Publications by authors named "Seth J Field"

32 Publications

Induction of membrane curvature by proteins involved in Golgi trafficking.

Adv Biol Regul 2020 01 16;75:100661. Epub 2019 Oct 16.

Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, CA, 92093, USA. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbior.2019.100661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056495PMC
January 2020

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

Dev Cell 2019 09 20;50(5):573-585.e5. Epub 2019 Jun 20.

Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2019.05.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583631PMC
September 2019

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

Genes (Basel) 2019 03 25;10(3). Epub 2019 Mar 25.

Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, 48080 Bilbao, Spain.

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 and , 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 , a Golgi protein that is also oncogenic, is induced by E2F1-3 and repressed by E2F7. Mutation of the E2F motifs present in the promoter region abrogates E2F1-mediated induction of a luciferase reporter construct. Furthermore, we identify a critical CREB/ATF element in the promoter that is required for its steady state and ATF2-induced expression. Interestingly, depletion of 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/genes10030247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471639PMC
March 2019

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

J Lipid Res 2019 04 4;60(4):747-752. Epub 2019 Feb 4.

Meyer Cancer Center Weill Cornell Medicine, New York, NY 10021. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1194/jlr.S092130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446698PMC
April 2019

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

J Med Chem 2018 12 28;61(23):10463-10472. Epub 2018 Nov 28.

Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography , University of California, San Diego , La Jolla , California 92093 , United States.

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 IC of ∼3 nM. Taken together, neolymphostin is the first reported example of a covalent kinase inhibitor from the bacterial domain of life.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.8b00975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688905PMC
December 2018

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

J Lipid Res 2019 02 28;60(2):269-275. Epub 2018 Sep 28.

Division of Endocrinology and Metabolism, Department of Medicine, University of California at San Diego, La Jolla, CA

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 -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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1194/jlr.R088328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358304PMC
February 2019

MYO18A: An unusual myosin.

Adv Biol Regul 2018 01 18;67:84-92. Epub 2017 Sep 18.

Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

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, βPIX, 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbior.2017.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5807147PMC
January 2018

Spelunking for lipids in caveolae.

Authors:
Seth J Field

J Biol Chem 2017 08;292(34):14308-14309

From the Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093. Electronic address:

Phosphatidylserine (PtdSer) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P) 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.H117.791400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5572928PMC
August 2017

Emerging themes of regulation at the Golgi.

Curr Opin Cell Biol 2017 04 16;45:17-23. Epub 2017 Feb 16.

Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, CA 92093-0707, USA. Electronic address:

The Golgi is generally recognized for its central role in the secretory pathway to orchestrate protein post-translational modification and trafficking of proteins and lipids to their final destination. Despite the common view of the Golgi as an inert sorting organelle, emerging data demonstrate that important signaling events occur at the Golgi, including those that regulate the trafficking function of the Golgi. The phosphatidylinositol-4-phosphate/GOLPH3/MYO18A/F-actin complex serves as a hub for signals that regulate Golgi trafficking function. Furthermore, the Golgi is increasingly appreciated for its important role in cell growth and in driving oncogenic transformation, as illuminated by the discovery that GOLPH3 and MYO18A are cancer drivers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ceb.2017.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5482773PMC
April 2017

GOLPH3 drives cell migration by promoting Golgi reorientation and directional trafficking to the leading edge.

Mol Biol Cell 2016 12 5;27(24):3828-3840. Epub 2016 Oct 5.

Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA 92093

The mechanism of directional cell migration remains an important problem, with relevance to cancer invasion and metastasis. GOLPH3 is a common oncogenic driver of human cancers, and is the first oncogene that functions at the Golgi in trafficking to the plasma membrane. Overexpression of GOLPH3 is reported to drive enhanced cell migration. Here we show that the phosphatidylinositol-4-phosphate/GOLPH3/myosin 18A/F-actin pathway that is critical for Golgi-to-plasma membrane trafficking is necessary and limiting for directional cell migration. By linking the Golgi to the actin cytoskeleton, GOLPH3 promotes reorientation of the Golgi toward the leading edge. GOLPH3 also promotes reorientation of lysosomes (but not other organelles) toward the leading edge. However, lysosome function is dispensable for migration and the GOLPH3 dependence of lysosome movement is indirect, via GOLPH3's effect on the Golgi. By driving reorientation of the Golgi to the leading edge and driving forward trafficking, particularly to the leading edge, overexpression of GOLPH3 drives trafficking to the leading edge of the cell, which is functionally important for directional cell migration. Our identification of a novel pathway for Golgi reorientation controlled by GOLPH3 provides new insight into the mechanism of directional cell migration with important implications for understanding GOLPH3's role in cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E16-01-0005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5170606PMC
December 2016

The GOLPH3 pathway regulates Golgi shape and function and is activated by DNA damage.

