DNA damage triggers Golgi dispersal via DNA-PK and GOLPH3.

The response to DNA damage, which regulates nuclear processes such as DNA repair, transcription, and cell cycle, has been studied thoroughly. However, the cytoplasmic response to DNA damage is poorly understood. Here, we demonstrate that DNA damage triggers dramatic reorganization of the Golgi, resulting in its dispersal throughout the cytoplasm. We further show that DNA-damage-induced Golgi dispersal requires GOLPH3/MYO18A/F-actin and the DNA damage protein kinase, DNA-PK. In response to DNA damage, DNA-PK phosphorylates GOLPH3, resulting in increased interaction with MYO18A, which applies a tensile force to the Golgi. Interference with the Golgi DNA damage response by depletion of DNA-PK, GOLPH3, or MYO18A reduces survival after DNA damage, whereas overexpression of GOLPH3, as is observed frequently in human cancers, confers resistance to killing by DNA-damaging agents. Identification of the DNA-damage-induced Golgi response reveals an unexpected pathway through DNA-PK, GOLPH3, and MYO18A that regulates cell survival following DNA damage.

GOLPH3 bridges phosphatidylinositol-4- phosphate and actomyosin to stretch and shape the Golgi to promote budding.

Golgi membranes, from yeast to humans, are uniquely enriched in phosphatidylinositol-4-phosphate (PtdIns(4)P), although the role of this lipid remains poorly understood. Using a proteomic lipid-binding screen, we identify the Golgi protein GOLPH3 (also called GPP34, GMx33, MIDAS, or yeast Vps74p) as a PtdIns(4)P-binding protein that depends on PtdIns(4)P for its Golgi localization. We further show that GOLPH3 binds the unconventional myosin MYO18A, thus connecting the Golgi to F-actin. We demonstrate that this linkage is necessary for normal Golgi trafficking and morphology. The evidence suggests that GOLPH3 binds to PtdIns(4)P-rich trans-Golgi membranes and MYO18A conveying a tensile force required for efficient tubule and vesicle formation. Consequently, this tensile force stretches the Golgi into the extended ribbon observed by fluorescence microscopy and the familiar flattened form observed by electron microscopy.

Cholesterol Efflux-Independent Modification of Lipid Rafts by AIBP (Apolipoprotein A-I Binding Protein).

OBJECTIVE: AIBP (apolipoprotein A-I binding protein) is an effective and selective regulator of lipid rafts modulating many metabolic pathways originating from the rafts, including inflammation. The mechanism of action was suggested to involve stimulation by AIBP of cholesterol efflux, depleting rafts of cholesterol, which is essential for lipid raft integrity. Here we describe a different mechanism contributing to the regulation of lipid rafts by AIBP. Approach and Results: We demonstrate that modulation of rafts by AIBP may not exclusively depend on the rate of cholesterol efflux or presence of the key regulator of the efflux, ABCA1 (ATP-binding cassette transporter A-I). AIBP interacted with phosphatidylinositol 3-phosphate, which was associated with increased abundance and activation of Cdc42 and rearrangement of the actin cytoskeleton. Cytoskeleton rearrangement was accompanied with reduction of the abundance of lipid rafts, without significant changes in the lipid composition of the rafts. The interaction of AIBP with phosphatidylinositol 3-phosphate was blocked by AIBP substrate, NADPH (nicotinamide adenine dinucleotide phosphate), and both NADPH and silencing of Cdc42 interfered with the ability of AIBP to regulate lipid rafts and cholesterol efflux. CONCLUSIONS: Our findings indicate that an underlying mechanism of regulation of lipid rafts by AIBP involves PIP-dependent rearrangement of the cytoskeleton.

GOLPH3: a Golgi phosphatidylinositol(4)phosphate effector that directs vesicle trafficking and drives cancer.

GOLPH3 is a peripheral membrane protein localized to the Golgi and its vesicles, but its purpose had been unclear. We found that GOLPH3 binds specifically to the phosphoinositide phosphatidylinositol(4)phosphate [PtdIns(4)P], which functions at the Golgi to promote vesicle exit for trafficking to the plasma membrane. PtdIns(4)P is enriched at the trans-Golgi and so recruits GOLPH3. Here, a GOLPH3 complex is formed when it binds to myosin18A (MYO18A), which binds F-actin. This complex generates a pulling force to extract vesicles from the Golgi; interference with this GOLPH3 complex results in dramatically reduced vesicle trafficking. The GOLPH3 complex has been identified as a driver of cancer in humans, likely through multiple mechanisms that activate secretory trafficking. In this review, we summarize the literature that identifies the nature of the GOLPH3 complex and its role in cancer. We also consider the GOLPH3 complex as a hub with the potential to reveal regulation of the Golgi and suggest the possibility of GOLPH3 complex inhibition as a therapeutic approach in cancer.

