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1.
PLoS One ; 19(3): e0298322, 2024.
Article in English | MEDLINE | ID: mdl-38502658

ABSTRACT

Many proteins in higher eukaryotes, especially those with crucial functions, have multiple isoforms with redundant roles providing protection against potential functional deficiencies in one isoform. However, these isoforms can also have some unique roles. Protein kinase B, also known as Akt, is one such protein that has three isoforms encoded on different genes. Due to high sequence similarity and the general lack of specific reagents, most studies on Akt generalize their findings and do not distinguish between the isoforms. Using an established chemical genetic strategy and a set of known Akt substrates, this work explores substrate specificity of Akt isoforms under steady state conditions in two commonly used cell lines. This strategy can be applied to study any Akt isoform-specific substrates of interest in any cell line of choice as long as the cell line can be transfected.


Subject(s)
Proto-Oncogene Proteins c-akt , Proto-Oncogene Proteins c-akt/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Cell Line
2.
Langmuir ; 39(6): 2291-2300, 2023 Feb 14.
Article in English | MEDLINE | ID: mdl-36716236

ABSTRACT

Metal-organic framework (MOF)-based membranes have been widely used in gas and liquid separation due to their porous structures and tunable compositions. Depending on the guest components, heterostructured MOFs can exhibit multiple functions. In the present work, we report a facile and rapid preparation of zeolitic imidazolate framework-8 (ZIF-8) and silver nanoparticle incorporated ZIF-8 (Ag/ZIF-8)-based membranes on stainless-steel mesh (SSM) through a "green" electrodeposition method. The SSM was first coated with a Zn-plated layer which contains mainly zinc hydroxide nitrate (Zn5(OH)8(NO3)2·2H2O) with a "leaf-like" morphology, providing anchoring points for the deposition of ZIF-8 and Ag/ZIF-8. It takes only 10 min to prepare a uniform coating of Zn5(OH)8(NO3)2·2H2O in aqueous conditions without the use of a strong base; this is by far the most efficient way of making zinc hydroxide nitrate nanocrystals. Following a similar electrodeposition approach, ZIF-8 and Ag/ZIF-8-coated SSM can be prepared within 20 min by applying a small current. The encapsulation of Ag does not alter the chemical composition nor the crystal structure of ZIF-8. The resulting ZIF-8 and Ag/ZIF-8-coated SSM have been tested for their effectiveness for rhodamine B dye removal in a fast vacuum filtration setting. Additionally, growth of E. coli was significantly inhibited after overnight incubation with Ag/ZIF-8-coated SSM. Overall, we demonstrate a fast synthesis procedure to make ZIF-8 and Ag/ZIF-8-coated SSM membranes for organic dye removal with excellent antimicrobial activity.

3.
FEBS Lett ; 594(6): 1088-1100, 2020 03.
Article in English | MEDLINE | ID: mdl-31736058

ABSTRACT

Cell surface translocation of the chaperone glucose-regulated protein 78 kDa (GRP78) is a key event that promotes cancer cell survival during endoplasmic reticulum (ER) stress. Here, we identify Gα-interacting vesicle-associated protein (GIV) - an enhancer of prosurvival signaling during ER stress - as a binding partner of GRP78. We show that GIV and GRP78 interact in an ER stress-dependent manner through their respective carboxyl terminal domains and that GIV aids in the localization of GRP78 to the plasma membrane. Kaplan-Meier analysis of disease-free survival in cancer patients shows poor prognosis for patients with high expression of both GIV and GRP78, further suggesting a vital role for these two proteins in enhancing cancer cell viability.


Subject(s)
Cell Membrane/metabolism , Endoplasmic Reticulum Stress , Heat-Shock Proteins/metabolism , Microfilament Proteins/metabolism , Neoplasm Proteins/metabolism , Neoplasms/metabolism , Vesicular Transport Proteins/metabolism , Animals , COS Cells , Cell Membrane/genetics , Cell Survival , Chlorocebus aethiops , Endoplasmic Reticulum Chaperone BiP , HeLa Cells , Heat-Shock Proteins/genetics , Humans , Microfilament Proteins/genetics , Neoplasm Proteins/genetics , Neoplasms/genetics , Neoplasms/pathology , Protein Transport , Vesicular Transport Proteins/genetics
4.
Indian J Tuberc ; 66(3): 358-363, 2019 Jul.
Article in English | MEDLINE | ID: mdl-31439180

