FLOWERING HTH1 is involved in CONSTANS-mediated flowering regulation in Arabidopsis

Abstract Flowering at the right time is essential for maximum reproductive fitness. In Arabidopsis thaliana, the CONSTANS (CO) protein facilitates the transition from the vegetative phase to the reproductive phase under long-day conditions. The formation of heterodimeric complexes between CO and DNA binding domain-containing transcription factors is important for the induction of day length-dependent flowering. Here, we report a myb-like helix turn helix (HTH) transcriptional regulator family protein as a new modulator of floral transition, which we have named FLOWERING HTH1 (FHTH1). We isolated FHTH1 as a CO-interacting protein by a yeast two-hybrid screen using an Arabidopsis transcription factor library. Our analysis showed that FHTH1 presented in the nucleus and the FHTH1-CO complex was formed in the same subcellular location. We also observed the expression of a FHTH1:GUS construct in the leaf vasculature, where CO exists. Transgenic plants overexpressing FHTH1 fused with the plant-specific repression domain SRDX showed a delayed flowering phenotype in long days, resembling the phenotype of the co mutant. Our results suggest that FHTH1 may contribute to CO-mediated photoperiodic flowering regulation.

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Arabidopsis B-BOX32 interacts with CONSTANS-LIKE3 to regulate flowering

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1616459114 ◽

2016 ◽

Vol 114 (1) ◽

pp. 172-177 ◽

Cited By ~ 22

Author(s):

Prateek Tripathi ◽

Marcela Carvallo ◽

Elizabeth E. Hamilton ◽

Sasha Preuss ◽

Steve A. Kay

Keyword(s):

Large Scale ◽

Early Morning ◽

Day Length ◽

Interacting Protein ◽

Protein Partners ◽

Biochemical Assays ◽

Environmental Variations ◽

Developmental Switch ◽

Two Hybrid

Plants have the ability to respond to seasonal environmental variations by monitoring day length to initiate flowering. The transition from vegetative to the reproductive stage is the critical developmental switch in flowering plants to ensure optimal fitness and/or yield. It has been previously reported that B-BOX32 (BBX32) has the potential to increase grain yield when ectopically expressed in soybean. In the present study, we performed a detailed molecular characterization of the Arabidopsis B-box domain gene BBX32. We showed that the circadian clock in Arabidopsis regulates BBX32 and expressed in the early morning. To understand the molecular mechanism of BBX32 regulation, we performed a large-scale yeast two-hybrid screen and identified CONSTANS-LIKE 3 (COL3)/BBX4 as one of its interacting protein partners. Using different genetic and biochemical assays, we have validated this interaction and shown that COL3 targets FT in the presence of BBX32 to regulate the flowering pathway. Based on these findings, we hypothesized that this BBX32-COL3 module could be an additional regulatory mechanism affecting the reproductive development in Arabidopsis that could be translated to crops for increased agricultural productivity.

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UXT chaperone prevents proteotoxicity by acting as an autophagy adaptor for p62-dependent aggrephagy

Nature Communications ◽

10.1038/s41467-021-22252-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Min Ji Yoon ◽

Boyoon Choi ◽

Eun Jin Kim ◽

Jiyeon Ohk ◽

Chansik Yang ◽

...

Keyword(s):

Motor Neurons ◽

Molecular Chaperones ◽

Ectopic Expression ◽

Protein Aggregates ◽

Yeast Two Hybrid ◽

Ubiquitinated Protein ◽

Interacting Protein ◽

Autophagy Pathway ◽

Cooperative Relationship ◽

Two Hybrid

Abstractp62/SQSTM1 is known to act as a key mediator in the selective autophagy of protein aggregates, or aggrephagy, by steering ubiquitinated protein aggregates towards the autophagy pathway. Here, we use a yeast two-hybrid screen to identify the prefoldin-like chaperone UXT as an interacting protein of p62. We show that UXT can bind to protein aggregates as well as the LB domain of p62, and, possibly by forming an oligomer, increase p62 clustering for its efficient targeting to protein aggregates, thereby promoting the formation of the p62 body and clearance of its cargo via autophagy. We also find that ectopic expression of human UXT delays SOD1(A4V)-induced degeneration of motor neurons in a Xenopus model system, and that specific disruption of the interaction between UXT and p62 suppresses UXT-mediated protection. Together, these results indicate that UXT functions as an autophagy adaptor of p62-dependent aggrephagy. Furthermore, our study illustrates a cooperative relationship between molecular chaperones and the aggrephagy machinery that efficiently removes misfolded protein aggregates.

