skip to main content

Title: Factors that affect protein abundance of a positive regulator of abscisic acid signalling, the basic leucine zipper transcription factor ABRE‐binding factor 2 (ABF2)

Most members of basic leucine zipper (bZIP) transcription factor (TF) subgroup A play important roles as positive effectors in abscisic acid (ABA) signaling during germination and/or in vegetative stress responses. In multiple plant species, one member, ABA insensitive 5 (ABI5), is a major TF that promotes seed maturation and blocks early seeding growth in response to ABA. Other members, referred to as either ABRE‐binding factors (ABFs), ABRE‐binding proteins (AREBs), or D3 protein‐binding factors (DPBFs), are implicated as major players in stress responses during vegetative growth. Studies on the proteolytic regulation of ABI5, ABF1, and ABF3 inArabidopsis thalianahave shown that the proteins have moderate degradation rates and accumulate in the presence of the proteasome inhibitor MG132. Exogenous ABA slows their degradation and the ubiquitin E3 ligase called KEEP ON GOING (KEG) is important for their degradation. However, there are some reported differences in degradation among subgroup A members. The conserved C‐terminal sequences (referred to as the C4 region) enhance degradation of ABI5 but stabilize ABF1 and ABF3. To better understand the proteolytic regulation of the ABI5/ABFs and determine whether there are differences between vegetative ABFs and ABI5, we studied the degradation of an additional family member, ABF2, and compared its in vitro degradation to that of ABI5. As previously seen for ABI5, ABF1, and ABF3, epitope‐tagged constitutively expressed ABF2 degrades in seedlings treated with cycloheximide and is stabilized following treatment with the proteasome inhibitor MG132. Tagged ABF2 protein accumulates when seedlings are treated with ABA, but its mRNA levels do not increase, suggesting that the protein is stabilized in the presence of ABA. ABF2 is also an in vitro ubiquitination substrate of the E3 ligase KEG and recombinant ABF2 is stable inkeglysates. ABF2 with a C4 deletion degrades more quickly in vitro than full‐length ABF2, as previously observed for ABF1 and ABF3, suggesting that the conserved C4 region contributes to its stability. In contrast to ABF2 and consistent with previously published work, ABI5 with C terminal deletions including an analogous C4 deletion is stabilized in vitro compared to full length ABI5. In vivo expression of an ABF1 C4 deletion protein appears to have reduced activity compared to equivalent levels of full length ABF1. Additional group A family members show similar proteolytic regulation by MG132 and ABA. Altogether, these results together with other work on ABI5 regulation suggest that the vegetative ABFs share proteolytic regulatory mechanisms that are not completely shared with ABI5.

more » « less
Author(s) / Creator(s):
 ;  ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Plant Direct
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Overexpression of ABA-INSENSITIVE5 binding proteins (AFPs) results in extreme ABA resistance of seeds and failure to acquire desiccation tolerance, at least in part through effects on chromatin modification. We tested the hypothesis that AFPs promote germination in Arabidopsis (Arabidopsis thaliana) by also functioning as adapters for E3 ligases that ubiquitinate ABI5, leading to its degradation. Interactions between AFPs and two well-characterized classes of E3 ligases targeting ABI5, DWD HYPERSENSITIVE TO ABA (DWA)s and KEEP ON GOING, were analyzed by yeast two-hybrid, bimolecular fluorescence complementation, and genetic assays. Although weak direct interactions were detected between AFPs and E3 ligases, loss of function for these E3 ligases did not impair ABA-resistance conferred by overexpression of the YFP–AFP2 fusion. Comparison of ABI5 and AFP2 levels in these lines showed that AFP2 accumulation increased during germination, but that ABI5 degradation followed germination, demonstrating that AFP2 overexpression reduces ABA sensitivity, thereby permitting germination prior to ABI5 degradation. Surprisingly, AFP2 overexpression in the dwa1 dwa2 mutant background produced the unusual combination of extreme ABA resistance and desiccation tolerance, creating an opportunity to separate the underlying biochemical characteristics of ABA sensitivity and desiccation tolerance. Our quantitative proteomics analysis identified at least three-fold more differentially accumulated seed proteins than previous studies. Comparison of dry seed proteomes of wild-type or dwa1 dwa2 mutants with or without AFP2 overexpression allowed us to separate and refine the changes in protein accumulation patterns associated with desiccation tolerance independently of ABA sensitivity, or vice versa, to a subset of cold-induced and defense stress-responsive proteins and signaling regulators.