ABSTRACT

OBJECTIVES: To study the clinical profile of cases, evaluation of comorbidities and problems encountered in initiation of second-line drugs for multidrug-resistant tuberculosis (MDR-TB) patients. METHODOLOGY: A prospective observational study was conducted on MDR patients admitted in drug resistance tuberculosis (DRTB) center of RDGMC Surasa Ujjain, a rural medical college, over a span of one year. RESULTS: Out of 130 admitted cases, majority (30%) were between 31 and 40 years of age. Males were predominant (70%). Females were significantly younger compared to males (p=0.00308). Most patients (83.8%) were underweight (body mass index (BMI)<18.5kg/m2). According to MDR-TB suspect criteria, majority were defaulter cases (39.23%). The anemia was the most common comorbidity (73.84%) among the study group followed by diabetes mellitus (9.23%), chronic obstructive pulmonary disease (COPD) (9.23%), 10 (7.69) asthma, 10 (7.69%) thyroid disease 9 (6.92%) followed by respiratory insufficiency 4 (3%), HIV 2 (1.5%), deep venous thrombosis (DVT) 2 (1.5%), renal failure 2 (1.5%), and hepatic failure 1 (0.76%). Majority had minimal lesion - 57 (43.8%), moderate - 38 (29.2%), and moderate advanced - 23 (17.7%) while far advanced was noted on X-rays in 12 (9.2%). A total of 91 (70%) cases had non-cavitary lesions and 39 (30%) had cavitary lesions, of which 27 were unilateral and 12 were bilateral. CONCLUSION: The males were predominant in our study however females were affected at a younger age compared to the males. Most of the patients had taken Anti tuberculosis treatment (ATT) from Revised National Tuberculosis Control Program (RNTCP) in which defaulter and relapse were the major contributors of MDR-TB cases in our study. Radiological extent of lesions of these patients was less than expectation. Management of comorbidities is essential for compliance to treatment. It necessitates prolonged hospitalization and requires frequent follow-up in the DRTB center.


Subject(s)
Antitubercular Agents/therapeutic use , Drug Resistance, Multiple, Bacterial , Patient Compliance , Tuberculosis, Multidrug-Resistant/epidemiology , Tuberculosis, Pulmonary/epidemiology , Adolescent , Adult , Aged , Antitubercular Agents/administration & dosage , Antitubercular Agents/pharmacology , Comorbidity , Female , Hospitalization , Humans , India/epidemiology , Male , Middle Aged , Mycobacterium tuberculosis/drug effects , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/etiology , Tuberculosis, Pulmonary/drug therapy , Tuberculosis, Pulmonary/etiology , Young Adult
5.
Mol Cell Biochem ; 453(1-2): 79-88, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30145643

ABSTRACT

Endoplasmic reticulum (ER) stress is a form of cellular stress that is experienced by cells both under normal physiological conditions such as in professional secretory cells and disease states such as cancer, diabetes, and neurodegeneration. Upon facing ER stress, cells activate a conserved signaling pathway called the unfolded protein response (UPR) to restore normal function by halting general protein translation, upregulating expression of chaperones, and promoting ER-associated degradation. However, if the stress is overwhelming and cells are not able to recover within a reasonable time frame, the UPR ultimately commits cells to programmed cell death. How cells make this life-or-death decision remains an exciting yet poorly understood phenomenon. Here, we show that Gα-interacting vesicle-associated protein (GIV) aka Girdin plays an important role in promoting cell survival during ER stress. Cells lacking GIV are impaired in activating the pro-survival Akt pathway upon induction of ER stress. These cells also show enhanced levels of the pro-apoptotic transcription factor, CCAAT/enhancer binding protein homologous protein (CHOP) as compared to control cells. Due to decreased pro-survival signals and a concomitant increase in pro-apoptotic signals, GIV-depleted cells show a significant reduction in cell survival upon prolonged ER stress which can be rescued by re-expression of GIV or by directly activating Akt in these cells. Together, this study shows a novel, cytoprotective role for GIV in ER-stressed cells and furthers our understanding of the mechanisms that contribute to cell survival during ER stress.