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HZwint-1, a novel human kinetochore component that interacts with HZW10

Journal of Cell Science ◽

10.1242/jcs.113.11.1939 ◽

2000 ◽

Vol 113 (11) ◽

pp. 1939-1950 ◽

Cited By ~ 6

Author(s):

D.A. Starr ◽

R. Saffery ◽

Z. Li ◽

A.E. Simpson ◽

K.H. Choo ◽

...

Keyword(s):

Hela Cells ◽

Coiled Coil ◽

Yeast Two Hybrid ◽

Interacting Protein ◽

Dicentric Chromosomes ◽

Centromere Function ◽

Coiled Coil Domain ◽

Gfp Fluorescence ◽

Two Hybrid ◽

Active Centromere

HZwint-1 (Human ZW10 interacting protein-1) was identified in a yeast two hybrid screen for proteins that interact with HZW10. HZwint-1 cDNA encodes a 43 kDa protein predicted to contain an extended coiled-coil domain. Immunofluorescence studies with sera raised against HZwint-1 protein revealed strong kinetochore staining in nocodazole-arrested chromosome spreads. This signal co-localizes at the kinetochore with HZW10, at a position slightly outside of the central part of the centromere as revealed by staining with a CREST serum. The kinetochore localization of HZwint-1 has been confirmed by following GFP fluorescence in HeLa cells transiently transfected with a plasmid encoding a GFP/HZwint-1 fusion protein. In cycling HeLa cells, HZwint-1 localizes to the kinetochore of prophase HeLa cells prior to HZW10 localization, and remains at the kinetochore until late in anaphase. This localization pattern, combined with the two-hybrid results, suggests that HZwint-1 may play a role in targeting HZW10 to the kinetochore at prometaphase. HZwint-1 was also found to localize to neocentromeres and to the active centromere of dicentric chromosomes. HZwint-1 thus appears to associate with all active centromeres, implying that it plays an important role in correct centromere function.

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Mammalian Homologue of the Caenorhabditis elegans UNC-76 Protein Involved in Axonal Outgrowth Is a Protein Kinase C ζ–interacting Protein

The Journal of Cell Biology ◽

10.1083/jcb.144.3.403 ◽

1999 ◽

Vol 144 (3) ◽

pp. 403-411 ◽

Cited By ~ 62

Author(s):

Shun'ichi Kuroda ◽

Noritaka Nakagawa ◽

Chiharu Tokunaga ◽

Kenji Tatematsu ◽

Katsuyuki Tanizawa

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Caenorhabditis Elegans ◽

Protein Kinase ◽

Rat Brain ◽

Axonal Outgrowth ◽

Yeast Two Hybrid ◽

Interacting Protein ◽

Kinase C ◽

Two Hybrid

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C ζ (PKCζ) as a bait, we have cloned a gene coding for a novel PKCζ-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH2-terminal variable region (V1) of PKCζ and weakly with that of PKCε. In the COS-7 cells coexpressing FEZ1 and PKCζ, FEZ1 was present mainly in the plasma membrane, associating with PKCζ and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCζ. When the constitutively active mutant of PKCζ was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCζ activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCζ stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCζ.

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Specific inhibition of transcriptional activity of the constitutive androstane receptor (CAR) by the splicing factor SF3a3

Biological Chemistry ◽

10.1515/bc.2008.149 ◽

2008 ◽

Vol 389 (10) ◽

Cited By ~ 1

Author(s):

Hye Jin Yun ◽

Jungsun Kwon ◽

Wongi Seol

Keyword(s):

Transcriptional Activity ◽

Constitutive Androstane Receptor ◽

Splicing Factor ◽

Yeast Two Hybrid ◽

Interacting Protein ◽

Functional Studies ◽

Reporter Activity ◽

Inhibitory Function ◽

Two Hybrid ◽

Hybrid Screening

Abstract The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily and plays an important role in the degradation of xenobiotics in the liver. Using yeast two-hybrid screening, we identified SF3a3, a 60-kDa subunit of the splicing factor 3a complex, as a specific CAR-interacting protein. We further confirmed their interaction by both co-immunoprecipitation and GST pull-down assay. Functional studies showed that overexpression of SF3a3 inhibited the reporter activity driven by a promoter containing CAR binding sequences by up to 50%, whereas reduced expression of SF3a3 activated the same reporter activity by approximately three-fold. The inhibitory function of SF3a3 is independent of the presence of TCPOBOP, a CAR ligand. These data suggest that SF3a3 functions as a co-repressor of CAR transcriptional activity, in addition to its canonical function.