Front Neurosci 2015 7;9:362. Epub 2015 Oct 7.

Division of Endocrinology and Metabolism, Department of Medicine, University of California San Diego, La Jolla, CA, USA.

The Golgi protein GOLPH3 binds to PtdIns(4)P and MYO18A, linking the Golgi to the actin cytoskeleton. The GOLPH3 pathway is essential for vesicular trafficking from the Golgi to the plasma membrane. A side effect of GOLPH3-dependent trafficking is to generate the extended ribbon shape of the Golgi. Perturbation of the pathway results in changes to both Golgi morphology and secretion, with functional consequences for the cell. The cellular response to DNA damage provides an example of GOLPH3-mediated regulation of the Golgi. Upon DNA damage, DNA-PK phosphorylation of GOLPH3 increases binding to MYO18A, activating the GOLPH3 pathway, which consequently results in Golgi fragmentation, reduced trafficking, and enhanced cell survival. The PtdIns(4)P/GOLPH3/MYO18A/F-actin pathway provides new insight into the relationship between Golgi morphology and function, and their regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnins.2015.00362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4595774PMC
October 2015

Biallelic mutations in SNX14 cause a syndromic form of cerebellar atrophy and lysosome-autophagosome dysfunction.

Nat Genet 2015 May 6;47(5):528-34. Epub 2015 Apr 6.

Department of Pediatric Neurology, Children's Hospital, Cairo University, Cairo, Egypt.

Pediatric-onset ataxias often present clinically as developmental delay and intellectual disability, with prominent cerebellar atrophy as a key neuroradiographic finding. Here we describe a new clinically distinguishable recessive syndrome in 12 families with cerebellar atrophy together with ataxia, coarsened facial features and intellectual disability, due to truncating mutations in the sorting nexin gene SNX14, encoding a ubiquitously expressed modular PX domain-containing sorting factor. We found SNX14 localized to lysosomes and associated with phosphatidylinositol (3,5)-bisphosphate, a key component of late endosomes/lysosomes. Patient-derived cells showed engorged lysosomes and a slower autophagosome clearance rate upon autophagy induction by starvation. Zebrafish morphants for snx14 showed dramatic loss of cerebellar parenchyma, accumulation of autophagosomes and activation of apoptosis. Our results characterize a unique ataxia syndrome due to biallelic SNX14 mutations leading to lysosome-autophagosome dysfunction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ng.3256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4414867PMC
May 2015

GOLPH3 links the Golgi, DNA damage, and cancer.

Cancer Res 2015 Feb 29;75(4):624-7. Epub 2015 Jan 29.

Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California.

GOLPH3 is the first example of an oncogene that functions in secretory trafficking at the Golgi. The discovery of GOLPH3's roles in both cancer and Golgi trafficking raises questions about how GOLPH3 and the Golgi contribute to cancer. Our recent investigation of the regulation of GOLPH3 revealed a surprising response by the Golgi upon DNA damage that is mediated by DNA-PK and GOLPH3. These results provide new insight into the DNA damage response with important implications for understanding the cellular response to standard cancer therapeutic agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-14-3081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4333064PMC
February 2015

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

Cell 2014 Jan;156(3):413-27

Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA 92093-0707, USA. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2013.12.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4018323PMC
January 2014

GOLPH3L antagonizes GOLPH3 to determine Golgi morphology.

Mol Biol Cell 2013 Mar 23;24(6):796-808. Epub 2013 Jan 23.

Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

GOLPH3 is a phosphatidylinositol-4-phosphate (PI4P) effector that plays an important role in maintaining Golgi architecture and anterograde trafficking. GOLPH3 does so through its ability to link trans-Golgi membranes to F-actin via its interaction with myosin 18A (MYO18A). GOLPH3 also is known to be an oncogene commonly amplified in human cancers. GOLPH3L is a GOLPH3 paralogue found in all vertebrate genomes, although previously it was largely uncharacterized. Here we demonstrate that although GOLPH3 is ubiquitously expressed in mammalian cells, GOLPH3L is present in only a subset of tissues and cell types, particularly secretory tissues. We show that, like GOLPH3, GOLPH3L binds to PI4P, localizes to the Golgi as a consequence of its PI4P binding, and is required for efficient anterograde trafficking. Surprisingly, however, we find that perturbations of GOLPH3L expression produce effects on Golgi morphology that are opposite to those of GOLPH3 and MYO18A. GOLPH3L differs critically from GOLPH3 in that it is largely unable to bind to MYO18A. Our data demonstrate that despite their similarities, unexpectedly, GOLPH3L antagonizes GOLPH3/MYO18A at the Golgi.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E12-07-0525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3596250PMC
March 2013

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

Dev Biol 2012 Dec 19;372(1):17-27. Epub 2012 Sep 19.