Subject(s)
Endoplasmic Reticulum Stress/physiology , Microfilament Proteins/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Transcription Factor CHOP/metabolism , Unfolded Protein Response , Vesicular Transport Proteins/metabolism , Cell Survival , HeLa Cells , Humans
6.
Biochem Biophys Res Commun ; 504(4): 753-758, 2018 10 12.
Article in English | MEDLINE | ID: mdl-30217452

ABSTRACT

Cyclin-dependent kinase 5 (CDK5) is a serine/threonine kinase essential for embryonic development whose overactivation has been implicated in several pathologies including neurodegeneration, cancer cell metastasis and type II diabetes. Therefore, it is important to investigate molecular mechanism(s) that mediate regulation of CDK5 activity. Here we identify and characterize a novel phosphoregulatory site on CDK5. Our mass spectrometry analysis identified seven putative phosphorylation sites on CDK5. Using phosphomimetic and non-phosphorylatable mutants, we determined that phosphorylation of S47, one of the identified sites, renders the kinase catalytically inactive. The inactivation of the kinase due to the phosphomimetic change at S47 results from inhibition of its interaction with its cognate activator, p35. We connect the effect of this regulatory event to a cellular phenotype by showing that the S47D CDK5 mutant inhibits cell migration and promotes cell proliferation. Together, these results have uncovered a potential physiological mechanism to regulate CDK5 activity. The evolutionary placement of a phosphorylatable residue (S/T) at this position not only in CDK5 but also in the majority of other CDK family members suggests that this phosphosite may represent a shared regulatory mechanism across the CDK family.


Subject(s)
Cyclin-Dependent Kinase 5/metabolism , Animals , COS Cells , Cell Movement/genetics , Cell Proliferation/genetics , Chlorocebus aethiops , Cyclin-Dependent Kinase 5/genetics , Enzyme Activation , Humans , Mutation , Phosphorylation , Phosphotransferases/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
7.
Curr Protoc Chem Biol ; 8(4): 265-298, 2016 Dec 07.
Article in English | MEDLINE | ID: mdl-27925669

ABSTRACT

Canonical signal transduction via heterotrimeric G proteins is spatiotemporally restricted, i.e., triggered exclusively at the plasma membrane, only by agonist activation of G protein-coupled receptors via a finite process that is terminated within a few hundred milliseconds. Recently, a rapidly emerging paradigm has revealed a noncanonical pathway for activation of heterotrimeric G proteins via the nonreceptor guanidine-nucleotide exchange factor, GIV/Girdin. Biochemical, biophysical, and functional studies evaluating this pathway have unraveled its unique properties and distinctive spatiotemporal features. As in the case of any new pathway/paradigm, these studies first required an in-depth optimization of tools/techniques and protocols, governed by rationale and fundamentals unique to the pathway, and more specifically to the large multimodular GIV protein. Here we provide the most up-to-date overview of protocols that have generated most of what we know today about noncanonical G protein activation by GIV and its relevance in health and disease. © 2016 by John Wiley & Sons, Inc.


Subject(s)
Fluorescent Antibody Technique/methods , Guanine Nucleotide Exchange Factors/analysis , Immunoblotting/methods , Immunoprecipitation/methods , Animals , Biophysics/methods , Guanine Nucleotide Exchange Factors/metabolism , Humans , Signal Transduction
8.
Proc Natl Acad Sci U S A ; 113(39): E5721-30, 2016 09 27.
Article in English | MEDLINE | ID: mdl-27621449