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Abstract 3600: HspA12B Promotes Angiogenesis through Suppressing AKAP12 and Up-Regulating VEGF Pathway

Circulation ◽

10.1161/circ.118.suppl_18.s_449 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Rebecca J Steagall ◽

Fang Hua ◽

Mahesh Thirunazukarasu ◽

Lijun Zhan ◽

Chuanfu Li ◽

...

Keyword(s):

Cancer Metastasis ◽

Yeast Two Hybrid ◽

Vegf Expression ◽

Tubule Formation ◽

Interacting Protein ◽

Over Expression ◽

Vegf Pathway ◽

Two Hybrid ◽

Hybrid Screening

We have previously shown that HspA12B, a member of HspA70 family subfamily 12, is a novel angiogenesis regulator that is preferentially expressed in endothelial cells (ECs) and required for angiogenesis in vitro . The mechanism by which HspA12B regulates angiogenesis, however, is unknown. In this study we identified AKAP12/SSeCKS as a HSPA12B-interacting protein through a yeast two-hybrid screening and confirmed the interaction by co-immunoprecipitation and co-localization. We observed that HspA12B negatively regulated the expression of AKAP12/SSeCKS, a cancer metastasis repressor that inhibits VEGF expression and angiogen-esis. In HUVEC, HspA12B knockdown increased AKAP12 levels, decreased VEGF by more than 75%, and down-regulated Akt and pAkt; whereas HspA12B over expression decreased AKAP12 and more than doubled VEGF levels. We further identified a 32-AA domain in AKAP12 that was capable of interacting with HspA12B. Overexpression of this 32-AA domain in HUVEC disrupted the HspA12B-AKAP12 interaction and decreased VEGF expression by more than 70%, suggesting the importance of HspA12B-AKAP12 interaction in regulating VEGF. We also observed that HspA12B expression was increased more than 2 folds in ECs by hypoxia or shearing stress, and induced in ischemic rat heart. Inhibition of HspA12B abolished hypoxia-induced tubule formation. Adeno-HspA12B promoted angiogenesis in DIVAA assay. We concluded that this is the first evidence that HspA12B promotes angiogenesis through regulating VEGF by way of suppressing AKAP12. Our finding is the first example of an EC-specific molecular chaperone acting as the regulator of angiogenesis.

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Delayed Flowering and Pollen Production in Male‐Sterile Sugarcane Subjected to Extended Day‐Length 1

Crop Science ◽

10.2135/cropsci1969.0011183x000900030007x ◽

1969 ◽

Vol 9 (3) ◽

pp. 279-282 ◽

Cited By ~ 5

Author(s):

Norman I. James

Keyword(s):

Day Length ◽

Pollen Production ◽

Male Sterile ◽

Extended Day ◽

Delayed Flowering

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The DEAD Box Protein DP103 Is a Regulator of Steroidogenic Factor-1

Molecular Endocrinology ◽

10.1210/mend.15.1.0580 ◽

2001 ◽

Vol 15 (1) ◽

pp. 69-79 ◽

Cited By ~ 43

Author(s):

Qinglin Ou ◽

Jean-François Mouillet ◽

Xiaomei Yan ◽

Christoph Dorn ◽

Peter A. Crawford ◽

...

Keyword(s):

Transcriptional Repression ◽

Transcriptional Activity ◽

Point Mutations ◽

Hypothalamic Nucleus ◽

Rna Helicases ◽

Interacting Protein ◽

Steroidogenic Factor 1 ◽

Dead Box ◽

The Dead ◽

Repression Domain

Abstract The nuclear receptor steroidogenic factor-1 (SF-1) is essential for development of the gonads, adrenal gland, and the ventromedial hypothalamic nucleus. It also regulates the expression of pivotal steroidogenic enzymes and other important proteins in the reproductive system. We sought to elucidate the mechanisms that govern the transcriptional activity of SF-1. We demonstrate here that a previously uncharacterized domain, located C-terminal to the DNA binding domain of SF-1, exhibits transcriptional repression function. Point mutations in this domain markedly potentiate the transcriptional activity of native SF-1. Using an SF-1 region that spans this proximal repression domain as bait in a yeast two-hybrid system, we cloned an SF-1 interacting protein that is homologous to human DP103, a member of the DEAD box family of putative RNA helicases. DP103 directly interacts with the proximal repression domain of SF-1, and mutations in this domain abrogate its interaction with DP103. DP103 is expressed predominantly in the testis and is also expressed at a lower level in other steroidogenic and nonsteroidogenic tissues. Functionally, DP103 exhibits a native transcriptional repression function that localizes to the C-terminal region of the protein and represses the activity of wild-type, but not mutant, SF-1. Together, the physical and functional interaction of DP103 with a previously unrecognized repression domain within SF-1 represents a novel mechanism for regulation of SF-1 activity.

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