scholarly journals Adenovirus RIDα regulates endosome maturation by mimicking GTP-Rab7

2007 ◽  
Vol 179 (5) ◽  
pp. 965-980 ◽  
Author(s):  
Ankur H. Shah ◽  
Nicholas L. Cianciola ◽  
Jeffrey L. Mills ◽  
Frank D. Sönnichsen ◽  
Cathleen Carlin
Keyword(s):  
Growth Factor Receptor ◽  
Dominant Negative ◽  
Receptor Internalization ◽  
Site Directed Mutagenesis ◽  
Oxysterol Binding Protein ◽  
Early Endosomes ◽  
Catalytically Active ◽  
Guanosine Triphosphatase ◽  
Endocytic Vesicles

The small guanosine triphosphatase Rab7 regulates late endocytic trafficking. Rab7-interacting lysosomal protein (RILP) and oxysterol-binding protein–related protein 1L (ORP1L) are guanosine triphosphate (GTP)–Rab7 effectors that instigate minus end–directed microtubule transport. We demonstrate that RILP and ORP1L both interact with the group C adenovirus protein known as receptor internalization and degradation α (RIDα), which was previously shown to clear the cell surface of several membrane proteins, including the epidermal growth factor receptor and Fas (Carlin, C.R., A.E. Tollefson, H.A. Brady, B.L. Hoffman, and W.S. Wold. 1989. Cell. 57:135–144; Shisler, J., C. Yang, B. Walter, C.F. Ware, and L.R. Gooding. 1997. J. Virol. 71:8299–8306). RIDα localizes to endocytic vesicles but is not homologous to Rab7 and is not catalytically active. We show that RIDα compensates for reduced Rab7 or dominant-negative (DN) Rab7(T22N) expression. In vitro, Cu2+ binding to RIDα residues His75 and His76 facilitates the RILP interaction. Site-directed mutagenesis of these His residues results in the loss of RIDα–RILP interaction and RIDα activity in cells. Additionally, expression of the RILP DN C-terminal region hinders RIDα activity during an acute adenovirus infection. We conclude that RIDα coordinates recruitment of these GTP-Rab7 effectors to compartments that would ordinarily be perceived as early endosomes, thereby promoting the degradation of selected cargo.

Phorbol ester promotes endocytosis by activating a factor involved in endosome fusion

1999 ◽  
Vol 112 (15) ◽  
pp. 2549-2557 ◽  
Author(s):  
A. Aballay ◽  
P.D. Stahl ◽  
L.S. Mayorga
Keyword(s):  
Phorbol Ester ◽  
Fluid Phase ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Early Endosomes ◽  
Zinc Depletion ◽  
Fluid Phase Endocytosis ◽  
Endocytic Vesicles

Previous studies indicate that a zinc- and phorbol ester-binding factor is necessary for in vitro endosome fusion and for the effect of Rab5 on endosome fusion. Rab5 is a small GTPase that regulates membrane fusion between early endosomes derived from either receptor-mediated endocytosis or fluid-phase endocytosis. In its GTP-bound form, Rab5 promotes endocytosis and enhances fusion among early endosomes. To determine if PMA stimulates endocytosis by activating a factor required for endosome fusion, we overexpressed wild-type Rab5, a dominant negative mutant (Rab5:S34N), and a GTPase deficient mutant (Rab5:Q79L) in BHK-21 cells. The phorbol ester PMA stimulates endocytosis and increases the number and the size of endocytic vesicles, even in the presence of Rab5:S34N. Zinc depletion with N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and addition of calphostin C (CPC), an inhibitor of PKC that interacts with zinc and phorbol ester binding motifs, inhibited both basal and Rab5-stimulated fluid phase endocytosis. These two reagents also inhibited the size and number of endocytic vesicles promoted by Rab5. These results suggest that PMA stimulates endocytosis by regulating the dynamics of the early endosome compartment.

scholarly journals Mouse Receptor Interacting Protein 3 Does Not Contain a Caspase-Recruiting or a Death Domain but Induces Apoptosis and Activates NF-κB