Treating cancer with phosphatidylinositol-3-kinase inhibitors: increasing efficacy and overcoming resistance.

The discovery of the phosphatidylinositol-3-kinase (PI3K) pathway was a major advance in understanding growth factor signaling. The high frequency of PI3K pathway mutations in many cancers has encouraged a new field targeting cancer driver mutations. Although there have been many successes, targeting PI3K itself has proven challenging, in part because of its multiple isoforms with distinct roles. Despite promising preclinical results, development of PI3K inhibitors as pharmacologic anticancer agents has been limited by modest single-agent efficacy and significant adverse effects. If we could overcome these limitations, PI3K inhibitors would be a powerful cancer-fighting tool. Data from phase III clinical trials yields insight into some of the problems with PI3K inhibitors. Recent advances have shed light on the mechanisms of tumor resistance to PI3K inhibitors via feedback pathways that cause elevated insulin levels that then activate the same PI3K pathways that are the targets of inhibition. Improving our understanding of the complex regulatory feedback pathways that activate in response to PI3K inhibition will reveal ways to increase the efficacy of PI3K inhibitors and reduce adverse effects, increasing the usefulness of this class as a treatment option for multiple cancer types.

Spelunking for lipids in caveolae.

Phosphatidylserine (PtdSer) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) have been implicated in the maintenance of caveolae, but direct evidence that these lipids are required for normal caveolar structure and dynamics in living cells has been lacking. A new study by Fairn and colleagues uses sophisticated tools to perturb specific lipids in living cells to assess the consequences for caveolae. This study demonstrates disparate roles for these lipids in the stability and mobility of caveolae and points the way for future work to understand how these lipids contribute to the biology of caveolae.

Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance.

Glucose flux through the hexosamine biosynthetic pathway leads to the post-translational modification of cytoplasmic and nuclear proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc). This tandem system serves as a nutrient sensor to couple systemic metabolic status to cellular regulation of signal transduction, transcription, and protein degradation. Here we show that O-GlcNAc transferase (OGT) harbours a previously unrecognized type of phosphoinositide-binding domain. After induction with insulin, phosphatidylinositol 3,4,5-trisphosphate recruits OGT from the nucleus to the plasma membrane, where the enzyme catalyses dynamic modification of the insulin signalling pathway by O-GlcNAc. This results in the alteration in phosphorylation of key signalling molecules and the attenuation of insulin signal transduction. Hepatic overexpression of OGT impairs the expression of insulin-responsive genes and causes insulin resistance and dyslipidaemia. These findings identify a molecular mechanism by which nutritional cues regulate insulin signalling through O-GlcNAc, and underscore the contribution of this modification to the aetiology of insulin resistance and type 2 diabetes.

Regulation of Drosophila mesoderm migration by phosphoinositides and the PH domain of the Rho GTP exchange factor Pebble.

The Drosophila RhoGEF Pebble (Pbl) is required for cytokinesis and migration of mesodermal cells. In a screen for genes that could suppress migration defects in pbl mutants we identified the phosphatidylinositol phosphate (PtdInsP) regulator pi5k59B. Genetic interaction tests with other PtdInsP regulators suggested that PtdIns(4,5)P2 levels are important for mesoderm migration when Pbl is depleted. Consistent with this, the leading front of migrating mesodermal cells was enriched for PtdIns(4,5)P2. Given that Pbl contains a Pleckstrin Homology (PH) domain, a known PtdInsP-binding motif, we examined PtdInsP-binding of Pbl and the importance of the PH domain for Pbl function. In vitro lipid blot assays showed that Pbl binds promiscuously to PtdInsPs, with binding strength associated with the degree of phosphorylation. Pbl was also able to bind lipid vesicles containing PtdIns(4,5)P2 but binding was strongly reduced upon deletion of the PH domain. Similarly, in vivo, loss of the PH domain prevented localisation of Pbl to the cell cortex and severely affected several aspects of early mesoderm development, including flattening of the invaginated tube onto the ectoderm, extension of protrusions, and dorsal migration to form a monolayer. Pbl lacking the PH domain could still localise to the cytokinetic furrow, however, and cytokinesis failure was reduced in pbl(ΔPH) mutants. Taken together, our results support a model in which interaction of the PH-domain of Pbl with PtdIns(4,5)P2 helps localise it to the plasma membrane which is important for mesoderm migration.