Research School of Biology, Australian National University, ACT 2601, Australia.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ydbio.2012.09.008DOI Listing
December 2012

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

J Biol Chem 2012 Aug 28;287(33):27637-47. Epub 2012 Jun 28.

Division of Infectious Diseases, Moores Cancer Center, University of California, San Diego, La Jolla, California 92093, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M112.346569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431621PMC
August 2012

Receptor tyrosine kinases and TLR/IL1Rs unexpectedly activate myeloid cell PI3kγ, a single convergent point promoting tumor inflammation and progression.

Cancer Cell 2011 Jun;19(6):715-27

Moores UCSD Cancer Center, University of California, San Diego, CA 92093, USA.

Tumor inflammation promotes angiogenesis, immunosuppression, and tumor growth, but the mechanisms controlling inflammatory cell recruitment to tumors are not well understood. We found that a range of chemoattractants activating G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and Toll-like/IL-1 receptors (TLR/IL1Rs) unexpectedly initiate tumor inflammation by activating the PI3-kinase isoform p110γ in Gr1+CD11b+ myeloid cells. Whereas GPCRs activate p110γ in a Ras/p101-dependent manner, RTKs and TLR/IL1Rs directly activate p110γ in a Ras/p87-dependent manner. Once activated, p110γ promotes inside-out activation of a single integrin, α4β1, causing myeloid cell invasion into tumors. Pharmacological or genetic blockade of p110γ suppressed inflammation, growth, and metastasis of implanted and spontaneous tumors, revealing an important therapeutic target in oncology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ccr.2011.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3144144PMC
June 2011

Regulation of oxysterol-binding protein Golgi localization through protein kinase D-mediated phosphorylation.

Mol Biol Cell 2010 Jul 5;21(13):2327-37. Epub 2010 May 5.

Departments of Pathology and Signal Transduction, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.

Protein kinase D (PKD) plays a critical role at the trans-Golgi network by regulating the fission of transport carriers destined for the plasma membrane. Two known Golgi-localized PKD substrates, PI4-kinase IIIbeta and the ceramide transfer protein CERT, mediate PKD signaling to influence vesicle trafficking to the plasma membrane and sphingomyelin synthesis, respectively. PKD is recruited and activated at the Golgi through interaction with diacylglycerol, a pool of which is generated as a by-product of sphingomyelin synthesis from ceramide. Here we identify a novel substrate of PKD at the Golgi, the oxysterol-binding protein OSBP. Using a substrate-directed phospho-specific antibody that recognizes the optimal PKD consensus motif, we show that PKD phosphorylates OSBP at Ser240 in vitro and in cells. We further show that OSBP phosphorylation occurs at the Golgi. Phosphorylation of OSBP by PKD does not modulate dimerization, sterol binding, or affinity for PI(4)P. Instead, phosphorylation attenuates OSBP Golgi localization in response to 25-hydroxycholesterol and cholesterol depletion, impairs CERT Golgi localization, and promotes Golgi fragmentation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.e10-02-0090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2893995PMC
July 2010

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

Cell 2009 Oct;139(2):337-51

Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA 92093-0707, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2009.07.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2779841PMC
October 2009

Mutations in INPP5E, encoding inositol polyphosphate-5-phosphatase E, link phosphatidyl inositol signaling to the ciliopathies.

Nat Genet 2009 Sep 9;41(9):1032-6. Epub 2009 Aug 9.

Neurogenetics Laboratory, Howard Hughes Medical Institute, Department of Neurosciences and Pediatrics, University of California, San Diego, La Jolla, USA.