1999 ◽  
Vol 19 (10) ◽  
pp. 6500-6508 ◽  
Author(s):  
Nanette J. Pazdernik ◽  
David B. Donner ◽  
Mark G. Goebl ◽  
Maureen A. Harrington
Keyword(s):  
Sequence Similarity ◽  
Kinase Domain ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Death Domain ◽  
Interacting Protein ◽  
C Terminus ◽  
Catalytically Active ◽  
Induced Apoptosis

ABSTRACT The death domain-containing receptor superfamily and their respective downstream mediators control whether or not cells initiate apoptosis or activate NF-κB, events critical for proper immune system function. A screen for upstream activators of NF-κB identified a novel serine-threonine kinase capable of activating NF-κB and inducing apoptosis. Based upon domain organization and sequence similarity, this novel kinase, named mRIP3 (mouse receptor interacting protein 3), appears to be a new RIP family member. RIP, RIP2, and mRIP3 contain an N-terminal kinase domain that share 30 to 40% homology. In contrast to the C-terminal death domain found in RIP or the C-terminal caspase-recruiting domain found in RIP2, the C-terminal tail of mRIP3 contains neither motif and is unique. Despite this feature, overexpression of the mRIP3 C terminus is sufficient to induce apoptosis, suggesting that mRIP3 uses a novel mechanism to induce death. mRIP3 also induced NF-κB activity which was inhibited by overexpression of either dominant-negative NIK or dominant-negative TRAF2. In vitro kinase assays demonstrate that mRIP3 is catalytically active and has autophosphorylation site(s) in the C-terminal domain, but the mRIP3 catalytic activity is not required for mRIP3 induced apoptosis and NF-κB activation. Unlike RIP and RIP2, mRIP3 mRNA is expressed in a subset of adult tissues and is thus likely to be a tissue-specific regulator of apoptosis and NF-κB activity. While the lack of a dominant-negative mutant precludes linking mRIP3 to a known upstream regulator, characterizing the expression pattern and the in vitro functions of mRIP3 provides insight into the mechanism(s) by which cells modulate the balance between survival and death in a cell-type-specific manner.

scholarly journals Oxysterol Binding Protein–related Protein 9 (ORP9) Is a Cholesterol Transfer Protein That Regulates Golgi Structure and Function

2009 ◽  
Vol 20 (5) ◽  
pp. 1388-1399 ◽  
Author(s):  
Mike Ngo ◽  
Neale D. Ridgway
Keyword(s):  
Secretory Pathway ◽  
Protein Transport ◽  
Binding Protein ◽  
Dominant Negative ◽  
Ph Domain ◽  
Oxysterol Binding Protein ◽  
Large Gene ◽  
Dominant Negative Variant

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large gene family that differentially localize to organellar membranes, reflecting a functional role in sterol signaling and/or transport. OSBP partitions between the endoplasmic reticulum (ER) and Golgi apparatus where it imparts sterol-dependent regulation of ceramide transport and sphingomyelin synthesis. ORP9L also is localized to the ER–Golgi, but its role in secretion and lipid transport is unknown. Here we demonstrate that ORP9L partitioning between the trans-Golgi/trans-Golgi network (TGN), and the ER is mediated by a phosphatidylinositol 4-phosphate (PI-4P)-specific PH domain and VAMP-associated protein (VAP), respectively. In vitro, both OSBP and ORP9L mediated PI-4P–dependent cholesterol transport between liposomes, suggesting their primary in vivo function is sterol transfer between the Golgi and ER. Depletion of ORP9L by RNAi caused Golgi fragmentation, inhibition of vesicular somatitus virus glycoprotein transport from the ER and accumulation of cholesterol in endosomes/lysosomes. Complete cessation of protein transport and cell growth inhibition was achieved by inducible overexpression of ORP9S, a dominant negative variant lacking the PH domain. We conclude that ORP9 maintains the integrity of the early secretory pathway by mediating transport of sterols between the ER and trans-Golgi/TGN.