ABSTRACT

We previously showed that guanine nucleotide-binding (G) protein α subunit (Gα)-interacting vesicle-associated protein (GIV), a guanine-nucleotide exchange factor (GEF), transactivates Gα activity-inhibiting polypeptide 1 (Gαi) proteins in response to growth factors, such as EGF, using a short C-terminal motif. Subsequent work demonstrated that GIV also binds Gαs and that inactive Gαs promotes maturation of endosomes and shuts down mitogenic MAPK-ERK1/2 signals from endosomes. However, the mechanism and consequences of dual coupling of GIV to two G proteins, Gαi and Gαs, remained unknown. Here we report that GIV is a bifunctional modulator of G proteins; it serves as a guanine nucleotide dissociation inhibitor (GDI) for Gαs using the same motif that allows it to serve as a GEF for Gαi. Upon EGF stimulation, GIV modulates Gαi and Gαs sequentially: first, a key phosphomodification favors the assembly of GIV-Gαi complexes and activates GIV's GEF function; then a second phosphomodification terminates GIV's GEF function, triggers the assembly of GIV-Gαs complexes, and activates GIV's GDI function. By comparing WT and GIV mutants, we demonstrate that GIV inhibits Gαs activity in cells responding to EGF. Consequently, the cAMP→PKA→cAMP response element-binding protein signaling axis is inhibited, the transit time of EGF receptor through early endosomes are accelerated, mitogenic MAPK-ERK1/2 signals are rapidly terminated, and proliferation is suppressed. These insights define a paradigm in G-protein signaling in which a pleiotropically acting modulator uses the same motif both to activate and to inhibit G proteins. Our findings also illuminate how such modulation of two opposing Gα proteins integrates downstream signals and cellular responses.


Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein alpha Subunits, Gs/metabolism , Microfilament Proteins/metabolism , Vesicular Transport Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Cell Proliferation/drug effects , Chemotaxis/drug effects , Cyclic AMP/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclin-Dependent Kinase 5/metabolism , Down-Regulation/drug effects , Endosomes/drug effects , Endosomes/metabolism , Epidermal Growth Factor/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , Fluorescence Resonance Energy Transfer , GTP-Binding Protein beta Subunits , GTP-Binding Protein gamma Subunits , Guanosine Triphosphate/metabolism , HeLa Cells , Humans , Microfilament Proteins/chemistry , Mutant Proteins/metabolism , Phosphorylation/drug effects , Protein Binding , Protein Kinase C-theta/metabolism , Signal Transduction/drug effects , Structure-Activity Relationship , Vesicular Transport Proteins/chemistry
9.
Proc Natl Acad Sci U S A ; 112(35): E4874-83, 2015 Sep 01.
Article in English | MEDLINE | ID: mdl-26286990

ABSTRACT

Signals propagated by receptor tyrosine kinases (RTKs) can drive cell migration and proliferation, two cellular processes that do not occur simultaneously--a phenomenon called "migration-proliferation dichotomy." We previously showed that epidermal growth factor (EGF) signaling is skewed to favor migration over proliferation via noncanonical transactivation of Gαi proteins by the guanine exchange factor (GEF) GIV. However, what turns on GIV-GEF downstream of growth factor RTKs remained unknown. Here we reveal the molecular mechanism by which phosphorylation of GIV by cyclin-dependent kinase 5 (CDK5) triggers GIV's ability to bind and activate Gαi in response to growth factors and modulate downstream signals to establish a dichotomy between migration and proliferation. We show that CDK5 binds and phosphorylates GIV at Ser1674 near its GEF motif. When Ser1674 is phosphorylated, GIV activates Gαi and enhances promigratory Akt signals. Phosphorylated GIV also binds Gαs and enhances endosomal maturation, which shortens the transit time of EGFR through early endosomes, thereby limiting mitogenic MAPK signals. Consequently, this phosphoevent triggers cells to preferentially migrate during wound healing and transmigration of cancer cells. When Ser1674 cannot be phosphorylated, GIV cannot bind either Gαi or Gαs, Akt signaling is suppressed, mitogenic signals are enhanced due to delayed transit time of EGFR through early endosomes, and cells preferentially proliferate. These results illuminate how GIV-GEF is turned on upon receptor activation, adds GIV to the repertoire of CDK5 substrates, and defines a mechanism by which this unusual CDK orchestrates migration-proliferation dichotomy during cancer invasion, wound healing, and development.


