early reports of cancer-specific mutations in p85α, a regulatory subunit of class I PI3K. Such mutations gained high significance by recent comprehensive genomic analyses of glioblastomas. Approximately 9% of these tumors harbor a mutation in p85α.The mutations cluster in the inter-SH2 domain of p85α,involving residues that interact VX-222 VCH222 with the C2 domain of the catalytic subunit p110α. The iSH2–C2 domain interaction has an inhibitory effect on enzyme activity, and the mutations in the iSH2 domain of p85α could weaken this interaction and release the inhibition of PI3K activity. A similar mechanism has been proposed for the gain-of-function mutations in the helical domain of p110α that alleviate an inhibitory interaction with the N-terminal SH2 domain of p85α. We have studied mutations in p85α.
Most of these were identified in a genomic characterization of glioblastoma PD0325901 and map to the iSH2 domain of p85; one was an engineered mutation that maps to the nSH2 domain of p85. These mutations show oncogenic potency in cell culture and elevated levels of downstream signaling and operate through the p110αisoform of the catalytic subunit of class I PI3K. Our observations extend recent studies of the p85α mutants using different cell systems by providing quantitative data on the oncogenic potency of the mutations and by presenting evidence that suggests a unique role of p110α for the p85 mutation-induced gain of function in PI3K activity. Results Cancer-Derived Mutations of p85 Induce Oncogenic Transformation and Increase Cell Proliferation. Fig.
1 lists recently identified p85 mutations and their map positions in the p85 sequence. The changes caused by the mutations in the protein sequence are summarized in Fig. S1. Most of the mutations are located in the iSH2 domain of p85. With the exception of the K379E mutation, they were first seen in human glioblastoma. To date, K379E has not been detected in human cancers; it is an engineered mutation designed to weaken the interaction between the nSH2 domain of p85 and the helical domain of p110α involving p110αresidue E545 by disrupting an inhibitory salt bridge. The mutant p85 proteins were expressed in chicken embryo fibroblasts with the replication-competent avian sarcoma retroviral vector , and expression was verified by Western blotting. The vector-mediated expression of exogenous p85 resulted in elevated levels of endogenous p110α.
After approximately 2 wk of incubation, foci of transformed cells appeared in the mutant-transfected cultures but not on plates transfected with WT p85. The mutant p85 proteins showed different efficiencies of transformation , as defined by the number of foci induced per microgram of transfected DNA. Two of the p85 deletion mutants, KS459delN and DKRMNS560del, displayed a particularly highEOT,comparable to that of theH1047Rmutant of p110α, which was used as a positive control. The nSH2 mutant, K379E, also belongs to this highly transforming category.R574fs and T576del transformed CEF with an intermediate efficiency, and the EOT of the remaining mutants was an order of magnitude lower than that of the highly transforming mutants.
These differences in EOT were maintained when the cell cultures were cotransfected with WT human p110α and therefore probably reflect inherent properties of the p85 mutants. These data suggest that cancer-derived mutants of p85 have oncogenic activity, which probably reflects a mutation-mediated gain of function in the catalytic subunit. The transforming mutants of p85 also conferred increased replicative ability to the host cells. Fig.4 documents this enhanced proliferation for the highly transforming mutant KS459delN. This enhance