Volume sensitivity of cation-Cl− cotransporters is modulated by the interaction of two kinases: Ste20-related proline-alanine-rich kinase and WNK4

2006 ◽  
Vol 290 (1) ◽  
pp. C134-C142 ◽  
Author(s):  
Kenneth B. E. Gagnon ◽  
Roger England ◽  
Eric Delpire
Keyword(s):  
Catalytic Activity ◽  
Xenopus Laevis Oocytes ◽  
Transport Function ◽  
Yeast Two Hybrid ◽  
Functional Interaction ◽  
Catalytic Domains ◽  
Volume Sensitivity ◽  
Two Hybrid

In the present study, we have demonstrated functional interaction between Ste20-related proline-alanine-rich kinase (SPAK), WNK4 [with no lysine (K)], and the widely expressed Na+-K+-2Cl− cotransporter type 1 (NKCC1). NKCC1 function, which we measured in Xenopus laevis oocytes under both isosmotic (basal) and hyperosmotic (stimulated) conditions, was unaffected when SPAK and WNK4 were expressed alone. In contrast, expression of both kinases with NKCC1 resulted in a significant increase in cotransporter activity and an insensitivity to external osmolarity or cell volume. NKCC1 activation is dependent on the catalytic activity of SPAK and likely also of WNK4, because mutations in their catalytic domains result in an absence of cotransporter stimulation. The results of our yeast two-hybrid experiments suggest that WNK4 does not interact directly with NKCC1 but does interact with SPAK. Functional experiments demonstrated that the binding of SPAK to WNK4 was also required because a SPAK-interaction-deficient WNK4 mutant (Phe997Ala) did not increase NKCC1 activity. We also have shown that the transport function of K+-Cl− cotransporter type 2 (KCC2), a neuron-specific KCl cotransporter, was diminished by the expression of both kinases under both isosmotic and hyposmotic conditions. Our data are consistent with WNK4 interacting with SPAK, which in turn phosphorylates and activates NKCC1 and phosphorylates and deactivates KCC2.

Apontic binds the translational repressor Bruno and is implicated in regulation of oskar mRNA translation

Development ◽  
1999 ◽  
Vol 126 (6) ◽  
pp. 1129-1138 ◽  
Author(s):  
Y.S. Lie ◽  
P.M. Macdonald
Keyword(s):  
Translational Control ◽  
Untranslated Region ◽  
Rna Binding ◽  
Mrna Translation ◽  
Posterior Pole ◽  
Translational Repression ◽  
Yeast Two Hybrid ◽  
Functional Interaction ◽  
Translational Repressor ◽  
Two Hybrid

The product of the oskar gene directs posterior patterning in the Drosophila oocyte, where it must be deployed specifically at the posterior pole. Proper expression relies on the coordinated localization and translational control of the oskar mRNA. Translational repression prior to localization of the transcript is mediated, in part, by the Bruno protein, which binds to discrete sites in the 3′ untranslated region of the oskar mRNA. To begin to understand how Bruno acts in translational repression, we performed a yeast two-hybrid screen to identify Bruno-interacting proteins. One interactor, described here, is the product of the apontic gene. Coimmunoprecipitation experiments lend biochemical support to the idea that Bruno and Apontic proteins physically interact in Drosophila. Genetic experiments using mutants defective in apontic and bruno reveal a functional interaction between these genes. Given this interaction, Apontic is likely to act together with Bruno in translational repression of oskar mRNA. Interestingly, Apontic, like Bruno, is an RNA-binding protein and specifically binds certain regions of the oskar mRNA 3′ untranslated region.

scholarly journals Neurotoxin Merging: A Strategy Deployed by the Venom of the Spider Cupiennius salei to Potentiate Toxicity on Insects

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 250 ◽  
Author(s):  
Benjamin Clémençon ◽  
Lucia Kuhn-Nentwig ◽  
Nicolas Langenegger ◽  
Lukas Kopp ◽  
Steve Peigneur ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Direct Interaction ◽  
Structural Type ◽  
Structure Type ◽  
Open Loop ◽  
Cytolytic Activity ◽  
Xenopus Laevis Oocytes ◽  
Cupiennius Salei

The venom of Cupiennius salei is composed of dozens of neurotoxins, with most of them supposed to act on ion channels. Some insecticidal monomeric neurotoxins contain an α-helical part besides their inhibitor cystine knot (ICK) motif (type 1). Other neurotoxins have, besides the ICK motif, an α-helical part of an open loop, resulting in a heterodimeric structure (type 2). Due to their low toxicity, it is difficult to understand the existence of type 2 peptides. Here, we show with the voltage clamp technique in oocytes of Xenopus laevis that a combined application of structural type 1 and type 2 neurotoxins has a much more pronounced cytolytic effect than each of the toxins alone. In biotests with Drosophila melanogaster, the combined effect of both neurotoxins was enhanced by 2 to 3 log units when compared to the components alone. Electrophysiological measurements of a type 2 peptide at 18 ion channel types, expressed in Xenopus laevis oocytes, showed no effect. Microscale thermophoresis data indicate a monomeric/heterodimeric peptide complex formation, thus a direct interaction between type 1 and type 2 peptides, leading to cell death. In conclusion, peptide mergers between both neurotoxins are the main cause for the high cytolytic activity of Cupiennius salei venom.

