Abstract 957: A Novel Interaction of Cardiac Troponin T with PKA Regulatory Subunits I and II

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
C. Amelia Sumandea ◽  
Mary L GarcÃa-CazarÃn ◽  
Catherine H Bozio ◽  
C. William Balke ◽  
R. John Solaro ◽  
...  
Keyword(s):  
Cardiac Troponin ◽  
Troponin T ◽  
Positive Interactions ◽  
Intracellular Camp ◽  
Cdna Clones ◽  
Type I ◽  
Adrenergic Stimulation ◽  
Yeast Two Hybrid ◽  
Rabbit Reticulocyte Lysate System ◽  
Two Hybrid

Increasing evidence suggests that cardiac myofilament activity is modulated by posttransla-tional modifications of cardiac troponin (cTn). In an attempt to identify novel proteins that associate with and modify cTn, we used the full-length human cardiac TnT as bait in the yeast two-hybrid system, and a human heart cDNA library as prey. Our screen of > 2×10 6 cDNA clones with hcTnT as bait, identified >1000 clones that grew on high-stringency plates, and 210 that turned the cells blue in the presence of α-gal, indicating positive interactions with hcTnT. DNA sequencing of the 210 clones led to the identification of 10 gene products: cTnI (identified in 180 clones), PKA-RIα, MLC2v, Dystrophin, PDZ and LIM domain 5, CGI-94, Muscle Creatine Kinase, ANF, Mitofusin-2 and an uncharacterized gene. HA-tagged clones (above) and myc-tagged cTnT were individually expressed using a rabbit reticulocyte lysate system and immunoprecipitated (IP) by agarose conjugated anti-myc and anti-HA antibodies. Therefore, the interaction of these proteins with cTnT was verified in a mammalian cellular milieu. Our finding suggests that cTnT, through its interaction with PKA regulatory domains (R), anchors PKA to the myofilament in close proximity to cTnI. This would explain the rapid, PKA dependent, phosphorylation of cTnI during increase intracellular cAMP levels following beta-adrenergic stimulation. While, PKA type I holoenzyme is predominantly cytoplasmic, the majority of PKA type II associates with cytoskeletal structures. Binding of PKA-RI and PKA-RII to intact and truncated forms of cTnT (amino acids 1–117; 118 –288; 1–180; 181–288) were determined by yeast two-hybrid and by IP assays. Our results demonstrate, for the first time, that both PKA-RI and RII associate with cTnT, and PKA-RI binds to a specific region of cTnT-1–180 fragment, whereas PKA-RII binds to a region of cTnT-181–288 fragment. PKA-dependent phosphorylation of cTnI-Ser 23/24 has profound effects on cardiac dynamics: modulating cross-bridge kinetics, energy consumption and calcium sensitivity. Therefore, it is important to have a mechanistic understanding of events that mediate and affect PKA-dependent cTnI phosphorylation.

scholarly journals Ebolavirus VP35 Interacts with the Cytoplasmic Dynein Light Chain 8

2009 ◽  
Vol 83 (13) ◽  
pp. 6952-6956 ◽  
Author(s):  
Toru Kubota ◽  
Mayumi Matsuoka ◽  
Tsung-Hsien Chang ◽  
Mike Bray ◽  
Steven Jones ◽  
...  
Keyword(s):  
Life Cycle ◽  
Light Chain ◽  
Viral Protein ◽  
Cytoplasmic Dynein ◽  
Type I ◽  
Dynein Light Chain ◽  
Yeast Two Hybrid ◽  
Binding Partners ◽  
Mapping Analysis ◽  
Two Hybrid

ABSTRACT The viral protein VP35 of ebolavirus (EBOV) is implicated to have diverse roles in the viral life cycle. We employed a yeast two-hybrid screen to search for VP35 binding partners and identified the cytoplasmic dynein light chain (DLC8) as a protein that interacts with VP35. Mapping analysis unraveled a consensus motif, SQTQT, within VP35 through which VP35 binds to DLC8. The disruption of DLC8 binding does not affect the ability of VP35 to inhibit type I IFN production. Given that VP35 from various EBOV species interacts with DLC8, this interaction may have a role in regulating the EBOV life cycle.