Signaling by the Granulocyte-Colony Stimulating Factor Receptor Involves Jak2-Dependent Phosphorylation of Gab2.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2173-2173
Author(s):  
Lin Wang ◽  
Jia Xue ◽  
Seth J. Corey ◽  
Lisa J. Robinson
Keyword(s):  
Fusion Protein ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Erk Phosphorylation ◽  
Stimulating Factor

Abstract Granulocyte colony stimulating factor (G-CSF) is the major cytokine involved in neutrophil production. G-CSF has pleiotropic effects on myeloid cells, initially stimulating proliferation but later promoting differentiation. The specific signaling pathways that mediate the diverse effects of G-CSF remain incompletely understood. Recently, the scaffolding molecule Grb2-associated binder protein 2 (Gab2) was shown to play an important role in G-CSF induced myeloid differentiation (Zhu et al. Blood 2004). Ligand stimulation of the G-CSF receptor results in the rapid phosphorylation of Gab2, but the identity of the responsible kinases and the molecular events dependent on Gab2 phosphorylation remain unclear. Because Janus kinases (Jaks) play a central role in G-CSF signaling, we investigated the involvement of Jaks in G-CSF-stimulated Gab2 phosphorylation using the hematologic DT40 cell line stably transduced with the human G-CSF receptor (DT40GR). Antisense Jak1 and Jak2 constructs expressed in DT40GR cells each produced a marked reduction in their target Jak protein, but only antisense Jak2 reduced G-CSF-stimulated Gab2 phosphorylation. To determine whether Gab2 phosphorylation required Jak2 kinase activity, dominant negative Jak2 mutants lacking catalytic activity were expressed in the DT40GR cells. Expression of dominant negative Jak2 inhibited Gab2 phosphorylation in response to G-CSF. Similarly, treatment with the Jak2-selective kinase inhibitor AG490 markedly reduced G-CSF-dependent Gab2 phosphorylation. Co-immunoprecipitation studies further demonstrated a G-CSF- and Gab2 phosphorylation-dependent association of Jak2 with Gab2 in vivo, which was detectable by 30 seconds after G-CSF stimulation. To determine whether Gab2 was a direct substrate of Jak2, we performed in vitro phosphorylation studies using Gab2-GST fusion protein substrates. Jak2 immunoprecipitated from G-CSF-stimulated cells, but not from control cells, phosphorylated the Gab2 fusion protein. To identify potential Jak2 tyrosine phosphorylation sites in Gab2, we used site-directed mutagenesis to produce three Gab2 tyrosine mutants. Tyrosines 409, 452, and 476 were each replaced by phenylalanine (Y409F, Y452F, and Y476F). The Y452F and Y476F mutations of Gab2 each inhibited G-CSF-stimulated Jak2-dependent phosphorylation of Gab2, both in stably-transfected DT40GR cells and in transiently-transfected 293 cells also transduced with the G-CSF receptor. In contrast, G-CSF-stimulated Gab2 phosphorylation appeared unaffected by the Y409F mutation. We also evaluated downstream events in G-CSF signaling in cells expressing these Gab2 tyrosine- mutants. Akt and Erk phosphorylation following G-CSF stimulation was inhibited by both the Y452F and Y476F Gab2 mutations, but was unaffected by the Y409F mutation. These results suggest that Jak2 may mediate G-CSF differentiation signals through Stat-independent mechanisms.

scholarly journals Upstream stimulatory factor activates the vasopressin promoter via multiple motifs, including a non-canonical E-box

2003 ◽  
Vol 369 (3) ◽  
pp. 549-561 ◽  
Author(s):  
Judy M. COULSON ◽  
Jodie L. EDGSON ◽  
Zoe V. MARSHALL-JONES ◽  
Robert MULGREW ◽  
John P. QUINN ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Upstream Stimulatory Factor ◽  
Mobility Shift ◽  
Transcriptional Initiation ◽  
E Box ◽  
E Boxes ◽  
Stimulatory Factor