Interaction of Plasminogen Activator Inhibitor-2 and Proteasome Subunit, Beta Type 1

2004 ◽  
Vol 36 (1) ◽  
pp. 42-46 ◽  
Author(s):  
Jing Fan ◽  
Yu-Qing Zhang ◽  
Ping Li ◽  
Min Hou ◽  
Li Tan ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Acid Fragment ◽  
Intracellular Proteins ◽  
Two Hybrid ◽  
Activator Inhibitor

Abstract The apoptosis protection by plasminogen activator inhibitor-2(PAI-2) is dependent on a 33 amino acid fragment between helix C and D of PAI-2 which is probably due to the interaction of PAI-2 with unknown intracellular proteins. In this study, we used the fragment between helix C and D of PAI-2 as bait to screen a HeLa cell cDNA library constructed during apoptosis in a yeast two-hybrid system and retrieved a clone encoding 241 amino acids of proteasome (prosome, macropain) subunit, beta type 1(PSMβ1) which plays important roles in NF-κB activation. GST-pulldown experiments confirmed the interaction between PAI-2 and PSMβ1 in vitro. These data suggest that the antiapoptosis activity of PAI-2 is probably related to its interation with PSMβ1.

scholarly journals PDZD8 Is a Novel Gag-Interacting Factor That Promotes Retroviral Infection

2010 ◽  
Vol 84 (17) ◽  
pp. 8990-8995 ◽  
Author(s):  
Matthew S. Henning ◽  
Scott G. Morham ◽  
Stephen P. Goff ◽  
Mojgan H. Naghavi
Keyword(s):  
Viral Entry ◽  
Coiled Coil ◽  
Yeast Two Hybrid ◽  
Retroviral Infection ◽  
Gag Protein ◽  
Cellular Factors ◽  
Immunodeficiency Virus ◽  
Two Hybrid ◽  
Hiv 1

ABSTRACT In a yeast two-hybrid screen for cellular factors that could interact with human immunodeficiency virus type 1 (HIV-1) Gag protein, we identified PDZD8 and confirmed the interaction by coimmunoprecipitation (co-IP). PDZD8 overexpression promoted the initiation of reverse transcription and increased infection by pseudotyped retroviruses independent of the route of viral entry, while transient knockdown of endogenous levels decreased HIV-1 infection. A mutant of PDZD8 lacking a predicted coiled-coil domain in its Gag-interacting region failed to bind Gag and promote HIV-1 infection, identifying the domain of PDZD8 required for mediating these effects. As such, we identify PDZD8 as a novel positive mediator of retroviral infection.

Functional interaction between the SSeCKS scaffolding protein and the cytoplasmic domain of β1,4-galactosyltransferase

2001 ◽  
Vol 114 (12) ◽  
pp. 2291-2300 ◽  
Author(s):  
Michael J. Wassler ◽  
Cynthia I. Foote ◽  
Irwin H. Gelman ◽  
Barry D. Shur
Keyword(s):  
Cytoplasmic Domain ◽  
Dominant Negative ◽  
Scaffolding Protein ◽  
Yeast Two Hybrid ◽  
Functional Interaction ◽  
Filament Dynamics ◽  
Golgi Region ◽  
Subcellular Domains ◽  
Induced Aggregation ◽  
Two Hybrid