Phosphotidylinositol (PtdIns) signaling is tightly regulated both spatially and temporally by subcellularly localized PtdIns kinases and phosphatases that dynamically alter downstream signaling events. Joubert syndrome is characterized by a specific midbrain-hindbrain malformation ('molar tooth sign'), variably associated retinal dystrophy, nephronophthisis, liver fibrosis and polydactyly and is included in the newly emerging group of 'ciliopathies'. In individuals with Joubert disease genetically linked to JBTS1, we identified mutations in the INPP5E gene, encoding inositol polyphosphate-5-phosphatase E, which hydrolyzes the 5-phosphate of PtdIns(3,4,5)P3 and PtdIns(4,5)P2. Mutations clustered in the phosphatase domain and impaired 5-phosphatase activity, resulting in altered cellular PtdIns ratios. INPP5E localized to cilia in major organs affected by Joubert syndrome, and mutations promoted premature destabilization of cilia in response to stimulation. These data link PtdIns signaling to the primary cilium, a cellular structure that is becoming increasingly recognized for its role in mediating cell signals and neuronal function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ng.423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2746682PMC
September 2009

E2F2 represses cell cycle regulators to maintain quiescence.

Cell Cycle 2008 Dec 10;7(24):3915-27. Epub 2008 Dec 10.

Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, Bilbao, Spain.

E2F transcription factors control diverse biological processes through regulation of target gene expression. However, the mechanism by which this regulation is established, and the relative contribution of each E2F member are still poorly defined. We have investigated the role of E2F2 in regulating cellular proliferation. We show that E2F2 is required for the normal G(0)/G(1) phase because targeted disruption of the E2F2 gene causes T cells to enter S phase early and to undergo accelerated cell division. A large set of E2F target genes involved in DNA replication and cell cycle progression (such as Mcm's, cyclins and Cdc2a) that are silent in G(0) and typically transcribed late in G(1) phase are already actively expressed in quiescent T cells and MEFs lacking E2F2. The classic E2F activators, E2F1 and E2F3, are largely dispensable for this process because compound loss of E2F1(-/-) and E2F2(-/-) produces a comparably shortened G(0)/G(1) phase, with early S phase entry. Likewise, shRNA knockdown of E2F3 does not alter significantly the E2F2(-/-) phenotype. Chromatin immunoprecipitation analysis indicates that in wild-type cells the promoters of the aberrantly early-transcribed genes are occupied by E2F2 in G(0), suggesting a direct role for E2F2 in transcriptional repression. We conclude that E2F2 functions to transcriptionally repress cell cycle genes to establish the G(0) state.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/cc.7.24.7379DOI Listing
December 2008

Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance.

Nature 2008 Feb;451(7181):964-9

Howard Hughes Medical Institute and Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature06668DOI Listing
February 2008

Deletion of the phosphoinositide 3-kinase p110gamma gene attenuates murine atherosclerosis.

Proc Natl Acad Sci U S A 2007 May 2;104(19):8077-82. Epub 2007 May 2.

Beth Israel Deaconess Medical Center (Signal Transduction and Cardiovascular Divisions), Boston, MA 02115, USA.

Inflammatory cell activation by chemokines requires intracellular signaling through phosphoinositide 3-kinase (PI3-kinase) and the PI3-kinase-dependent protein serine/threonine kinase Akt. Atherosclerosis is a chronic inflammatory process driven by oxidatively modified (atherogenic) lipoproteins, chemokines, and other agonists that activate PI3-kinase. Here we show that macrophage PI3-kinase/Akt is activated by oxidized low-density lipoprotein, inflammatory chemokines, and angiotensin II. This activation is markedly reduced or absent in macrophages lacking p110gamma, the catalytic subunit of class Ib PI3-kinase. We further demonstrate activation of macrophage/foam cell PI3-kinase/Akt in atherosclerotic plaques from apolipoprotein E (apoE)-null mice, which manifest an aggressive form of atherosclerosis, whereas activation of PI3-kinase/Akt was undetectable in lesions from apoE-null mice lacking p110gamma despite the presence of class Ia PI3-kinase. Moreover, plaques were significantly smaller in apoE-/-p110gamma-/- mice than in apoE-/-p110gamma+/+ or apoE-/-p110gamma+/-mice at all ages studied. In marked contrast to the embryonic lethality seen in mice lacking class Ia PI3-kinase, germ-line deletion of p110gamma results in mice that exhibit normal viability, longevity, and fertility, with relatively well tolerated defects in innate immune and inflammatory responses that may play a role in diseases such as atherosclerosis and multiple sclerosis. Our results not only shed mechanistic light on inflammatory signaling during atherogenesis, but further identify p110gamma as a possible target for pharmacological intervention in the primary and secondary prevention of human atherosclerotic cardiovascular disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0702663104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1864909PMC
May 2007

Modification of protein sub-nuclear localization by synthetic phosphoinositides: evidence for nuclear phosphoinositide signaling mechanisms.