We have described previously a complex E-box enhancer (-147) of the vasopressin promoter in small-cell lung cancer (SCLC) extracts [Coulson, Fiskerstrand, Woll and Quinn, (1999) Biochem. J. 344, 961—970]. Upstream stimulatory factor (USF) heterodimers were one of the complexes binding to this site in vitro. We now report that USF overexpression in non-SCLC (NSCLC) cells can functionally activate vasopressin promoter-driven reporters that are otherwise inactive in this type of lung cancer cell. Site-directed mutagenesis and electrophoretic mobility-shift analysis demonstrate that although the −147 E-box contributes, none of the previously predicted E-boxes (-147, −135, −34) wholly account for this USF-mediated activation in NSCLC. 5′ Deletion showed the key promoter region as −52 to +42; however, USF-2 binding was not reliant on the −34 E-box, but on a novel adjacent CACGGG non-canonical E-box at −42 (motif E). This mediated USF binding in both SCLC and USF-2-transfected NSCLC cells. Mutation of motif E or the non-canonical TATA box abolished activity, implying both are required for transcriptional initiation on overexpression of USF-2. Co-transfected dominant negative USF confirmed that binding was required through motif E for function, but that the classical activation domain of USF was not essential. USF-2 bound motif E with 10-fold lower affinity than the −147 E-box. In NSCLC, endogenous USF-2 expression is low, and this basal level appears to be insufficient to activate transcription of arginine vasopressin (AVP). In summary, we have demonstrated a novel mechanism for USF activation, which contributes to differential vasopressin expression in lung cancer.

scholarly journals Elevated Endosomal Cholesterol Levels in Niemann-Pick Cells Inhibit Rab4 and Perturb Membrane Recycling

2004 ◽  
Vol 15 (10) ◽  
pp. 4500-4511 ◽  
Author(s):  
Amit Choudhury ◽  
Deepak K. Sharma ◽  
David L. Marks ◽  
Richard E. Pagano
Keyword(s):  
Dominant Negative ◽  
Cholesterol Depletion ◽  
Membrane Recycling ◽  
Normal Human Skin ◽  
Lipid Storage Disease ◽  
Cholesterol Levels ◽  
Early Endosomes ◽  
Niemann Pick ◽  
Gdp Dissociation Inhibitor

In normal human skin fibroblasts (HSFs), fluorescent glycosphingolipid analogues are endocytosed and sorted into two pools, one that is recycled to the plasma membrane and one that is transported to the Golgi complex. Here, we investigated glycosphingolipid recycling in Niemann-Pick type A and C lipid storage disease fibroblasts (NPFs). Cells were incubated with a fluorescent analogue of lactosylceramide (LacCer) at 16°C to label early endosomes (EEs), shifted to 37°C, and lipid recycling was quantified. Using dominant negative rabs, we showed that, in normal HSFs, LacCer recycling was rapid (t1/2 ∼8 min) and mainly rab4-dependent. In NPFs, LacCer recycling was delayed (t1/2 ∼30–40 min), and rab4-dependent recycling was absent, whereas rab11-dependent recycling predominated. Transferrin recycling via the rab4 pathway was similarly perturbed in NPFs. Compared with normal HSFs, EEs in NPFs showed high cholesterol levels and an altered organization of rab4. In vitro extraction of rab4 (but not rab11) with GDP dissociation inhibitor was severely attenuated in NPF endosomal fractions. This impairment was reversed with cholesterol depletion of isolated endosomes or with high-salt treatment of endosomes. These data suggest that abnormal membrane recycling in NPFs results from specific inhibition of rab4 function by excess cholesterol in EEs.

scholarly journals Coordinated regulation of AP2 uncoating from clathrin-coated vesicles by rab5 and hRME-6

2008 ◽  
Vol 183 (3) ◽  
pp. 499-511 ◽  
Author(s):  
Sophia Semerdjieva ◽  
Barry Shortt ◽  
Emma Maxwell ◽  
Sukhdeep Singh ◽  
Paul Fonarev ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Coated Vesicles ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Steady State Levels ◽  
Endocytic Vesicles ◽  
Interfering Rna