The β1,4-galactosyltransferase family contains at least seven unique gene products, of which β1,4-galactosyltransferase I (GalT) is the most exhaustively studied. GalT exists in the Golgi complex, similar to many other glycosyltransferases, as well as on the cell surface, where it functions as a signaling receptor for extracellular glycoside ligands. When expressed on the surface, GalT associates with the cytoskeleton and, upon ligand-induced aggregation, induces cell-type specific intracellular signal cascades. In an effort to define the mechanisms by which surface GalT exerts these intracellular effects, we used the yeast two-hybrid system to identify proteins that specifically interact with the GalT cytoplasmic domain. The yeast two-hybrid screen identified two distinct clones (1.12 and 2.52) that showed identity to portions of SSeCKS (Src Suppressed CKinase Substrate). SSeCKS is a previously defined kinase and cytoskeleton scaffolding protein whose subcellular distribution and functions are remarkably similar to those attributed to GalT. Both SSeCKS and GalT have been localized to the perinuclear/Golgi region as well as to filopodia/lamellipodia. SSeCKS and GalT have been implicated in regulating cell growth, actin filament dynamics, and cell spreading. Interestingly, 1.12 and 2.52-GFP constructs were localized to subcellular domains that correlated with the two purported subcellular distributions for GalT; 2.52 being confined to the Golgi, whereas 1.12 localized primarily to filopodia. Coimmunoprecipitation assays demonstrate stable binding between the GalT cytoplasmic domain and the 1.12 and 2.52 domains of SSeCKS in appropriately transfected cells. Similar assays demonstrate binding between the endogenous GalT and SSeCKS proteins also. Coimmunoprecipitation assays were performed in both directions and produced similar results (i.e. using either anti-GalT domain or anti-SSeCKS domain antibodies as the precipitating reagent). A functional interaction between the GalT cytoplasmic domain and SSeCKS was illustrated by the ability of either the 1.12 or 2.52 SSeCKS domain to restore a normal adhesive phenotype in cells overexpressing the TL-GFP dominant negative construct. TL-GFP is composed of the GalT cytoplasmic and transmembrane domains fused to GFP, and leads to a loss of cell adhesion on laminin by displacing the endogenous GalT from its cytoskeleton binding sites. This is the first reported interaction between a glycosyltransferase and a scaffolding protein, and suggests that SSeCKS serve to integrate the various functions ascribed to the GalT cytoplasmic domain.

scholarly journals The Second Catalytic Domain of Protein Tyrosine Phosphatase δ (PTPδ) Binds to and Inhibits the First Catalytic Domain of PTPς

1998 ◽  
Vol 18 (5) ◽  
pp. 2608-2616 ◽  
Author(s):  
Megan J. Wallace ◽  
Christopher Fladd ◽  
Jane Batt ◽  
Daniela Rotin
Keyword(s):  
Mammalian Cells ◽  
Catalytic Domain ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Regulatory Function ◽  
Yeast Two Hybrid ◽  
Protein Tyrosine ◽  
Catalytic Domains ◽  
Two Hybrid

ABSTRACT The LAR family protein tyrosine phosphatases (PTPs), including LAR, PTPδ, and PTPς, are transmembrane proteins composed of a cell adhesion molecule-like ectodomain and two cytoplasmic catalytic domains: active D1 and inactive D2. We performed a yeast two-hybrid screen with the first catalytic domain of PTPς (PTPς-D1) as bait to identify interacting regulatory proteins. Using this screen, we identified the second catalytic domain of PTPδ (PTPδ-D2) as an interactor of PTPς-D1. Both yeast two-hybrid binding assays and coprecipitation from mammalian cells revealed strong binding between PTPς-D1 and PTPδ-D2, an association which required the presence of the wedge sequence in PTPς-D1, a sequence recently shown to mediate D1-D1 homodimerization in the phosphatase RPTPα. This interaction was not reciprocal, as PTPδ-D1 did not bind PTPς-D2. Addition of a glutathione S-transferase (GST)–PTPδ-D2 fusion protein (but not GST alone) to GST–PTPς-D1 led to ∼50% inhibition of the catalytic activity of PTPς-D1, as determined by an in vitro phosphatase assay againstp-nitrophenylphosphate. A similar inhibition of PTPς-D1 activity was obtained with coimmunoprecipitated PTPδ-D2. Interestingly, the second catalytic domains of LAR (LAR-D2) and PTPς (PTPς-D2), very similar in sequence to PTPδ-D2, bound poorly to PTPς-D1. PTPδ-D1 and LAR-D1 were also able to bind PTPδ-D2, but more weakly than PTPς-D1, with a binding hierarchy of PTPς-D1>>PTPδ-D1>LAR-D1. These results suggest that association between PTPς-D1 and PTPδ-D2, possibly via receptor heterodimerization, provides a negative regulatory function and that the second catalytic domains of this and likely other receptor PTPs, which are often inactive, may function instead to regulate the activity of the first catalytic domains.

scholarly journals Vpx proteins of SIVmac239 and HIV-2ROD interact with the cytoskeletal protein α-actinin 1

