Oscar Vadas

Phosphoinositide 3-Kinase δ Gene Mutation Predisposes to Respiratory Infection and Airway Damage

Answers from Exomes Exome sequencing, which targets only the protein-coding regions of the genome, has the potential to identify the underlying genetic causes of rare inherited diseases. Angulo et al. (p. 866, published online 17 October; see Perspective by Conley and Fruman) performed exome sequencing of individuals from seven unrelated families with severe, recurrent respiratory infections. The patients carried the same mutation in the gene coding for the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ). The mutation caused aberrant activation of this kinase, which plays a key role in immune cell signaling. Drugs inhibiting PI3Kδ are already in clinical trials for other disorders. Gene sequencing of unrelated patients with recurrent airway infections identifies a common underlying mutation. [Also see Perspective by Conley and Fruman] Genetic mutations cause primary immunodeficiencies (PIDs) that predispose to infections. Here, we describe activated PI3K-δ syndrome (APDS), a PID associated with a dominant gain-of-function mutation in which lysine replaced glutamic acid at residue 1021 (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 families, but not among 3346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased immunoglobulin M, and reduced immunoglobulin G2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110δ. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110δ inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, which suggested a therapeutic approach for patients with APDS.

Structural Basis for Activation and Inhibition of Class I Phosphoinositide 3-Kinases

The regulatory interactions between PI3Ks and their binding partners could be exploited for therapies. Phosphoinositide 3-kinases (PI3Ks) phosphorylate a hydroxyl group on phosphoinositide lipids. The 3-phosphorylated inositol lipids act as membrane-resident second messengers, recruiting downstream signaling components that control cell growth, proliferation, differentiation, survival, and motility. The best studied of the PI3Ks, the class I enzymes, are heterodimers with a catalytic and a regulatory subunit and have been implicated in many human diseases. Class I PI3Ks can be stimulated downstream of receptor tyrosine kinases and heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors as well as small G proteins of the Ras superfamily. Both the catalytic and regulatory subunits have a multidomain organization. Crystal structures, biochemical analysis, and oncogenic mutations in PI3Ks have shown that interdomain interactions are not static but undergo regulated conformational cycles, resulting in enzyme activation or inhibition. This Review, which contains 7 figures and 104 references, highlights the molecular details of how their regulatory partners selectively inhibit and activate PI3K isoforms. Phosphoinositide 3-kinases (PI3Ks) are implicated in a broad spectrum of cellular activities, such as growth, proliferation, differentiation, migration, and metabolism. Activation of class I PI3Ks by mutation or overexpression correlates with the development and maintenance of various human cancers. These PI3Ks are heterodimers, and the activity of the catalytic subunits is tightly controlled by the associated regulatory subunits. Although the same p85 regulatory subunits associate with all class IA PI3Ks, the functional outcome depends on the isotype of the catalytic subunit. New PI3K partners that affect the signaling by the PI3K heterodimers have been uncovered, including phosphate and tensin homolog (PTEN), cyclic adenosine monophosphate–dependent protein kinase (PKA), and nonstructural protein 1. Interactions with PI3K regulators modulate the intrinsic membrane affinity and either the rate of phosphoryl transfer or product release. Crystal structures for the class I and class III PI3Ks in complexes with associated regulators and inhibitors have contributed to developing isoform-specific inhibitors and have shed light on the numerous regulatory mechanisms controlling PI3K activation and inhibition.

Structure of Lipid Kinase p110β/p85β Elucidates an Unusual SH2-Domain-Mediated Inhibitory Mechanism

Summary Phosphoinositide 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 RTKs 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.

Molecular determinants of PI3Kγ-mediated activation downstream of G-protein-coupled receptors (GPCRs).