Adv Enzyme Regul 2005 30;45:171-85. Epub 2005 Sep 30.

Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

PtdInsPs are critical signaling molecules that regulate diverse cellular functions. One method to study PtdInsP biology involves using synthetic PtdInsP analogs to activate endogenous PtdInsP-mediated events in living cells. Such methodology has been successfully employed to explore the role of several PtdInsP-biological outcomes in the cytoplasm. However, this strategy has not previously been used to examine the function of PtdInsPs in the nucleus of live cells, primarily because there has not been a well-defined PtdInsP-binding protein to provide functional nuclear readouts. Here we have shown that synthetic PtdIns(5)P analogs access and function in the nucleus. We have found that these molecules modify the sub-nuclear localization of PHD finger-containing proteins in live cells and in real time. This work demonstrates that synthetic PtdInsPs and PtdInsP derivatives may be powerful tools for probing nuclear PtdInsP functions. Finally, our work supports a model that endogenous PtdInsPs regulate sub-nuclear localization and function of endogenous nuclear PtdInsP-binding proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.advenzreg.2005.02.010DOI Listing
February 2006

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

Curr Biol 2005 Aug;15(15):1407-12

Division of Signal Transduction, Beth Israel-Deaconess Medical Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2005.06.059DOI Listing
August 2005

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

J Cell Biol 2005 Aug 25;170(3):455-64. Epub 2005 Jul 25.

Department of Systems Biology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.200503088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2171479PMC
August 2005

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

J Cell Biol 2004 Jul 12;166(2):205-11. Epub 2004 Jul 12.

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St., Rm. 819, Worcester, MA 01605, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.200404150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2172303PMC
July 2004

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

J Clin Invest 2004 May;113(10):1398-407

Department of Genetics, Physical Anthropology and Animal Physiology, Faculty of Sciences, University of the Basque Country, Bilbao, Spain.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI18879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC406522PMC
May 2004

Identification and characterization of a phosphoinositide phosphate kinase homolog.

J Biol Chem 2004 Mar 30;279(12):11672-9. Epub 2003 Dec 30.

Beth Israel Deaconess Medical Center, Divisions of Signal Transduction, Cardiovascular Medicine, and Hematology-Oncology, Boston, Massachusetts 02215, USA.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) plays a central role in regulating the actin cytoskeleton as a substrate for phosphoinositide 3-kinase and phospholipase C as well as by binding directly to proteins that control the processes of actin monomer sequestration, filament severing, capping, nucleation, cross-linking, and bundling (Ma, L., Cantley, L. C., Janmey, P. A., and Kirschner, M. W. (1998) J. Cell Biol. 140, 1125-1136; Hinchliffe, K. (2000) Curr. Biol. 10, R104-R1051). Three related phosphatidylinositol 4-phosphate 5-kinases (PI(4)P 5-kinases) have been identified in mammalian cells (types Ialpha, Ibeta, and Igamma) and appear to play distinct roles in actin remodeling. Here we have identified a fourth member of this family by searching the human genome and EST data bases. This new protein, which we have designated phosphatidylinositol phosphate kinase homolog (PIPKH), is expressed at relatively high levels in brain and testis. Immunoprecipitates of PIPKH expressed in mammalian cells contain PI(4)P 5-kinase activity, but this activity is not affected by mutations in residues that inactivate other type I PI(4)P 5-kinases. We show that the PI(4)P 5-kinase activity in PIPKH immunoprecipitates can be explained by the ability of PIPKH to heterodimerize with other type I PI(4)P 5-kinases. Transfection of 293t cells with PIPKH resulted in >8-fold increase in total phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) without a significant net increase in total PI(4,5)P(2). When coexpressed with PIPKH, green fluorescent protein (GFP) fusion construct of the pleckstrin homology domain from Bruton's tyrosine kinase (GFP-BTK-PH) localized in intracellular vesicular structures, suggesting an unusual intracellular site of PI(3,4,5)P(3) production. Finally, expression of PIPKH induced the reorganization of actin from predominantly stress fibers to predominantly foci and comets similar to those observed previously in cells infected with the intracellular pathogen Listeria or transfected with recombinant PIPKIalpha. These results suggest that PIPKH acts as a scaffold to localize and regulate type I PI(4)P 5-kinases and the synthesis of PI(3,4,5)P(3).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M309721200DOI Listing
March 2004
-->