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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.

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.

Phosphoinositide 3-kinases (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 on 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.

The Phosphoinositide 3-Kinase (PI3K) pathway has been extensively studied in tumors due its roles in promoting cellular growth and proliferation [1]. The most common PI3K mutations are in the PIK3CA gene encoding the p110α catalytic subunit, including the “hotspot” activating mutations E545K and H1047R that can lead to constitutive signaling of the pathway [1]. Consequently, activation of the serine/threonine kinase Akt can promote proliferative and cell growth pathways through regulation of mTOR and other intermediates [1]. In addition to driving tumorigenesis, hotspot PIK3CA mutations have also been shown to drive a wide spectrum of non-malignant over-growth disorders collectively termed the PIK3CA-Related Overgrowth Spectrum [2]. More recently, mutations in PIK3CA have been identified in venous malformations (VMs) [3], the most frequent form of vascular malformations with a frequency of about 1 in 5000 people in the general population [4]. These painful and often disfiguring lesions are characterized by endothelial cell overgrowth, loss of supporting mural cells, and a disorganized extracellular matrix resulting in dilated and distended vessels in a variety of tissues, with common occurrence in the cutaneous layer of the skin [4].

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.

Received: December 24, 2010 , Accepted: December 30, 2010 , Published: December 30, 2010 // The PI3K pathway is frequently activated in tumors, most commonly through p110α mutation or PTEN deletion. In contrast to p110α, p110β is oncogenic when over-expressed in the wild-type state, suggesting that its regulation by p85 is different than that of p110α. In this perspective, we summarize recent data concerning the regulation of p110β, which shows that wild-type p110β acts like an oncogenic mutant of p110α. We also discuss the significance of this altered regulation in tumor models of PTEN deletion, as well as the potential implications of the unique p110β regulation on GPCR-driven tumorigenesis.

Class I PI3-kinases signal downstream of receptor tyrosine kinases and G protein-coupled receptors and have been implicated in tumorigenesis. Although the oncogenic potential of the PI3-kinase subunit p110α requires its mutational activation, other p110 isoforms can induce transformation when overexpressed in the wild-type state. In wild-type p110α, N345 in the C2 domain forms hydrogen bonds with D560 and N564 in the inter-SH2 (iSH2) domain of p85, and mutations of p110α or p85 that disrupt this interface lead to increased basal activity and transformation. Sequence analysis reveals that N345 in p110α aligns with K342 in p110β. This difference makes wild-type p110β analogous to a previously described oncogenic mutant, p110α-N345K. We now show that p110β is inhibited by p85 to a lesser extent than p110α and is not differentially inhibited by wild-type p85 versus p85 mutants that disrupt the C2–iSH2 domain interface. Similar results were seen in soft agar and focus-formation assays, where p110β was similar to p110α-N345K in transforming potential. Inhibition of p110β by p85 was enhanced by a K342N mutation in p110β, which led to decreased activity in vitro, decreased basal Akt and ribosomal protein S6 kinase (S6K1) activation, and decreased transformation in NIH 3T3 cells. Moreover, unlike wild-type p110β, p110β-K342N was differentially regulated by wild-type and mutant p85, suggesting that the inhibitory C2–iSH2 interface is functional in this mutant. This study shows that the enhanced transforming potential of p110β is the result of its decreased inhibition by p85, due to the disruption of an inhibitory C2–iSH2 domain interface.

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.

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.

Phosphoinositide 3 kinase gamma (PI3Kgamma) 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 Gbetagamma heterodimers we mapped these regulatory interactions using hydrogen deuterium exchange mass spectrometry. We identify residues in both the p110gamma and p101 subunits that contribute critical interactions with Gbetagamma heterodimers leading to PI3Kgamma activation. Mutating Gbetagamma interaction sites of either p110gamma or p101 ablates G protein coupled receptor mediated signaling to p110gamma/p101 in cells and severely affects chemotaxis and cell transformation induced by PI3Kgamma 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 p110gamma. Lipid interaction massively exposes that same helical site which is then stabilized by Gbetagamma. Membrane elicited conformational change of the helical domain could help prepare the enzyme for Gbetagamma 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 Gbetagamma heterodimers; and the beta adrenergic receptor 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.