Role of phosphatidylinositol 4-phosphate (PI4P) and its binding protein GOLPH3 in hepatitis C virus secretion.

Hepatitis C virus (HCV) RNA replicates within the ribonucleoprotein complex, assembled on the endoplasmic reticulum (ER)-derived membranous structures closely juxtaposed to the lipid droplets that facilitate the post-replicative events of virion assembly and maturation. It is widely believed that the assembled virions piggy-back onto the very low density lipoprotein particles for secretion. Lipid phosphoinositides are important modulators of intracellular trafficking. Golgi-localized phosphatidylinositol 4-phosphate (PI4P) recruits proteins involved in Golgi trafficking to the Golgi membrane and promotes anterograde transport of secretory proteins. Here, we sought to investigate the role of Golgi-localized PI4P in the HCV secretion process. Depletion of the Golgi-specific PI4P pool by Golgi-targeted PI4P phosphatase hSac1 K2A led to significant reduction in HCV secretion without any effect on replication. We then examined the functional role of a newly identified PI4P binding protein GOLPH3 in the viral secretion process. GOLPH3 is shown to maintain a tensile force on the Golgi, required for vesicle budding via its interaction with an unconventional myosin, MYO18A. Silencing GOLPH3 led to a dramatic reduction in HCV virion secretion, as did the silencing of MYO18A. The reduction in virion secretion was accompanied by a concomitant accumulation of intracellular virions, suggesting a stall in virion egress. HCV-infected cells displayed a fragmented and dispersed Golgi pattern, implicating involvement in virion morphogenesis. These studies establish the role of PI4P and its interacting protein GOLPH3 in HCV secretion and strengthen the significance of the Golgi secretory pathway in this process.

A multienzyme S-nitrosylation cascade regulates cholesterol homeostasis.

Accumulating evidence suggests that protein S-nitrosylation is enzymatically regulated and that specificity in S-nitrosylation derives from dedicated S-nitrosylases and denitrosylases that conjugate and remove S-nitrosothiols, respectively. Here, we report that mice deficient in the protein denitrosylase SCoR2 (S-nitroso-Coenzyme A Reductase 2; AKR1A1) exhibit marked reductions in serum cholesterol due to reduced secretion of the cholesterol-regulating protein PCSK9. SCoR2 associates with endoplasmic reticulum (ER) secretory machinery to control an S-nitrosylation cascade involving ER cargo-selection proteins SAR1 and SURF4, which moonlight as S-nitrosylases. SAR1 acts as a SURF4 nitrosylase and SURF4 as a PCSK9 nitrosylase to inhibit PCSK9 secretion, while SCoR2 counteracts nitrosylase activity by promoting PCSK9 denitrosylation. Inhibition of PCSK9 by an NO-based drug requires nitrosylase activity, and small-molecule inhibition of SCoR2 phenocopies the PCSK9-mediated reductions in cholesterol observed in SCoR2-deficient mice. Our results reveal enzymatic machinery controlling cholesterol levels through S-nitrosylation and suggest a distinct treatment paradigm for cardiovascular disease.

The p85 regulatory subunit of phosphoinositide 3-kinase down-regulates IRS-1 signaling via the formation of a sequestration complex.

Phosphoinositide (PI) 3-kinase is required for most insulin and insulin-like growth factor (IGF) 1-dependent cellular responses. The p85 regulatory subunit of PI 3-kinase is required to mediate the insulin-dependent recruitment of PI 3-kinase to the plasma membrane, yet mice with reduced p85 expression have increased insulin sensitivity. To further understand the role of p85, we examined IGF-1-dependent translocation of p85alpha by using a green fluorescence protein (GFP)-tagged p85alpha (EGFP-p85alpha). In response to IGF-1, but not to PDGF signaling, EGFP-p85alpha translocates to discrete foci in the cell. These foci contain the insulin receptor substrate (IRS) 1 adaptor molecule, and their formation requires the binding of p85 to IRS-1. Surprisingly, monomeric p85 is preferentially localized to these foci compared with the p85-p110 dimer, and these foci are not sites of phosphatidylinositol-3,4,5-trisphosphate production. Ultrastructural analysis reveals that p85-IRS-1 foci are cytosolic protein complexes devoid of membrane. These results suggest a mechanism of signal down-regulation of IRS-1 that is mediated by monomeric p85 through the formation of a sequestration complex between p85 and IRS-1.