Subject(s)
Cell Movement , Cell Proliferation , Cyclin-Dependent Kinase 5/metabolism , Microfilament Proteins/metabolism , Vesicular Transport Proteins/metabolism , Amino Acid Sequence , Animals , ErbB Receptors/metabolism , Humans , Microfilament Proteins/chemistry , Molecular Sequence Data , Morphogenesis , Phosphorylation , Protein Transport , Sequence Homology, Amino Acid , Signal Transduction , Vesicular Transport Proteins/chemistry , Wound Healing
10.
Mol Biol Cell ; 24(17): 2727-38, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23864707

ABSTRACT

Traffic from the endoplasmic reticulum (ER) to the Golgi complex is initiated when the activated form of the GTPase Sar1p recruits the Sec23p-Sec24p complex to ER membranes. The Sec23p-Sec24p complex, which forms the inner shell of the COPII coat, sorts cargo into ER-derived vesicles. The coat inner shell recruits the Sec13p-Sec31p complex, leading to coat polymerization and vesicle budding. Recent studies revealed that the Sec23p subunit sequentially interacts with three different binding partners to direct a COPII vesicle to the Golgi. One of these binding partners is the serine/threonine kinase Hrr25p. Hrr25p phosphorylates the COPII coat, driving the membrane-bound pool into the cytosol. The phosphorylated coat cannot rebind to the ER to initiate a new round of vesicle budding unless it is dephosphorylated. Here we screen all known protein phosphatases in yeast to identify one whose loss of function alters the cellular distribution of COPII coat subunits. This screen identifies the PP2A-like phosphatase Sit4p as a regulator of COPII coat dephosphorylation. Hyperphosphorylated coat subunits accumulate in the sit4Δ mutant in vivo. In vitro, Sit4p dephosphorylates COPII coat subunits. Consistent with a role in coat recycling, Sit4p and its mammalian orthologue, PP6, regulate traffic from the ER to the Golgi complex.


Subject(s)
COP-Coated Vesicles/metabolism , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Protein Phosphatase 2/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Vesicular Transport Proteins/metabolism , Animals , COS Cells , Chlorocebus aethiops , HeLa Cells , Humans , Membrane Proteins/metabolism , Phosphorylation , Saccharomyces cerevisiae/metabolism
11.
Nature ; 473(7346): 181-6, 2011 May 12.
Article in English | MEDLINE | ID: mdl-21532587

ABSTRACT

How the directionality of vesicle traffic is achieved remains an important unanswered question in cell biology. The Sec23p/Sec24p coat complex sorts the fusion machinery (SNAREs) into vesicles as they bud from the endoplasmic reticulum (ER). Vesicle tethering to the Golgi begins when the tethering factor TRAPPI binds to Sec23p. Where the coat is released and how this event relates to membrane fusion is unknown. Here we use a yeast transport assay to demonstrate that an ER-derived vesicle retains its coat until it reaches the Golgi. A Golgi-associated kinase, Hrr25p (CK1δ orthologue), then phosphorylates the Sec23p/Sec24p complex. Coat phosphorylation and dephosphorylation are needed for vesicle fusion and budding, respectively. Additionally, we show that Sec23p interacts in a sequential manner with different binding partners, including TRAPPI and Hrr25p, to ensure the directionality of ER-Golgi traffic and prevent the back-fusion of a COPII vesicle with the ER. These events are conserved in mammalian cells.


Subject(s)
Casein Kinase I/metabolism , Golgi Apparatus/metabolism , Saccharomyces cerevisiae/metabolism , Animals , COP-Coated Vesicles/metabolism , Endoplasmic Reticulum/metabolism , Rats , SNARE Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Vesicular Transport Proteins/metabolism
12.
Nat Rev Mol Cell Biol ; 11(11): 759-63, 2010 11.
Article in English | MEDLINE | ID: mdl-20966969

ABSTRACT

Transport protein particle (TRAPP; also known as trafficking protein particle), a multimeric guanine nucleotide-exchange factor for the yeast GTPase Ypt1 and its mammalian homologue, RAB1, regulates multiple membrane trafficking pathways. TRAPP complexes exist in three forms, each of which activates Ypt1 or RAB1 through a common core of subunits and regulates complex localization through distinct subunits. Whereas TRAPPI and TRAPPII tether coated vesicles during endoplasmic reticulum to Golgi and intra-Golgi traffic, respectively, TRAPPIII has recently been shown to be required for autophagy. These advances illustrate how the TRAPP complexes link Ypt1 and RAB1 activation to distinct membrane-tethering events.