    more » « less
  2. Abstract

    Osteosarcoma is a highly aggressive malignancy for which treatment has remained essentially unchanged for years. Our previous studies found that the F‐box protein SKP2 is overexpressed in osteosarcoma, acting as a proto‐oncogene; p27Kip1(p27) is an inhibitor of cyclin‐dependent kinases and a downstream substrate of SKP2‐mediated ubiquitination. Overexpression of SKP2 and underexpression of p27 are common characteristics of cancer cells. The SCFSKP2E3 ligase ubiquitinates Thr187‐phosphorylated p27 for proteasome degradation, which can be abolished by a Thr187Ala knock‐in (p27T187AKI) mutation.RB1andTP53are two major tumor suppressors commonly coinactivated in osteosarcoma. We generated a mouse model with a double knockout (DKO) ofRb1andTrp53within cells of the osteoblastic lineage, which developed osteosarcoma with full penetrance. When p27T187AKI mice were crossed on to the DKO background, p27T187Aprotein was found to accumulate in osteosarcoma tumor tissues. Furthermore, p27T187Apromoted apoptosis in DKO tumors, slowed disease progression, and significantly prolonged overall survival. RNA sequencing analysis also linked the SCFSKP2–p27T187Aaxis to potentially reduced cancer stemness. Given thatRB1andTP53loss or coinactivation is common in human osteosarcoma, our study suggests that inhibiting the SKP2–p27 axis may represent a desirable therapeutic strategy for this cancer.

    more » « less
  3. Abstract

    The plant hypersensitive response (HR), a rapid cell death at the point of pathogenesis, is mediated by nucleotide‐binding site, leucine‐rich repeat (NLR) resistance proteins (R‐proteins) that recognize the presence of specific pathogen‐derived proteins. Rp1‐D21 is an autoactive maize NLR R‐protein that triggers HR spontaneously. We previously mapped loci associated with variation in the strength of HR induced by Rp1‐D21. Here we identify the E3 ligaseZmMIEL1as the causal gene at a chromosome 10 modifier locus. TransientZmMIEL1expression inNicotiana benthamianareduced HR induced by Rp1‐D21, while suppression ofZmMIEL1expression in maize carryingRp1‐D21increased HR. ZmMIEL1 also suppressed HR induced by another autoactive NLR, theArabidopsisR‐protein RPM1D505V, inN. benthamiana. We demonstrated that ZmMIEL1 is a functional E3 ligase and that the effect of ZmMIEL1 was dependent on the proteasome but also that levels of Rp1‐D21 and RPM1D505V were not reduced when coexpressed with ZmMIEL1 in theN. benthamianasystem. By comparison to a similar system inArabidopsis, we identify ZmMYB83 as a potential target of ZmMIEL1. Suppression ofZmMYB83expression in maize lines carryingRp1‐D21suppressed HR. Suppression ofZmMIEL1expression caused an increase inZmMYB83transcript and protein levels inN. benthamianaand maize. Using coimmunoprecipitation and bimolecular fluorescence complementation assays, we demonstrated that ZmMIEL1 and ZmMYB83 physically interacted. Additionally,ZmMYB83andZmMIEL1regulated the expression of a set of maize very long chain fatty acid (VLCFA) biosynthetic genes that may be involved in regulating HR.

    more » « less
  4. Protein degradation through the Ubiquitin (Ub)-26S Proteasome System (UPS) is a major gene expression regulatory pathway in plants. In this pathway, the 76-amino acid Ub proteins are covalently linked onto a large array of UPS substrates with the help of three enzymes (E1 activating, E2 conjugating, and E3 ligating enzymes) and direct them for turnover in the 26S proteasome complex. The S-phase Kinase-associated Protein 1 (Skp1), CUL1, F-box (FBX) protein (SCF) complexes have been identified as the largest E3 ligase group in plants due to the dramatic number expansion of the FBX genes in plant genomes. Since it is the FBX proteins that recognize and determine the specificity of SCF substrates, much effort has been done to characterize their genomic, physiological, and biochemical roles in the past two decades of functional genomic studies. However, the sheer size and high sequence diversity of the FBX gene family demands new approaches to uncover unknown functions. In this work, we first identified 82 known FBX members that have been functionally characterized up to date in Arabidopsis thaliana . Through comparing the genomic structure, evolutionary selection, expression patterns, domain compositions, and functional activities between known and unknown FBX gene members, we developed a neural network machine learning approach to predict whether an unknown FBX member is likely functionally active in Arabidopsis, thereby facilitating its future functional characterization. 
    more » « less
  5. null (Ed.)
    Glycosyltransferase OGT catalyzes the conjugation of O-linked β-D-N-acetylglucosamine (O-GlcNAc) to Ser and Thr residues of the cellular proteins and regulates many key processes in the cell. Here, we report the identification of OGT as a ubiquitination target of HECT-type E3 ubiquitin (UB) ligase E6AP, whose overexpression in HEK293 cells would induce the degradation of OGT. We also found that the expression of E6AP in HeLa cells with the endogenous expression of the E6 protein of the human papillomavirus (HPV) would accelerate OGT degradation by the proteasome and suppress O-GlcNAc modification of OGT substrates in the cell. Overall, our study establishes a new mechanism of OGT regulation by the ubiquitin–proteasome system (UPS) that mediates the crosstalk between protein ubiquitination and O-GlcNAcylation pathways underlying diverse cellular processes. 
    more » « less