The 30-kD Domain of Protein 4.1 Mediates Its Binding to the Carboxyl Terminus of pICln, a Protein Involved in Cellular Volume Regulation

Blood ◽  
1998 ◽  
Vol 92 (4) ◽  
pp. 1442-1447 ◽  
Author(s):  
Chieh-Ju C. Tang ◽  
Tang K. Tang
Keyword(s):  
Hybrid System ◽  
Volume Regulation ◽  
Carboxyl Terminus ◽  
Cdna Clones ◽  
Yeast Two Hybrid ◽  
Protein 4.1 ◽  
Yeast Two Hybrid System ◽  
Cellular Volume ◽  
Direct Association ◽  
Two Hybrid

Abstract Erythrocyte protein 4.1 (P4.1) is an 80-kD cytoskeletal protein that is important for the maintenance of the structural integrity and flexibility of the red blood cell membrane. Limited chymotryptic digestion of erythroid P4.1 yields 4 structural domains corresponding to the 30-, 16-, 10-, and 22/24-kD domains. Using a yeast two-hybrid system, we isolated cDNA clones encoding pICln that specifically interacts with the 30-kD domain of P4.1. In this report, we show that the carboxyl-terminus (amino acid residues 103-237) of pICln binds to the 30-kD domain of P4.1 in a yeast two-hybrid system. The direct association between the 30-kD domain of P4.1 and pICln was further confirmed by the following findings: (1) the S35-methione–labeled pICln specifically bound to both GST/P4.1-80 (80 kD) and GST/P4.1-30 (30 kD) fusion proteins, but not to the proteins that lack the 30-kD domain; (2) coimmunoprecipitation analysis of the cell extracts from transfected SiHa cells showed that pICln and P4.1 associate in transfected cells. It was reported that pICln can form a complex with actin and may play a role involved in cellular volume regulation. The direct association between P4.1 and pICln suggests that pICln may link P4.1-bound cytoskeletal elements to an unidentified volume-sensitive chloride channel. © 1998 by The American Society of Hematology.

Screening and Verification of Host Proteins Interacting with Neospora Caninum AMA1 Using a Yeast Two-hybrid System

2020 ◽  
Author(s):  
Baoyi Wu ◽  
Chunmei Jin ◽  
Yinan Wang ◽  
Yan Zhang ◽  
Tianyu Zheng ◽  
...  
Keyword(s):  
Neospora Caninum ◽  
Transmembrane Protein ◽  
Type I ◽  
Filamin A ◽  
Biological Functions ◽  
Yeast Two Hybrid ◽  
Apical Membrane Antigen 1 ◽  
Invasion Mechanism ◽  
First Time ◽  
Two Hybrid

Abstract Background: Neospora caninum apical membrane antigen 1 (NcAMA1) is a conservative type I transmembrane protein that is secreted by the microneme to the surface of the parasite, and is a key component of the invasion mechanism. In order to explore further the biological functions of NcAMA1 in the process of parasite invasion, we conducted research on NcAMA1 and its interacting partners. Methods: In this study, Vero cell yeast two-hybrid (Y2H) cDNA library was constructed. Using the constructed recombinant vector pGBKT7-NcAMA1 as bait, the Y2H system was used to screen the proteins that interact with NcAMA1. In addition, the interaction between NcAMA1 and the screened transmembrane emp24 domain trafficking protein 2 （Tmed2）was further verified by one-to-one Y2H experiments and pull-down, and the role of Tmed2 protein in the process of N. caninum invasion was initially verified by RNA silencing and antibody blocking experiments.Results: Our results show that, through the Y2H experiment, we have identified two proteins that interact with NcAMA1, which are the Chlorocebus sabaeus filamin A, alpha and Chlorocebus sabaeus Tmed2. When the expression of Tmed2 protein decreased or blocked, the invasion rate of N. caninum was increased.Conclusions: These findings give us a deeper understanding of the biological functions of NcAMA1, and for the first time suggest that Tmed2 may be involved in the process invasion by of N. caninum, inhibiting the invasion of parasites by interacting with the protein secreted by N. caninum.

scholarly journals Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits.

