Hashem A. Dbouk

Somatic mutations in p85α promote tumorigenesis through class IA PI3K activation

Members of the mammalian phosphoinositide-3-OH kinase (PI3K) family of proteins are critical regulators of various cellular process including cell survival, growth, proliferation, and motility. Oncogenic activating mutations in the p110alpha catalytic subunit of the heterodimeric p110/p85 PI3K enzyme are frequent in human cancers. Here we show the presence of frequent mutations in p85alpha in colon cancer, a majority of which occurs in the inter-Src homology-2 (iSH2) domain. These mutations uncouple and retain p85alphas p110-stabilizing activity, while abrogating its p110-inhibitory activity. The p85alpha mutants promote cell survival, AKT activation, anchorage-independent cell growth, and oncogenesis in a p110-dependent manner.

Connexins: a myriad of functions extending beyond assembly of gap junction channels.

Connexins constitute a large family of trans-membrane proteins that allow intercellular communication and the transfer of ions and small signaling molecules between cells. Recent studies have revealed complex translational and post-translational mechanisms that regulate connexin synthesis, maturation, membrane transport and degradation that in turn modulate gap junction intercellular communication. With the growing myriad of connexin interacting proteins, including cytoskeletal elements, junctional proteins, and enzymes, gap junctions are now perceived, not only as channels between neighboring cells, but as signaling complexes that regulate cell function and transformation. Connexins have also been shown to form functional hemichannels and have roles altogether independent of channel functions, where they exert their effects on proliferation and other aspects of life and death of the cell through mostly-undefined mechanisms. This review provides an updated overview of current knowledge of connexins and their interacting proteins, and it describes connexin modulation in disease and tumorigenesis.

Class IA PI3K p110β Subunit Promotes Autophagy through Rab5 Small GTPase in Response to Growth Factor Limitation

Autophagy is an evolutionarily conserved membrane trafficking process. Induction of autophagy in response to nutrient limitation or cellular stress occurs by similar mechanisms in organisms from yeast to mammals. Unlike yeast, metazoan cells rely more on growth factor signaling for a wide variety of cellular activities including nutrient uptake. How growth factor availability regulates autophagy is poorly understood. Here we show that, upon growth factor limitation, the p110β catalytic subunit of the class IA phosphoinositide 3-kinases (PI3Ks) dissociates from growth factor receptor complexes and increases its interaction with the small GTPase Rab5. This p110β-Rab5 association maintains Rab5 in its guanosine triphosphate (GTP)-bound state and enhances the Rab5-Vps34 interaction that promotes autophagy. p110β mutants that fail to interact with Rab5 are defective in autophagy promotion. Hence, in mammalian cells, p110β acts as a molecular sensor for growth factor availability and induces autophagy by activating a Rab5-mediated signaling cascade.

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.

Characterization of a Tumor-Associated Activating Mutation of the p110β PI 3-Kinase

The PI3-kinase pathway is commonly activated in tumors, most often by loss of PTEN lipid phosphatase activity or the amplification or mutation of p110α. Oncogenic mutants have commonly been found in p110α, but rarely in any of the other catalytic subunits of class I PI3-kinases. We here characterize a p110β helical domain mutation, E633K, first identified in a Her2-positive breast cancer. The mutation increases basal p110β activity, but does not affect activation of p85/p110β dimers by phosphopeptides or Gβγ. Expression of the mutant causes increases in Akt and S6K1 activation, transformation, chemotaxis, proliferation and survival in low serum. E633 is conserved among class I PI3 Ks, and its mutation in p110β is also activating. Interestingly, the E633K mutant occurs near a region that interacts with membranes in activated PI 3-kinases, and its mutation abrogates the requirement for an intact Ras-binding domain in p110β-mediated transformation. We propose that the E633K mutant activates p110β by enhancing its basal association with membranes. This study presents the first analysis of an activating oncogenic mutation of p110β.

G protein-coupled receptors and the regulation of autophagy.

Autophagy is an important catabolic cellular process that eliminates damaged and unnecessary cytoplasmic proteins and organelles. Basal autophagy occurs during normal physiological conditions, but the activity of this process can be significantly altered in human diseases. Thus, defining the regulatory inputs and signals that control autophagy is essential. Nutrients are key modulators of autophagy. Although autophagy is generally accepted to be regulated in a cell-autonomous fashion, recent studies suggest that nutrients can modulate autophagy in a systemic manner by inducing the secretion of hormones and neurotransmitters that regulate G protein-coupled receptors (GPCRs). Emerging studies show that GPCRs also regulate autophagy by directly detecting extracellular nutrients. We review the role of GPCRs in autophagy regulation, highlighting their potential as therapeutic drug targets.

