Genetic mutations cause primary immunodeficiencies (PIDs), which predispose to infections. Here we describe Activated PI3K-δ Syndrome (APDS), a PID associated with a dominant gain-offunction mutation E1021K in the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated

The p110α catalytic subunit (PIK3CA) is one of the most frequently mutated genes in cancer. We have examined the activation of the wild-type p110α/p85α and a spectrum of oncogenic mutants using hydrogen/deuterium exchange mass spectrometry (HDX-MS). We find that for the wild-type enzyme, the natural transition from an inactive cytosolic conformation to an activated form on membranes entails four distinct events. Analysis of oncogenic mutations shows that all up-regulate the enzyme by enhancing one or more of these dynamic events. We provide the first insight into the activation mechanism by mutations in the linker between the adapter-binding domain (ABD) and the Ras-binding domain (RBD) (G106V and G118D). These mutations, which are common in endometrial cancers, enhance two of the natural activation events: movement of the ABD and ABD-RBD linker relative to the rest of the catalytic subunit and breaking the C2-iSH2 interface on binding membranes. C2 domain mutants (N345K and C420R) also mimic these events, even in the absence of membranes. A third event is breaking the nSH2-helical domain contact caused by phosphotyrosine-containing peptides binding to the enzyme, which is mimicked by a helical domain mutation (E545K). Interaction of the C lobe of the kinase domain with membranes is the fourth activation event, and is potentiated by kinase domain mutations (e.g., H1047R). All mutations increased lipid binding and basal activity, even mutants distant from the membrane surface. Our results elucidate a unifying mechanism in which diverse PIK3CA mutations stimulate lipid kinase activity by facilitating allosteric motions required for catalysis on membranes.H/D exchange | PI3 kinase | signaling T he oncogenic potential of phosphoinositide 3-kinases (PI3Ks) was first established when it was shown that an activated form of the PI3K isoform p110α (v-P3K) was encoded by an avian sarcoma virus and that this viral gene and its cellular counterpart can cause cell transformation (1). The advent of large-scale sequencing established that cancer-linked somatic mutations of p110α are among the most common mutations associated with human tumors (2-4). Tumor-associated mutations appear throughout the sequence of p110α (Fig. 1A). The majority of these are in hotspots in the helical (E545K) or kinase (H1047R) domains. The synergy of these two mutations in activating p110α suggests they act independently, and both structural and functional studies indicate they work by distinct mechanisms (5-7). The presence of both of these mutations in colorectal tumors, compared with the singly mutated form, strongly correlates with decreased survival (8). However, it is not clear how other mutations throughout the sequence activate p110α, nor is it clear whether there is any unifying principle governing all of the oncogenic mutations. It is becoming increasingly apparent that the p110α mutations do not act alone in tumor development. Mice with the most common p110α mutation (H1047R) expressed in ovaries develop hyperplasias, but not tumors, wher...

SummaryPhosphoinositide 3-kinases (PI3Ks) are essential for cell growth, migration, and survival. The structure of a p110β/p85β complex identifies an inhibitory function for the C-terminal SH2 domain (cSH2) of the p85 regulatory subunit. Mutagenesis of a cSH2 contact residue activates downstream signaling in cells. This inhibitory contact ties up the C-terminal region of the p110β catalytic subunit, which is essential for lipid kinase activity. In vitro, p110β basal activity is tightly restrained by contacts with three p85 domains: the cSH2, nSH2, and iSH2. RTK phosphopeptides relieve inhibition by nSH2 and cSH2 using completely different mechanisms. The binding site for the RTK's pYXXM motif is exposed on the cSH2, requiring an extended RTK motif to reach and disrupt the inhibitory contact with p110β. This contrasts with the nSH2 where the pY-binding site itself forms the inhibitory contact. This establishes an unusual mechanism by which p85 SH2 domains contribute to RTK signaling specificities.