2005 ◽  
Vol 52 (1) ◽  
pp. 117-128 ◽  
Author(s):  
Frantz Liszewska ◽  
Dali Gaganidze ◽  
Agnieszka Sirko
Keyword(s):  
Escherichia Coli ◽  
Hybrid System ◽  
Nicotiana Plumbaginifolia ◽  
Cdna Clones ◽  
Serine Acetyltransferase ◽  
Yeast Two Hybrid ◽  
Cysteine Synthase ◽  
Yeast Two Hybrid System ◽  
Potential Signal ◽  
Two Hybrid

We applied the yeast two-hybrid system for screening of a cDNA library of Nicotiana plumbaginifolia for clones encoding plant proteins interacting with two proteins of Escherichia coli: serine acetyltransferase (SAT, the product of cysE gene) and O-acetylserine (thiol)lyase A, also termed cysteine synthase (OASTL-A, the product of cysK gene). Two plant cDNA clones were identified when using the cysE gene as a bait. These clones encode a probable cytosolic isoform of OASTL and an organellar isoform of SAT, respectively, as indicated by evolutionary trees. The second clone, encoding SAT, was identified independently also as a "prey" when using cysK as a bait. Our results reveal the possibility of applying the two-hybrid system for cloning of plant cDNAs encoding enzymes of the cysteine synthase complex in the two-hybrid system. Additionally, using genome walking sequences located upstream of the sat1 cDNA were identified. Subsequently, in silico analyses were performed aiming towards identification of the potential signal peptide and possible location of the deduced mature protein encoded by sat1.

scholarly journals The nonhelical tail domain of keratin 14 promotes filament bundling and enhances the mechanical properties of keratin intermediate filaments in vitro

2001 ◽  
Vol 155 (5) ◽  
pp. 747-754 ◽  
Author(s):  
Olivier Bousquet ◽  
Linglei Ma ◽  
Soichiro Yamada ◽  
Changhong Gu ◽  
Toshihiro Idei ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Mechanical Strain ◽  
Type I ◽  
Basal Cells ◽  
Tail Domain ◽  
Yeast Two Hybrid ◽  
Keratin Filaments ◽  
Keratin Intermediate Filaments ◽  
Two Hybrid

Keratin filaments arise from the copolymerization of type I and II sequences, and form a pancytoplasmic network that provides vital mechanical support to epithelial cells. Keratins 5 and 14 are expressed as a pair in basal cells of stratified epithelia, where they occur as bundled arrays of filaments. In vitro, bundles of K5–K14 filaments can be induced in the absence of cross-linkers, and exhibit enhanced resistance to mechanical strain. This property is not exhibited by copolymers of K5 and tailless K14, in which the nonhelical tail domain has been removed, or copolymers of K5 and K19, a type I keratin featuring a short tail domain. The purified K14 tail domain binds keratin filaments in vitro with specificity (kD ∼2 μM). When transiently expressed in cultured cells, the K14 tail domain associates with endogenous keratin filaments. Utilization of the K14 tail domain as a bait in a yeast two-hybrid screen pulls out type I keratin sequences from a skin cDNA library. These data suggest that the tail domain of K14 contributes to the ability of K5–K14 filaments to self-organize into large bundles showing enhanced mechanical resilience in vitro.

scholarly journals FOXL2 Interacts with Steroidogenic Factor-1 (SF-1) and Represses SF-1-Induced CYP17 Transcription in Granulosa Cells

2010 ◽  
Vol 24 (5) ◽  
pp. 1024-1036 ◽  
Author(s):  
Mira Park ◽  
Eunkyoung Shin ◽  
Miae Won ◽  
Jae-Hong Kim ◽  
Hayoung Go ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Transcriptional Activation ◽  
Chromatin Immunoprecipitation ◽  
Ovarian Follicle ◽  
Type I ◽  
Yeast Two Hybrid ◽  
Steroidogenic Enzyme ◽  
Steroidogenic Factor 1 ◽  
Ovarian Follicle Development ◽  
Two Hybrid

Abstract Mutations in FOXL2 are responsible for blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) type I, in which affected women exhibit premature ovarian failure. FOXL2-null mice showed defects in granulosa cell development during folliculogenesis. We screened a rat ovarian yeast two-hybrid cDNA library to identify FOXL2-interacting proteins and found steroidogenic factor-1 (SF-1). Here, we show that human FOXL2 and SF-1 proteins interact in human granulosa cells and that FOXL2 negatively regulates the transcriptional activation of a steroidogenic enzyme, CYP17, by SF-1. Furthermore, FOXL2 mutants found in blepharophimosis-ptosis-epicanthus inversus syndrome type I patients lost the ability to repress CYP17 induction mediated by SF-1. Chromatin immunoprecipitation and EMSA results further revealed that FOXL2 inhibited the binding of SF-1 to the CYP17 promoter, whereas the FOXL2 mutants failed to block this interaction. Therefore, this study identifies a novel regulatory role for FOXL2 on a key steroidogenic enzyme and provides a possible mechanism by which mutations in FOXL2 disrupt normal ovarian follicle development.