Here we investigate the role of rab5 and its cognate exchange factors rabex-5 and hRME-6 in the regulation of AP2 uncoating from endocytic clathrin-coated vesicles (CCVs). In vitro, we show that the rate of AP2 uncoating from CCVs is dependent on the level of functional rab5. In vivo, overexpression of dominant-negative rab5S34N, or small interfering RNA (siRNA)–mediated depletion of hRME-6, but not rabex-5, resulted in increased steady-state levels of AP2 associated with endocytic vesicles, which is consistent with reduced uncoating efficiency. hRME-6 guanine nucleotide exchange factor activity requires hRME-6 binding to α-adaptin ear, which displaces the ear-associated μ2 kinase AAK1. siRNA-mediated depletion of hRME-6 increases phospho-μ2 levels, and expression of a phosphomimetic μ2 mutant increases levels of endocytic vesicle-associated AP2. Depletion of hRME-6 or rab5S35N expression also increases the levels of phosphoinositide 4,5-bisphosphate (PtdIns(4,5)P2) associated with endocytic vesicles. These data are consistent with a model in which hRME-6 and rab5 regulate AP2 uncoating in vivo by coordinately regulating μ2 dephosphorylation and PtdIns(4,5)P2 levels in CCVs.

scholarly journals Identification of a druggable protein-protein interaction site between mutant p53 and its stabilizing chaperone DNAJA1

2020 ◽  
pp. jbc.RA120.014749
Author(s):  
Xin Tong ◽  
Dandan Xu ◽  
Rama K. Mishra ◽  
Ryan D Jones ◽  
Leyu Sun ◽  
...  
Keyword(s):  
Small Molecule ◽  
Tp53 Gene ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Dominant Negative Effect ◽  
Mutant P53 ◽  
Missense Mutations ◽  
Oncogenic Function

The TP53 gene is the most frequently mutated gene in human cancers, and the majority of TP53 mutations are missense mutations. As a result, these mutant p53 (mutp53) either directly lose wild-type p53 (wtp53) tumor suppressor function or exhibit a dominant negative effect over wtp53. In addition, some mutp53 have acquired new oncogenic function (gain of function). Therefore, targeting mutp53 for its degradation, may serve as a promising strategy for cancer prevention and therapy. Based on our previous finding that farnesylated DNAJA1 is a crucial chaperone in maintaining mutp53 stabilization, and by using an in silico approach, we built 3-D homology models of human DNAJA1 and mutp53R175H proteins, identified the interacting pocket in the DNAJA1-mutp53R175H complex, and found one critical druggable small molecule binding  site in the DNAJA1 glycine/phenylalanine rich region. We confirmed that the interacting pocket in the DNAJA1-mutp53R175H complex was crucial for stabilizing mutp53R175H using a site-directed mutagenesis approach. We further screened a drug-like library to identify a promising small molecule hit (GY1-22) against the interacting pocket in DNAJA1-mutp53R175H complex. The GY1-22 compound displayed an effective activity against DNAJA1-mutp53R175H complex. Treatment with GY1-22 significantly reduced mutp53 protein levels, enhanced Waf1p21 expression, suppressed cyclin D1 expression, and inhibited mutp53-driven pancreatic cancer growth both in vitro and in vivo. Together, our results indicate that the interacting pocket in the DNAJA1-mutp53R175H complex is critical for mutp53’s stability and oncogenic function, and DNAJA1 is a robust therapeutic target for developing the efficient small molecule inhibitors against oncogenic mutp53.

scholarly journals Localized PtdIns 3,5-P2 synthesis to regulate early endosome dynamics and fusion

2006 ◽  
Vol 291 (2) ◽  
pp. C393-C404 ◽  
Author(s):  
Ognian C. Ikonomov ◽  
Diego Sbrissa ◽  
Assia Shisheva
Keyword(s):  
Morphological Changes ◽  
Degradation Pathway ◽  
Dominant Negative ◽  
Functional Requirements ◽  
Cos Cells ◽  
Cytoplasmic Vacuoles ◽  
Early Endosomes ◽  
Gradual Development ◽  
Early Expression