Induction of membrane curvature by proteins involved in Golgi trafficking.

The Golgi apparatus serves a key role in processing and sorting lipids and proteins for delivery to their final cellular destinations. Vesicle exit from the Golgi initiates with directional deformation of the lipid bilayer to produce a bulge. Several mechanisms have been described by which lipids and proteins can induce directional membrane curvature to promote vesicle budding. Here we review some of the mechanisms implicated in inducing membrane curvature at the Golgi to promote vesicular trafficking to various cellular destinations.

Lipid research picks up speed on the slopes of Taos.

The 2002 Keystone Symposium on "Regulation of Cellular Responses by Lipid Mediators" provided a lively and active forum to discuss research in lipid signaling. This meeting review can provide only a glimpse into the diversity of research presented. Here we have chosen to highlight a group of exciting presentations describing novel features of the temporal and spatial regulation of phosphoinositides and their downstream targets.

A novel phosphatidylinositol(3,4,5)P3 pathway in fission yeast.

The mammalian tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), inhibits cell growth and survival by dephosphorylating phosphatidylinositol-(3,4,5)-trisphosphate (PI[3,4,5]P3). We have found a homologue of PTEN in the fission yeast, Schizosaccharomyces pombe (ptn1). This was an unexpected finding because yeast (S. pombe and Saccharomyces cerevisiae) lack the class I phosphoinositide 3-kinases that generate PI(3,4,5)P3 in higher eukaryotes. Indeed, PI(3,4,5)P3 has not been detected in yeast. Surprisingly, upon deletion of ptn1 in S. pombe, PI(3,4,5)P3 became detectable at levels comparable to those in mammalian cells, indicating that a pathway exists for synthesis of this lipid and that the S. pombe ptn1, like mammalian PTEN, suppresses PI(3,4,5)P3 levels. By examining various mutants, we show that synthesis of PI(3,4,5)P3 in S. pombe requires the class III phosphoinositide 3-kinase, vps34p, and the phosphatidylinositol-4-phosphate 5-kinase, its3p, but does not require the phosphatidylinositol-3-phosphate 5-kinase, fab1p. These studies suggest that a pathway for PI(3,4,5)P3 synthesis downstream of a class III phosphoinositide 3-kinase evolved before the appearance of class I phosphoinositide 3-kinases.

Golgi Oncoprotein GOLPH3 Gene Expression Is Regulated by Functional E2F and CREB/ATF Promoter Elements.

The Golgi organelle duplicates its protein and lipid content to segregate evenly between two daughter cells after mitosis. However, how Golgi biogenesis is regulated during interphase remains largely unknown. Here we show that messenger RNA (mRNA) expression of GOLPH3 and GOLGA2, two genes encoding Golgi proteins, is induced specifically in G1 phase, suggesting a link between cell cycle regulation and Golgi growth. We have examined the role of E2F transcription factors, critical regulators of G1 to S progression of the cell cycle, in the expression of Golgi proteins during interphase. We show that promoter activity for GOLPH3, a Golgi protein that is also oncogenic, is induced by E2F1-3 and repressed by E2F7. Mutation of the E2F motifs present in the GOLPH3 promoter region abrogates E2F1-mediated induction of a GOLPH3 luciferase reporter construct. Furthermore, we identify a critical CREB/ATF element in the GOLPH3 promoter that is required for its steady state and ATF2-induced expression. Interestingly, depletion of GOLPH3 with small interfering RNA (siRNA) delays the G1 to S transition in synchronized U2OS cells. Taken together, our results reveal a link between cell cycle regulation and Golgi function, and suggest that E2F-mediated regulation of Golgi genes is required for the timely progression of the cell cycle.

Efficient Golgi Forward Trafficking Requires GOLPH3-Driven, PI4P-Dependent Membrane Curvature.