2004 ◽  
Vol 85 (11) ◽  
pp. 3291-3303 ◽  
Author(s):  
Sandra M. Mueller ◽  
Ronny Jung ◽  
Sigrid Weiler ◽  
Sabine M. Lang
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Cytoskeletal Protein ◽  
Yeast Two Hybrid ◽  
Nuclear Localization Signals ◽  
Quiescent Cells ◽  
Immunodeficiency Virus ◽  
Sequence Elements ◽  
Two Hybrid

vpx genes of human immunodeficiency virus type 2 (HIV-2) and immunodeficiency viruses from macaques (SIVmac), sooty mangabeys (SIVsm) and red-capped mangabeys (SIVrcm) encode a 112 aa protein that is packed into virion particles via interaction with the p6 domain of p55gag. Vpx localizes to the nucleus when expressed in the absence of other viral proteins. Moreover, Vpx is necessary for efficient nuclear import of the pre-integration complex (PIC) and critical for virus replication in quiescent cells, such as terminally differentiated macrophages and memory T cells. Vpx does not contain sequence elements that are homologous to previously characterized nuclear localization signals (NLSs). Therefore, it is likely that Vpx-dependent import of the PIC is mediated by interaction of Vpx with cellular proteins that do not belong to the classical import pathways. By using a yeast two-hybrid screen, α-actinin 1, a cytoskeletal protein, was identified to interact with SIVmac239 Vpx. Interestingly, deletion of the proline-rich C-terminal domain (aa 101–112) of Vpx, which is important for nuclear localization, resulted in loss of interaction with α-actinin 1. These findings suggest that the interaction with α-actinin 1 may play an important role in the transport of Vpx to the nucleus and in Vpx-mediated nuclear import of the PIC.

scholarly journals The 32-Kilodalton Subunit of Replication Protein A Interacts with Menin, the Product of the MEN1 Tumor Suppressor Gene

2003 ◽  
Vol 23 (2) ◽  
pp. 493-509 ◽  
Author(s):  
Karen E. Sukhodolets ◽  
Alison B. Hickman ◽  
Sunita K. Agarwal ◽  
Maxim V. Sukhodolets ◽  
Victor H. Obungu ◽  
...  
Keyword(s):  
Multiple Endocrine Neoplasia ◽  
Multiple Endocrine Neoplasia Type ◽  
Protein A ◽  
Suppressor Gene ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Endocrine Neoplasia ◽  
Two Hybrid

ABSTRACT Menin is a 70-kDa protein encoded by MEN1, the tumor suppressor gene disrupted in multiple endocrine neoplasia type 1. In a yeast two-hybrid system based on reconstitution of Ras signaling, menin was found to interact with the 32-kDa subunit (RPA2) of replication protein A (RPA), a heterotrimeric protein required for DNA replication, recombination, and repair. The menin-RPA2 interaction was confirmed in a conventional yeast two-hybrid system and by direct interaction between purified proteins. Menin-RPA2 binding was inhibited by a number of menin missense mutations found in individuals with multiple endocrine neoplasia type 1, and the interacting regions were mapped to the N-terminal portion of menin and amino acids 43 to 171 of RPA2. This region of RPA2 contains a weak single-stranded DNA-binding domain, but menin had no detectable effect on RPA-DNA binding in vitro. Menin bound preferentially in vitro to free RPA2 rather than the RPA heterotrimer or a subcomplex consisting of RPA2 bound to the 14-kDa subunit (RPA3). However, the 70-kDa subunit (RPA1) was coprecipitated from HeLa cell extracts along with RPA2 by menin-specific antibodies, suggesting that menin binds to the RPA heterotrimer or a novel RPA1-RPA2-containing complex in vivo. This finding was consistent with the extensive overlap in the nuclear localization patterns of endogenous menin, RPA2, and RPA1 observed by immunofluorescence.

scholarly journals Herpes simplex virus type 2 membrane protein UL56 associates with the kinesin motor protein KIF1A

2005 ◽  
Vol 86 (3) ◽  
pp. 527-533 ◽  
Author(s):  
Tetsuo Koshizuka ◽  
Yasushi Kawaguchi ◽  
Yukihiro Nishiyama
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Yeast Two Hybrid ◽  
Infected Cells ◽  
Simplex Virus ◽  
Two Hybrid

The herpes simplex virus UL56 gene product is a C-terminal-anchored, type II membrane protein of unknown function. UL56 was found to interact with KIF1A, a member of the kinesin-3 family, in a yeast two-hybrid screen and a GST pull-down assay. KIF1A mediates the transport of synaptic vesicle precursors and is essential for the function and viability of neurons. When overexpressed, KIF1A co-localized with full-sized UL56, but no clear co-localization was observed when co-expressed with the UL56 mutant protein lacking its C-terminal transmembrane domain (TMD). Although the C-terminal TMD was not essential for the interaction with KIF1A in the yeast two-hybrid screen and GST pull-down assays, these results indicate that the C-terminal TMD, as well as aa 69–217, of UL56 are important for the interaction with KIF1A in vivo. The hypothesis that the UL56 protein affects vesicular trafficking in infected cells, potentially by acting as a receptor for motor proteins in neurons, is discussed.

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