Significance Pathology of many diseases depends on signaling by phosphoinositide 3-kinase gamma (PI3Kγ), the lipid kinase that is exquisitely adapted to activation downstream of heterotrimeric G-protein–coupled receptors (GPCRs). Using hydrogen–deuterium exchange mass spectrometry, we demonstrate the mechanism by which the p110γ catalytic subunit and its p101 regulatory subunit interact with G-protein Gβγ heterodimers liberated upon GPCR activation. We identify residues in both p110γ and p101 interacting with Gβγ heterodimers on membranes. This enabled us to generate Gβγ-insensitive p110γ and p101 variants that eliminate activation of PI3Kγ by Gβγs without affecting the enzyme’s basal activity or its activation by the small G-protein Ras. Ablating the interaction of PI3Kγ with Gβγ heterodimers attenuates signaling, chemotaxis, and transformation driven by a GPCR agonist in cell lines. Phosphoinositide 3-kinase gamma (PI3Kγ) has profound roles downstream of G-protein–coupled receptors in inflammation, cardiac function, and tumor progression. To gain insight into how the enzyme’s activity is shaped by association with its p101 adaptor subunit, lipid membranes, and Gβγ heterodimers, we mapped these regulatory interactions using hydrogen–deuterium exchange mass spectrometry. We identify residues in both the p110γ and p101 subunits that contribute critical interactions with Gβγ heterodimers, leading to PI3Kγ activation. Mutating Gβγ-interaction sites of either p110γ or p101 ablates G-protein–coupled receptor-mediated signaling to p110γ/p101 in cells and severely affects chemotaxis and cell transformation induced by PI3Kγ overexpression. Hydrogen–deuterium exchange mass spectrometry shows that association with the p101 regulatory subunit causes substantial protection of the RBD-C2 linker as well as the helical domain of p110γ. Lipid interaction massively exposes that same helical site, which is then stabilized by Gβγ. Membrane-elicited conformational change of the helical domain could help prepare the enzyme for Gβγ binding. Our studies and others identify the helical domain of the class I PI3Ks as a hub for diverse regulatory interactions that include the p101, p87 (also known as p84), and p85 adaptor subunits; Rab5 and Gβγ heterodimers; and the β-adrenergic receptor kinase.

Dynamics of the Phosphoinositide 3-Kinase p110δ Interaction with p85α and Membranes Reveals Aspects of Regulation Distinct from p110α

Summary Phosphoinositide 3-kinase δ is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85α. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110δ with p85α. Both nSH2 and cSH2 domains of p85α contribute to full inhibition of p110δ, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110δ. The cSH2 inhibits p110β and p110δ, but not p110α, implying that p110α is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes.

Abstract IA02: G-proteins regulating PI3Ks and PI4KIIIβ regulating a G-protein

Dysregulation of phosphoinositide levels is associated with many cancer phenotypes. A variety of mechanisms activate phosphoinositide 3-kinases (PI3Ks), including association with G-proteins. Increased activity of PI 4-kinases (PI4Ks) also has been associated with cancer phenotypes. Although PI4Ks associate with G proteins, the relationship with enzyme activity is more complex. To understand regulation of these enzymes, we have used hydrogen/deuterium exchange mass spectrometry (HDX-MS) in conjunction with X-ray crystallography. The method enables us to explore not only three-dimensional structures but also the dynamics of the activation process and to map key regulatory interactions with G-proteins. PI3Ks and PI4Ks, along with PI3K-related protein kinases such as TOR are members of a closely related family that all have a common core structure, consisting of a helical domain closely associated with a kinase domain. The helical domains have important roles both in stabilizing the structures and forming regulatory interactions. For example, PI3Kγ is activated by associating with Gβγ heterodimers released upon activation of GPCRs, and HDX-MS shows how the helical domain of the enzyme interacts with the p101 regulatory subunit and with Gβγ heterodimers. When the p110γ/p101 complex interacts with lipid membranes a conformational change takes place that prepares the helical domain for association with prenylated Gβγ heterodimers on membranes. Type IIIβ PI4K (PI4KB) synthesizes PI4P on Golgi membranes. The enzyme is important for processes that require rapid expansion and remodeling of PI4P-containing membranes, including replication of a range of viruses. Its activity has also been associated with decreased cell-cell adhesion in invasive cancer cell lines. Using both HDX-MS and X-ray crystallography we have shown how the helical domain of PI4KB interacts with the Ras superfamily G protein Rab11. In this unique interaction, PI4KB grasps the Rab11 but leaves the switch regions of the G protein unencumbered and free to interact with Rab11 effectors. Consistent with this type of interaction, PI4K can interact with complexes of Rab11 bound to both GDP and GTP. Furthermore, the structure of the complex of the Rab11 effector FIP3 bound to the Rab11-PI4KB complex shows that PI4KB does not perturb Rab11 effector binding and suggests that PI4KB may be able to recruit both Rab11 and its downstream effectors to Golgi membranes. Citation Format: John E. Burke, Alison J. Inglis, Oscar Vadas, Olga Perisic, Glenn R. Masson, Stephen H. McLaughlin, Roger L. Williams. G-proteins regulating PI3Ks and PI4KIIIβ regulating a G-protein. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA02.