Connexins (Cx), gap junction (GJ) proteins, are regarded as tumor suppressors, and Cx43 expression is often down regulated in breast tumors. We assessed the effect of Cx43 over-expression in 2D and 3D cultures of two breast adenocarcinoma cell lines: MCF-7 and MDA-MB-231. While Cx43 over-expression decreased proliferation of 2D and 3D cultures of MCF-7 by 56% and 80% respectively, MDA-MB-231 growth was not altered in 2D cultures, but exhibited 35% reduction in 3D cultures. C-terminus truncated Cx43 did not alter proliferation. Untransfected MCF-7 cells formed spherical aggregates in 3D cultures, and MDA-MB-231 cells formed stellar aggregates. However, MCF-7 cells over-expressing Cx43 formed smaller sized clusters and Cx43 expressing MDA-MB-231 cells lost their stellar morphology. Extravasation ability of both MCF-7 and MDA-MB-231 cells was reduced by 60% and 30% respectively. On the other hand, silencing Cx43 in MCF10A cells, nonneoplastic human mammary cell line, increased proliferation in both 2D and 3D cultures, and disrupted acinar morphology. Although Cx43 over-expression did not affect total levels of β-catenin, α-catenin and ZO-2, it decreased nuclear levels of β-catenin in 2D and 3D cultures of MCF-7 cells, and in 3D cultures of MDA-MB-231 cells. Cx43 associated at the membrane with α-catenin, β-catenin and ZO-2 in 2D and 3D cultures of MCF-7 cells, and only in 3D conditions in MDA-MB-231 cells. This study suggests that Cx43 exerts tumor suppressive effects in a context-dependent manner where GJ assembly with α-catenin, β-catenin and ZO-2 may be implicated in reducing growth rate, invasiveness, and, malignant phenotype of 2D and 3D cultures of MCF-7 cells, and 3D cultures of MDA-MB-231 cells, by sequestering β-catenin away from nucleus.

Synergistic activation by heterotrimeric guanine nucleotide binding protein (G protein) coupled receptors (GPCRs) and receptor tyrosine kinases distinguishes p110beta from other class IA phosphoinositide 3 kinases (PI3Ks). Activation of p110beta is specifically implicated in various physiological and pathophysiological processes such as the growth of tumors deficient in phosphatase and tensin homolog deleted from chromosome 10 (PTEN). To determine the specific contribution of GPCR signaling to p110beta dependent functions we identified the site in p110beta that binds to the Gbetagamma subunit of G proteins. Mutation of this site eliminated Gbetagamma dependent activation of PI3Kbeta (a dimer of p110beta and the p85 regulatory subunit) in vitro and in cells without affecting basal activity or phosphotyrosine peptide mediated activation. Disrupting the p110beta Gbetagamma interaction by mutation or with a cell permeable peptide inhibitor blocked the transforming capacity of PI3Kbeta in fibroblasts and reduced the proliferation chemotaxis and invasiveness of PTEN null tumor cells in culture. Our data suggest that specifically targeting GPCR signaling to PI3Kbeta could provide a therapeutic approach for tumors that depend on p110beta for growth and metastasis.

Familial Mediterranean fever (FMF) is the earliest known autoinflammatory disease, characterized by symptoms such as arthritis, peritonitis, pleuritis, erysipelas-like erythema, and most importantly amyloidosis. This disease is very common in populations of the Mediterranean area, and due to its high carrier frequency and occurrence rate in these populations, it has been the focus of much research work. Such research has allowed greater insights into the genetics of FMF, leading to the discovery of the responsible gene in 1997 and the determination of mutations and their effect on the phenotype of patients, as well as the interactions and roles of the pyrin protein, which seems to have various roles in regulation of innate immunity, inflammation, and apoptosis. Colchicine has been used as preventive treatment since 1972, and recent studies have allowed the determination of its mode of action.

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.