The 30-kD Domain of Protein 4.1 Mediates Its Binding to the Carboxyl Terminus of pICln, a Protein Involved in Cellular Volume Regulation

Blood ◽  
1998 ◽  
Vol 92 (4) ◽  
pp. 1442-1447 ◽  
Author(s):  
Chieh-Ju C. Tang ◽  
Tang K. Tang
Keyword(s):  
Hybrid System ◽  
Volume Regulation ◽  
Carboxyl Terminus ◽  
Cdna Clones ◽  
Yeast Two Hybrid ◽  
Protein 4.1 ◽  
Yeast Two Hybrid System ◽  
Cellular Volume ◽  
Direct Association ◽  
Two Hybrid

Erythrocyte protein 4.1 (P4.1) is an 80-kD cytoskeletal protein that is important for the maintenance of the structural integrity and flexibility of the red blood cell membrane. Limited chymotryptic digestion of erythroid P4.1 yields 4 structural domains corresponding to the 30-, 16-, 10-, and 22/24-kD domains. Using a yeast two-hybrid system, we isolated cDNA clones encoding pICln that specifically interacts with the 30-kD domain of P4.1. In this report, we show that the carboxyl-terminus (amino acid residues 103-237) of pICln binds to the 30-kD domain of P4.1 in a yeast two-hybrid system. The direct association between the 30-kD domain of P4.1 and pICln was further confirmed by the following findings: (1) the S35-methione–labeled pICln specifically bound to both GST/P4.1-80 (80 kD) and GST/P4.1-30 (30 kD) fusion proteins, but not to the proteins that lack the 30-kD domain; (2) coimmunoprecipitation analysis of the cell extracts from transfected SiHa cells showed that pICln and P4.1 associate in transfected cells. It was reported that pICln can form a complex with actin and may play a role involved in cellular volume regulation. The direct association between P4.1 and pICln suggests that pICln may link P4.1-bound cytoskeletal elements to an unidentified volume-sensitive chloride channel. © 1998 by The American Society of Hematology.

scholarly journals Yeast Two-Hybrid Screen Identifies PKA-Riα Interacting Proteins during Mouse Spermiogenesis

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1941
Author(s):  
Kunyu Shi ◽  
Lele Yang ◽  
Xueqing Zhuang ◽  
Lan Zhang ◽  
Huayu Qi
Keyword(s):  
Protein Synthesis ◽  
Molecular Mechanisms ◽  
Regulation Of Gene Expression ◽  
Protein Translation ◽  
Regulatory Subunit ◽  
Type I ◽  
Interacting Proteins ◽  
Spermatogenic Cells ◽  
Yeast Two Hybrid ◽  
Two Hybrid

cAMP-dependent protein kinase (PKA) signaling plays various roles during mammalian spermatogenesis, ranging from the regulation of gene expression to the modulation of sperm motility. However, the molecular mechanisms that govern the multifaceted functions of PKA during spermatogenesis remain largely unclear. We previously found that PKA regulatory subunit I α (RIα) and catalytic subunit α (Cα) co-sediment with polyribosomal fractions of mouse testis lysate on sucrose gradient and the stimulation of PKA activity facilitates protein synthesis in post-meiotic elongating spermatids, indicating that type I PKA is intricately associated with protein translation machinery and regulates protein synthesis during mouse spermiogenesis. Since PKA activity is often regulated by interacting proteins that form complexes with its regulatory subunits, the identification of PKA-RIα interacting proteins in post-meiotic spermatogenic cells will facilitate our understanding of its regulatory roles in protein synthesis and spermiogenesis. In the present study, we applied a yeast two-hybrid screen to identify PKA-Riα-binding proteins using a cDNA library generated from mouse round and elongating spermatids. Numerous proteins were found to potentially interact with PKA-RIα, including proteostasis modulators, metabolic enzymes, cytoskeletal regulators, and mitochondrial proteins, many of which are specifically expressed in testes. Consistently, the examination of MENA (mouse ENA/VASP homolog) in developing mouse testes suggested that post-meiotic spermatogenic cells express a short isoform of MENA that interacts with PKA-RIα in yeast two-hybrid assay. The identification of PKA-RIα interacting proteins provides us solid basis to further explore how PKA signaling regulates protein synthesis and cellular morphogenesis during mouse spermatogenesis.

Sign in / Sign up

Export Citation Format

Share Document