Vesicle budding for Golgi-to-plasma membrane trafficking is a key step in secretion. Proteins that induce curvature of the Golgi membrane are predicted to be required, by analogy to vesicle budding from other membranes. Here, we demonstrate that GOLPH3, upon binding to the phosphoinositide PI4P, induces curvature of synthetic membranes in vitro and the Golgi in cells. Moreover, efficient Golgi-to-plasma membrane trafficking critically depends on the ability of GOLPH3 to curve the Golgi membrane. Interestingly, uncoupling of GOLPH3 from its binding partner MYO18A results in extensive curvature of Golgi membranes, producing dramatic tubulation of the Golgi, but does not support forward trafficking. Thus, forward trafficking from the Golgi to the plasma membrane requires the ability of GOLPH3 both to induce Golgi membrane curvature and to recruit MYO18A. These data provide fundamental insight into the mechanism of Golgi trafficking and into the function of the unique Golgi secretory oncoproteins GOLPH3 and MYO18A.

PtdIns(4,5)P2 functions at the cleavage furrow during cytokinesis.

Phosphoinositides play important roles in regulating the cytoskeleton and vesicle trafficking, potentially important processes at the cleavage furrow. However, it remains unclear which, if any, of the phosphoinositides play a role during cytokinesis. A systematic analysis to determine if any of the phosphoinositides might be present or of functional importance at the cleavage furrow has not been published. Several studies hint at a possible role for one or more phosphoinositides at the cleavage furrow. The best of these are genetic data identifying mutations in phosphoinositide-modifying enzymes (a PtdIns(4)P-5-kinase in S. pombe and a PI-4-kinase in D. melanogaster) that interfere with cytokinesis. The genetic nature of these experiments leaves questions as to how direct may be their contribution to cytokinesis. Here we show that a single phosphoinositide, PtdIns(4,5)P2, specifically accumulates at the furrow. Interference with PtdIns(4,5)P2 interferes with adhesion of the plasma membrane to the contractile ring at the furrow. Finally, four distinct interventions to specifically interfere with PtdIns(4,5)P2 each impair cytokinesis. We conclude that PtdIns(4,5)P2 is present at the cleavage furrow and is required for normal cytokinesis at least in part because of a role in adhesion between the contractile ring and the plasma membrane.

MYO18A: An unusual myosin.

MYO18A is a divergent member of the myosin family characterized by the presence of an amino-terminal PDZ domain. MYO18A has been found in a few different complexes involved in intracellular transport processes. MYO18A is found in a complex with LURAP1 and MRCK that functions in retrograde treadmilling of actin. It also has been found in a complex with PAK2, ßPIX, and GIT1, functioning to transport that protein complex from focal adhesions to the leading edge. Finally, a high proportion of MYO18A is found in complex with GOLPH3 at the trans Golgi, where it functions to promote vesicle budding for Golgi-to-plasma membrane trafficking. Interestingly, MYO18A has been implicated as a cancer driver, as have other components of the GOLPH3 pathway. It remains uncertain as to whether or not MYO18A has intrinsic motor activity. While many questions remain, MYO18A is a fascinatingly unique myosin that is essential in higher organisms.

Neolymphostin A Is a Covalent Phosphoinositide 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Dual Inhibitor That Employs an Unusual Electrophilic Vinylogous Ester.

Using a novel chemistry-based assay for identifying electrophilic natural products in unprocessed extracts, we identified the PI3-kinase/mTOR dual inhibitor neolymphostin A from Salinispora arenicola CNY-486. The method further showed that the vinylogous ester substituent on the neolymphostin core was the exact site for enzyme conjugation. Tandem MS/MS experiments on PI3Kα treated with the inhibitor revealed that neolymphostin covalently modified Lys802 with a shift in mass of +306 amu, corresponding to addition of the inhibitor and elimination of methanol. The binding pose of the inhibitor bound to PI3Kα was modeled, and hydrogen-deuterium exchange mass spectrometry experiments supported this model. Against a panel of kinases, neolymphostin showed good selectivity for PI3-kinase and mTOR. In addition, the natural product blocked AKT phosphorylation in live cells with an IC50 of ∼3 nM. Taken together, neolymphostin is the first reported example of a covalent kinase inhibitor from the bacterial domain of life.

Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice.

E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.