Carcinoembryonic antigen and vascular endothelial growth factors are among the most important prognostic markers of colorectal cancer. Testing for these markers independently has been of limited value in screening for this tumor. The aim of this study is to determine the importance of simultaneous blood CEA and VEGF level determinations in diagnosis of colorectal cancer. Thirty-six patients diagnosed with colorectal cancer along with eight healthy controls were tested by ELISA for CEA and VEGF levels in serum and plasma, respectively. The positive predictive value of these markers was 95.4% for CEA and 89.5% for VEGF, and for combined CEA and VEGF was also high at 88%. Combined CEA and VEGF blood level assay constitutes a useful panel in detecting patients with colorectal cancer. Positive results allow selection of a subgroup of patients with a high tumor risk; therefore, such tests comprise valuable tumor diagnostic tests to add to current detection methods.

Of the class IA PI3Ks, p110β is the only member to simultaneously signal downstream of both Receptor Tyrosine Kinases (RTKs) and G Protein-Coupled Receptors (GPCRs). While the mechanism of RTK-mediated activation of p85/p110 heterodimers has been characterized [1, 2], defining the mechanism of Gβγ-mediated activation of p110β downstream of GPCRs has been challenging due to the transient nature of the interaction. The binding of p110β to Gβγ is weak, but is reinforced by interactions with lipid membranes, where Gβγ resides. A combination of sequence analysis and hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) experiments has enabled us to map the regions of interaction on both p110β and Gβγ [3]. Furthermore, HDX-MS data analysis provided a unique insight into the mechanism of p110β activation by Gβγ, showing that p110β kinase domain makes stronger interaction with membranes in the presence of Gβγ. The regulatory p85 subunit inhibits basal activity of p110β, largely by the inhibitory contacts that the nSH2 and the cSH2 domains of p85 make with the p110β subunit [1, 2]. The nSH2 contacts the helical domain of p110β close to the residues essential for Gβγ binding. Whether the nSH2 has to disengage from p110β to allow Gβγ binding is still uncertain. In vitro, p110β/p85 truncation constructs lacking either the nSH2 or both SH2 domains are still activated by Gγ. In contrast, activation of p110β/p85 constructs by tyrosyl phosphorylated phosphopeptides require the presence of at least one p85 SH2 domain. Thus, Gβγ and phosphopeptides activate the enzyme via distinct mechanisms, resulting in synergistic activation when both activators are present. Whether there is an allosteric component of activation in addition to membrane recruitment mediated by Gβγ remains an open question. Based on the current results and on the structure of a membrane interacting heterotrimeric G-protein, we can begin to model how the activated p110β would interact with membranes. In this model, Gβγ makes no direct contact with the kinase domain of p110β, suggesting a mechanism where Gβγ stimulates p110β activity by increasing its membrane residence time. A similar mechanism has been proposed for the other Gβγ-sensitive PI3K, p110γ [4]. The crystal structure of p110β in a complex with Gβγ will answer many remaining questions. Wild-type p110β is transforming when over-expressed in fibroblasts [5], and it is the major p110 isoform required for driving PTEN−/− tumors [6]. However, it has not been previously possible to determine whether GPCR activation of p110β/p85 was required in these cases. Using a mutant p110β or a cell-permeable peptide, both of which block GPCR- mediated but not RTK-mediated activation of p110β/p85, we showed that GPCR inputs to p110β/p85 are required for transformation, proliferation, chemotaxis, and invasion driven by either p110β over-expression or stimulation with GPCR ligands [3]. Furthermore, a requirement for GPCR activation of p110β/p85 was seen in the growth of PTEN−/− cell lines, but not PTEN+/+ cells. Surprisingly, the peptide inhibitor of p110β/Gβγ binding blocked the growth of PTEN−/− cells, whereas the p110β-specific kinase inhibitor, TGX-221, did not. This suggests a role for a Gβγ-mediated scaffolding function of p110β in proliferation. This is consistent with previous studies showing that some functions of p110β are kinase independent [7, 8]. Our work suggests that in some tumors, inhibitors specifically targeting the Gβγ-p110β interaction might be more potent than inhibitors targeting p110β catalytic activity. Our study also suggests that the identification of the GPCRs that drive PTEN−/− tumors could provide an important alternative therapeutic approach for the treatment of these tumors. Understanding the regulation of p110β catalytic activity, as well as defining its scaffolding functions, will be important in developing drugs that target its functions in human disease.