (B) Activation of Akt in wild-type and mutant p110γ-expressing cells in media containing 3% serum. CEF that were transfected with wild-type, kinase-inactive mutants (D964A and lipid-kinase inactive), or Ras binding-inactive mutant (K255E) of p110γ were overlaid with nutrient agar containing 3% serum for 10 days. The agar was then removed, the cells were harvested and lysed, and the proteins were analyzed as described above.

Fig. 4.

Differential effect of Ras-binding mutations on the transforming activity of p110 isoforms. CEF were transfected with RCAS vectors encoding the wild-type or Ras-binding mutants (p110α-H1047R/K227E, p110β-K230E, p110δ-K223E, and p110γ-K255E) of p110 isoforms. The cultures were overlaid with nutrient agar and fixed and stained with crystal violet on day 10.

Fig. 5.

Rapamycin inhibits transformation induced by p110 isoforms. CEF were inoculated (100 μl) with 10, 100, or 1,000-fold dilutions of retroviral vectors expressing the indicated p110 isoforms or v-Jun. The cells were overlaid with nutrient agar supplemented with 1 ng/ml rapamycin (+) or solvent (–) only. After 2 weeks, the cultures were fixed and stained with crystal violet.