Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110alpha and are disrupted in oncogenic p85 mutants

Abstract

We previously proposed a model of Class IA PI3K regulation in which p85 inhibition of p110alpha requires (i) an inhibitory contact between the p85 nSH2 domain and the p110alpha helical domain, and (ii) a contact between the p85 nSH2 and iSH2 domains that orients the nSH2 so as to inhibit p110alpha. We proposed that oncogenic truncations of p85 fail to inhibit p110 due to a loss of the iSH2-nSH2 contact. However, we now find that within the context of a minimal regulatory fragment of p85 (the nSH2-iSH2 fragment, termed p85ni), the nSH2 domain rotates much more freely (tau(c) approximately 12.7 ns) than it could if it were interacting rigidly with the iSH2 domain. These data are not compatible with our previous model. We therefore tested an alternative model in which oncogenic p85 truncations destabilize an interface between the p110alpha C2 domain (residue N345) and the p85 iSH2 domain (residues D560 and N564). p85ni-D560K/N564K shows reduced inhibition of p110alpha, similar to the truncated p85ni-572(STOP). Conversely, wild-type p85ni poorly inhibits p110alphaN345K. Strikingly, the p110alphaN345K mutant is inhibited to the same extent by the wild-type or truncated p85ni, suggesting that mutation of p110alpha-N345 is not additive with the p85ni-572(STOP) mutation. Similarly, the D560K/N564K mutation is not additive with the p85ni-572(STOP) mutant for downstream signaling or cellular transformation. Thus, our data suggests that mutations at the C2-iSH2 domain contact and truncations of the iSH2 domain, which are found in human tumors, both act by disrupting the C2-iSH2 domain interface.

Fig. 1.

Disruption of C2-iSH2 domain contacts is redundant with oncogenic truncation of p85ni. (A) Two micrograms of purified GST or wild-type or mutant GST-p85ni was immobilized on glutathione-Sepharose beads, and incubated with lysates from HEK293T cells transfected with myc-p110α or myc-p110αN345K. The beads were washed and bound proteins were blotted with anti-myc antibody. (B) Recombinant wild-type or mutant p110α was incubated for 1 h at 4 °C without or with wild-type or mutant p85ni and assayed for lipid activity at 22 °C as described. The data are the mean ± SEM of triplicate determinations from three experiments.

Fig. 2.

Truncation mutants and C2-iSH2 contact mutants of p85 are redundant for activation of downstream signaling. (A) HEK293T cells were transiently transfected with myc-p110α and wild-type or mutant p85ni. Cells were incubated without or with insulin, lysed, and blotted for p85 and p110α, for total and phosphorylated forms of Akt, and for phosphorylated 4EBP1, and PRAS40 as shown. The data are representative of five separate experiments (B) Cells were transfected and stimulated as above and blotted for pSer9-GSK3, p85ni and p110α. The data are representative of three separate experiments. (C) HEK293E cells were transiently transfected with HA-S6K1, myc-p110α and wild-type or mutant p85ni. Anti-HA immunoprecipitates were blotted for total and phosphorylated S6K1 and for myc-p110α (which binds to HA-p85ni). Lysates were blotted for p85ni. The ratio of pT389/total S6K1 was determined using a LICOR Odyssey imaging system, and is the mean ± SD from three experiments. (D) HEK293T cells were transiently transfected with HA-Akt, myc-p110α and wild-type or mutant HA-p85ni. Anti-HA immunoprecipitates were blotted for total and phosphorylated Akt and for myc-p110α (which binds to HA-p85ni). Lysates were blotted for p85ni. The data are representative of two separate experiments.

Fig. 3.

Effects of truncation mutants and C2-iSH2 contact mutants of p85 on PIP3 production in vivo. HeLa cells were transfected with myc-p110α and wild-type or mutant p85ni. The cells were rendered quiescent overnight, fixed, and stained with anti-PIP3 antibody. Cells were imaged using a Nikon 60 × 1.4 NA objective and a Roper cooled-CCD camera. Quantitation of PIP3 was performed as described in ref. . The data are the mean ± SD from two independent experiments.

Fig. 4.

Effects of truncation mutants and C2-iSH2 contact mutants of p85 on transformation. (A) NIH 3T3 cells were transiently transfected with myc-p110α and wild-type or mutant p85ni. The cells were plated in soft agar as described and colonies were counted after 3 weeks. Colony counts were normalized to the number produced by cells expressing p85ni572^STOP. The data are the mean ± SEM from three experiments. (B) NIH 3T3 cells were transiently transfected with p110α-N345K and wild-type or mutant p85ni. Colony counts were normalized to the number produced by cells expressing p85ni572^STOP. The data are the mean ± SD from 2–3 experiments.