Actions of the protein kinase WNK1 on endothelial cells are differentially mediated by its substrate kinases OSR1 and SPAK.

Significance With no lysine (K) (WNK)1, which is mutated in pseudohypoaldosteronism type II (PHAII) autosomal dominant hypertension, is a large, complex enzyme essential for development, blood pressure control, and many cellular functions. WNK1 signaling is largely mediated by two downstream protein kinases, OSR1 (oxidative stress responsive 1) and SPAK (STE20/SPS1-related proline-, alanine-rich kinase), sometimes considered redundant in terms of WNK1 function. This study characterizes an essential contribution of WNK1 in angiogenesis and presents a mechanism of clear bifurcation in WNK1-dependent functions between OSR1 and SPAK, with SPAK regulating WNK1 effects on proliferation and OSR1 mediating effects on invasion. Our work also identifies a previously unidentified link between WNK1 and the zinc-finger transcription factor Slug, with implications in cancer biology. This study also suggests potential mechanisms for cardiovascular defects associated with PHAII. The with no lysine (K) (WNK) family of enzymes is best known for control of blood pressure through regulation of the function and membrane localization of ion cotransporters. In mice, global as well as endothelial-specific WNK1 gene disruption results in embryonic lethality due to angiogenic and cardiovascular defects. WNK1−/− embryos can be rescued by endothelial-specific expression of a constitutively active form of the WNK1 substrate protein kinase OSR1 (oxidative stress responsive 1). Using human umbilical vein endothelial cells (HUVECs), we explored mechanisms underlying the requirement of WNK1–OSR1 signaling for vascular development. WNK1 is required for cord formation in HUVECs, but the actions of the two major WNK1 effectors, OSR1 and its close relative SPAK (STE20/SPS1-related proline-, alanine-rich kinase), are distinct. SPAK is important for endothelial cell proliferation, whereas OSR1 is required for HUVEC chemotaxis and invasion. We also identified the zinc-finger transcription factor Slug in WNK1-mediated control of endothelial functions. Our study identifies a separation of functions for the WNK1-activated protein kinases OSR1 and SPAK in mediating proliferation, invasion, and gene expression in endothelial cells and an unanticipated link between WNK1 and Slug that is important for angiogenesis.

Novel approaches to inhibitor design for the p110β phosphoinositide 3-kinase

Phosphoinositide (PI) 3-kinases are essential regulators of cellular proliferation, survival, metabolism, and motility that are frequently dysregulated in human disease. The design of inhibitors to target the PI 3-kinase/mTOR pathway is a major area of investigation by both academic laboratories and the pharmaceutical industry. This review focuses on the Class IA PI 3-kinase p110β, which plays a unique role in thrombogenesis and in the growth of tumors with deletion or loss-of-function mutation of the Phosphatase and Tensin Homolog (PTEN) lipid phosphatase. Several p110β-selective inhibitors that target the ATP-binding site in the kinase domain have been identified. However, recent discoveries regarding the regulatory mechanisms that control p110β activity suggest alternative strategies by which to disrupt signaling by this PI 3-kinase isoform. This review summarizes the current status of p110β-specific inhibitors and discusses how these new insights into p110 regulation might be used to devise novel pharmacological inhibitors.

Hypertension: the missing WNKs

The With no Lysine [K] (WNK) family of enzymes are central in the regulation of blood pressure. WNKs have been implicated in hereditary hypertension disorders, mainly through control of the activity and levels of ion cotransporters and channels. Actions of WNKs in the kidney have been heavily investigated, and recent studies have provided insight into not only the regulation of these enzymes but also how mutations in WNKs and their interacting partners contribute to hypertensive disorders. Defining the roles of WNKs in the cardiovascular system will provide clues about additional mechanisms by which WNKs can regulate blood pressure. This review summarizes recent developments in the regulation of the WNK signaling cascade and its role in regulation of blood pressure.

Identification of the Rab5 Binding Site in p110β: Assays for PI3Kβ Binding to Rab5

Isoform-specific signaling by Class IA PI 3-kinases depends in part on the interactions between distinct catalytic subunits and upstream regulatory proteins. From among the class IA catalytic subunits (p110α, p110β, and p110δ), p110β has unique properties. Unlike the other family members, p110β directly binds to Gβγ subunits, downstream from activated G-protein coupled receptors, and to activated Rab5. Furthermore, the Ras-binding domain (RBD) of p110β binds to Rac and Cdc42 but not to Ras. Defining mutations that specifically disrupt these regulatory interactions is critical for defining their role in p110β signaling. This chapter describes the approach that was used to identify the Rab5 binding site in p110β, and discusses methods for the analysis of p110β-Rab5 interactions.