Subject(s)
Guanine Nucleotide Exchange Factors/metabolism , Vesicular Transport Proteins/metabolism , rab1 GTP-Binding Proteins/metabolism , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Guanine Nucleotide Exchange Factors/genetics , Humans , Intercellular Signaling Peptides and Proteins , Models, Biological , Mutation , Vesicular Transport Proteins/genetics
13.
Proc Natl Acad Sci U S A ; 107(17): 7811-6, 2010 Apr 27.
Article in English | MEDLINE | ID: mdl-20375281

ABSTRACT

Macroautophagy (hereafter autophagy) is a ubiquitous process in eukaryotic cells that is integrally involved in various aspects of cellular and organismal physiology. The morphological hallmark of autophagy is the formation of double-membrane cytosolic vesicles, autophagosomes, which sequester cytoplasmic cargo and deliver it to the lysosome or vacuole. Thus, autophagy involves dynamic membrane mobilization, yet the source of the lipid that forms the autophagosomes and the mechanism of membrane delivery are poorly characterized. The TRAPP complexes are multimeric guanine nucleotide exchange factors (GEFs) that activate the Rab GTPase Ypt1, which is required for secretion. Here we describe another form of this complex (TRAPPIII) that acts as an autophagy-specific GEF for Ypt1. The Trs85 subunit of the TRAPPIII complex directs this Ypt1 GEF to the phagophore assembly site (PAS) that is involved in autophagosome formation. Consistent with the observation that a Ypt1 GEF is directed to the PAS, we find that Ypt1 is essential for autophagy. This is an example of a Rab GEF that is specifically targeted for canonical autophagosome formation.


Subject(s)
Autophagy/physiology , Guanine Nucleotide Exchange Factors/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/physiology , Vesicular Transport Proteins/metabolism , rab GTP-Binding Proteins/metabolism , Chromatography, Gel , DNA Primers/genetics , Immunoblotting , Immunoprecipitation , Microscopy, Fluorescence , Phagosomes/metabolism
14.
Mol Biol Cell ; 20(5): 1324-39, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19116316

ABSTRACT

The E3 ubiquitin ligase atrophin interacting protein 4 (AIP4) mediates ubiquitination and down-regulation of the chemokine receptor CXCR4. AIP4 belongs to the Nedd4-like homologous to E6-AP carboxy terminus domain family of E3 ubiquitin ligases, which typically bind proline-rich motifs within target proteins via the WW domains. The intracellular domains of CXCR4 lack canonical WW domain binding motifs; thus, whether AIP4 is targeted to CXCR4 directly or indirectly via an adaptor protein remains unknown. Here, we show that AIP4 can interact directly with CXCR4 via a novel noncanonical WW domain-mediated interaction involving serine residues 324 and 325 within the carboxy-terminal tail of CXCR4. These serine residues are critical for mediating agonist-promoted binding of AIP4 and subsequent ubiquitination and degradation of CXCR4. These residues are phosphorylated upon agonist activation and phosphomimetic mutants show enhanced binding to AIP4, suggesting a mechanism whereby phosphorylation mediates the interaction between CXCR4 and AIP4. Our data reveal a novel noncanonical WW domain-mediated interaction involving phosphorylated serine residues in the absence of any proline residues and suggest a novel mechanism whereby an E3 ubiquitin ligase is targeted directly to an activated G protein-coupled receptor.


Subject(s)
Receptors, CXCR4/metabolism , Repressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Binding Sites , Cell Line , Cell Membrane/metabolism , Humans , Phosphorylation , Protein Interaction Mapping , Protein Structure, Tertiary , Receptors, CXCR4/chemistry , Receptors, G-Protein-Coupled/metabolism , Repressor Proteins/analysis , Repressor Proteins/chemistry , Ubiquitin-Protein Ligases/analysis , Ubiquitin-Protein Ligases/chemistry
15.
Cancer Res ; 68(13): 5226-35, 2008 Jul 01.
Article in English | MEDLINE | ID: mdl-18593923