Perturbations in the intracellular PtdIns 3,5-P2 pool or the downstream transmission of PtdIns 3,5-P2 signals often result in a gradual development of gross morphological changes in the pleiomorphic multivesicular endosomes, culminating with the appearance of cytoplasmic vacuoles. To identify the onset of PtdIns 3,5-P2 functional requirements along the endocytic system, in this study we characterized the morphological changes associated with early expression of the dominant-negative kinase-deficient form (K1831E) of the PtdIns 3,5-P2-producing kinase PIKfyve, before the formation of cytoplasmic vacuoles in transfected COS cells. Enlarged PIKfyveK1831E-positive vesicles co-localizing with dilated EEA1- and Rab5aWT-positive perinuclear endosomes were observed (WT, wild type). This was dependent on the presence of active forms of Rab5 and the generation of PtdIns 3-P-enriched platforms on early endosomess. Because PIKfyveWT did not substantially colocalize with EEA1- or Rab5-positive endosomes in COS cells, the dynamic PIKfyve-catalyzed PtdIns 3-to-PtdIns 3,5-P2 switch was suggested to drive away PIKfyveWT from early endosomes toward later compartments. Late endosomes/lysosomes marked by LAMP1 or Rab7 were dislocated from their typical perinuclear position upon PIKfyveK1831E early expression. Cytosols derived from cells stably expressing PIKfyveK1831E stimulated endosome fusion in vitro, whereas PIKfyveWT-enriched cytosols had the opposite effect, consistent with PtdIns 3,5-P2 production negatively regulating the endosome fusion. Together, our data indicate that PtdIns 3,5-P2 defines specific endosome platforms at the onset of the degradation pathway to regulate the complex process of membrane remodeling and dynamics.

scholarly journals Mutant EGFR is a preferred SMURF2 substrate of ubiquitination: role in enhanced receptor stability and TKI sensitivity

2020 ◽  
Author(s):  
Paramita Ray ◽  
Krishnan Raghunathan ◽  
Aarif Ahsan ◽  
Uday Sankar Allam ◽  
Shirish Shukla ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
Super Resolution ◽  
Receptor Internalization ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Steady State Levels ◽  
Epidermal Growth ◽  
Optical Reconstruction ◽  
Super Resolution Microscopy

ABSTRACTWe previously reported that differential protein degradation of TKI-sensitive [L858R, del(E746-A750)] and resistant (T790M) epidermal growth factor receptor (EGFR) mutants upon erlotinib treatment correlates with drug sensitivity. However, the molecular mechanism remains unclear. We also reported SMAD ubiquitination regulatory factor 2 (SMURF2) ligase activity is important in stabilizing EGFR. Here, using in vitro and in vivo ubiquitination assays, mass spectrometry, and super-resolution microscopy, we show SMURF2-EGFR functional interaction is critical in receptor stability and TKI sensitivity. We found that L858R/T790M EGFR is a preferred substrate of SMURF2-UBCH5 (an E3-E2) complex-mediated K63-linked polyubiquitination, which preferentially stabilizes mutant receptor. We identified four lysine (K) residues (K721, 846, 1037 and 1164) as the sites of ubiquitination and replacement of K to acetylation-mimicking asparagine (Q) at K1037 position in L858R/T790M background converts the stable protein sensitive to erlotinib-induced degradation. Using STochastic Optical Reconstruction Microscopy (STORM) imaging, we show that SMURF2 presence allows longer membrane retention of activated EGFR upon EGF treatment, whereas, siRNA-mediated SMURF2 knockdown fastens receptor endocytosis and lysosome enrichment. In an erlotinib-sensitive PC9 cells, SMURF2 overexpression increased EGFR levels with improved erlotinib tolerance, whereas, SMURF2 knockdown decreased EGFR steady state levels in NCI-H1975 and PC9-AR cells to overcome erlotinib and AZD-9291 resistance respectively. Additionally, by genetically altering the SMURF2-UBCH5 complex formation destabilized EGFR. Together, we propose that SMURF2-mediated preferential polyubiquitination of L858R/T790M EGFR may be competing with acetylation-mediated receptor internalization to provide enhanced receptor stability and that disruption of the E3-E2 complex may be an attractive alternate to overcome TKI resistance.

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