ABSTRACT

High expression of Notch-1 and Jagged-1 mRNA correlates with poor prognosis in breast cancer. Elucidating the cross-talk between Notch and other major breast cancer pathways is necessary to determine which patients may benefit from Notch inhibitors, which agents should be combined with them, and which biomarkers indicate Notch activity in vivo. We explored expression of Notch receptors and ligands in clinical specimens, as well as activity, regulation, and effectors of Notch signaling using cell lines and xenografts. Ductal and lobular carcinomas commonly expressed Notch-1, Notch-4, and Jagged-1 at variable levels. However, in breast cancer cell lines, Notch-induced transcriptional activity did not correlate with Notch receptor levels and was highest in estrogen receptor alpha-negative (ERalpha(-)), Her2/Neu nonoverexpressing cells. In ERalpha(+) cells, estradiol inhibited Notch activity and Notch-1(IC) nuclear levels and affected Notch-1 cellular distribution. Tamoxifen and raloxifene blocked this effect, reactivating Notch. Notch-1 induced Notch-4. Notch-4 expression in clinical specimens correlated with proliferation (Ki67). In MDA-MB231 (ERalpha(-)) cells, Notch-1 knockdown or gamma-secretase inhibition decreased cyclins A and B1, causing G(2) arrest, p53-independent induction of NOXA, and death. In T47D:A18 (ERalpha(+)) cells, the same targets were affected, and Notch inhibition potentiated the effects of tamoxifen. In vivo, gamma-secretase inhibitor treatment arrested the growth of MDA-MB231 tumors and, in combination with tamoxifen, caused regression of T47D:A18 tumors. Our data indicate that combinations of antiestrogens and Notch inhibitors may be effective in ERalpha(+) breast cancers and that Notch signaling is a potential therapeutic target in ERalpha(-) breast cancers.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Drug Delivery Systems , Estrogen Receptor alpha/physiology , Receptor Cross-Talk/physiology , Receptors, Notch/physiology , Animals , Antineoplastic Agents, Hormonal/administration & dosage , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Proliferation , Estradiol/administration & dosage , Estradiol/pharmacology , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor alpha/metabolism , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , Neoplasms, Hormone-Dependent/drug therapy , Proto-Oncogene Proteins/metabolism , RNA, Small Interfering/administration & dosage , Receptor, Notch1/metabolism , Receptor, Notch4 , Receptors, Notch/antagonists & inhibitors , Receptors, Notch/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
16.
J Biol Chem ; 282(51): 36971-9, 2007 Dec 21.
Article in English | MEDLINE | ID: mdl-17947233

ABSTRACT

The chemokine receptor CXCR4 is rapidly targeted for lysosomal degradation by the E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4). Although it is known that AIP4 mediates ubiquitination and degradation of CXCR4 and that perturbations in these events contribute to disease, the mechanisms mediating AIP4-dependent regulation of CXCR4 degradation remain poorly understood. Here we show that AIP4 directly interacts with the amino-terminal half of nonvisual arrestin-2 via its WW domains. We show that depletion of arrestin-2 by small interfering RNA blocks agonist-promoted degradation of CXCR4 by preventing CXCR4 trafficking from early endosomes to lysosomes. Surprisingly, CXCR4 internalization and ubiquitination remain intact, suggesting that the interaction between arrestin-2 and AIP4 is not required for ubiquitination of the receptor at the plasma membrane but perhaps for a later post-internalization event. Accordingly, we show that activation of CXCR4 promotes the interaction between AIP4 and arrestin-2 that is consistent with a time when AIP4 co-localizes with arrestin-2 on endocytic vesicles. Taken together, our data suggest that the AIP4.arrestin-2 complex functions on endosomes to regulate sorting of CXCR4 into the degradative pathway.


Subject(s)
Arrestins/metabolism , Endosomes/metabolism , Receptors, CXCR4/metabolism , Repressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Ubiquitination/physiology , Arrestins/antagonists & inhibitors , Arrestins/genetics , Cell Line , Cell Membrane/genetics , Cell Membrane/metabolism , Endocytosis/drug effects , Endocytosis/physiology , Endosomes/genetics , Humans , Protein Binding/drug effects , Protein Binding/physiology , Protein Structure, Tertiary/physiology , RNA, Small Interfering/pharmacology , Receptors, CXCR4/genetics , Repressor Proteins/genetics , Transport Vesicles/genetics , Transport Vesicles/metabolism , Ubiquitin/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitination/drug effects , beta-Arrestins
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