Dimerization of the Human E3 Ligase CHIP via a Coiled-coil Domain Is Essential for Its Activity

122

113

The NH 2 -terminal sequence of steroid receptors is highly variable between different receptors and in the same receptor from different species. In this study, a primary sequence homology comparison identified a 14-amino acid NH 2 -terminal motif of the human androgen receptor (AR) that is common to AR from all species reported, including the lower vertebrates. The evolutionarily conserved motif is unique to AR, with the exception of a partial sequence in the glucocorticoid receptor of higher species. The presence of the conserved motif in AR and the glucocorticoid receptor and its absence in other steroid receptors suggests convergent evolution. The function of the AR NH 2 -terminal conserved motif was suggested from a yeast two-hybrid screen that identified the COOH terminus of the Hsp70-interacting protein (CHIP) as a binding partner. We found that CHIP functions as a negative regulator of AR transcriptional activity by promoting AR degradation. In support of this, two mutations in the AR NH 2 -terminal conserved motif previously identified in the transgenic adenocarcinoma of mouse prostate model reduced the interaction between CHIP and AR. Our results suggest that the AR NH 2 -terminal domain contains an evolutionarily conserved motif that functions to limit AR transcriptional activity. Moreover, we demonstrate that the combination of comparative sequence alignment and yeast two-hybrid screening using short conserved peptides as bait provides an effective strategy to probe the structure-function relationships of steroid receptor NH 2 -terminal domains and other intrinsically unstructured transcriptional regulatory proteins.Steroid receptors depend on multiple domains for their function as ligand-dependent transcriptional activators. The DNAand ligand-binding domains have been studied extensively and have a high degree of structural and functional conservation. In contrast, the largely unstructured NH 2 -terminal domains (1-3) are highly variable in size and sequence, and the molecular mechanisms that contribute to transactivation are not well understood. The size of the NH 2 -terminal region of steroid receptors increases with evolutionary expansion (4, 5), from estrogen receptor-␣ (185 amino acid residues) to the glucocorticoid receptor (GR 1 ; 420 residues), androgen receptor (AR; 558 residues), progesterone receptor (B form; 566 residues), and mineralocorticoid receptor (602 residues). In contrast, transcription factors such as p53, NF-B, and VP16 typically have transcriptional activation domains of Ͻ100 residues.

Section: Fig 3 Interaction Of Chip With the Ar Nh 2 -Terminal Consementioning

confidence: 99%

An Androgen Receptor NH2-terminal Conserved Motif Interacts with the COOH Terminus of the Hsp70-interacting Protein (CHIP)

He¹,

Bai²,

Hnat³

et al. 2004

122

113

“…Constructs PRK5-HA-ubiquitin-WT, pRK5-HA-Ubiquitin-K48R, pRK5-HA-Ubiquitin-K63R, and pCI-HA-Nedd4-1 were procured from Addgene. The full-length coding sequence and three deletion mutants of human CHIP (NM_005861) viz., CHIP-TPR (1-134), CHIP-⌬TPR (135-303), CHIP-⌬Ubox (1-189), and CHIP-Ubox (216 -303) were PCR amplified from HEK293 cDNA while CHIP-⌬CC (⌬128 -229) was generated following a previous protocol (44) and inserted into pIRES-hrGFP-1a-Flag. The QuickChange XL Site-directed Mutagenesis kit (Stratagene) was used to generate the CHIP-H260Q (H260Q) and CHIP-K30A (K30A) mutants.…”

Section: Plasmids and Recombinant Proteins-pgz21dx-gfp-pten-mentioning

confidence: 99%

The Chaperone-assisted E3 Ligase C Terminus of Hsc70-interacting Protein (CHIP) Targets PTEN for Proteasomal Degradation

Ahmed

Deb

Paul

et al. 2012

140

138

Background: PTEN is targeted by multiple E3 ligases but that does not clearly decipher the rigid control of its level and activity. Results: CHIP interacts with PTEN and promotes its proteasomal degradation. Conclusion: CHIP acts as a bridge between the chaperone system and the degradation machinery for PTEN. Significance: Stabilization of PTEN by targeting CHIP can be a novel therapeutic approach in cancer regulation.

“…The U-box proteins hCHIP (45) and yeast Prp19 (46) form a dimer and tetramer, respectively, via coiled-coil regions. Other RING/U-box proteins utilize a different secondary element for homo-and hetero-oligomerization, such as ␣-helices for the RING BRCA1/BARD heterodimer (47,48) and the RAG1 (49) and AtPUB14 homodimers (50).…”

Section: Discussionmentioning

confidence: 99%

BRIZ1 and BRIZ2 Proteins Form a Heteromeric E3 Ligase Complex Required for Seed Germination and Post-germination Growth in Arabidopsis thaliana

Hsia

Callis

2010

Ubiquitin pathway E3 ligases are an important component conferring specificity and regulation in ubiquitin attachment to substrate proteins. The Arabidopsis thaliana RING (Really Interesting New Gene) domain-containing proteins BRIZ1 and BRIZ2 are essential for normal seed germination and post-germination growth. Loss of either BRIZ1 (At2g42160) or BRIZ2 (At2g26000) results in a severe phenotype. Heterozygous parents produce progeny that segregate 3:1 for wild-type:growtharrested seedlings. Homozygous T-DNA insertion lines are recovered for BRIZ1 and BRIZ2 after introduction of a transgene containing the respective coding sequence, demonstrating that disruption of BRIZ1 or BRIZ2 in the T-DNA insertion lines is responsible for the observed phenotype. Both proteins have multiple predicted domains in addition to the RING domain as follows: a BRAP2 (BRCA1-Associated Protein 2), a ZnF UBP (Zinc Finger Ubiquitin Binding protein), and a coiled-coil domain. In vitro, both BRIZ1 and BRIZ2 are active as E3 ligases but only BRIZ2 binds ubiquitin. In vitro synthesized and purified recombinant BRIZ1 and BRIZ2 preferentially form heterooligomers rather than homo-oligomers, and the coiled-coil domain is necessary and sufficient for this interaction. BRIZ1 and BRIZ2 co-purify after expression in tobacco leaves, which also requires the coiled-coil domain. BRIZ1 and BRIZ2 coding regions with substitutions in the RING domain are inactive in vitro and, after introduction, fail to complement their respective mutant lines. In our current model, BRIZ1 and BRIZ2 together are required for formation of a functional ubiquitin E3 ligase in vivo, and this complex is required for germination and early seedling growth.The ubiquitin pathway is a protein modification pathway that regulates many processes such as cell cycle, signal transduction, translational regulation, and chromatin remodeling (1-4). This pathway utilizes three enzymes for attachment of ubiquitin to its target. An E1 (ubiquitin-activating enzyme) forms a thioester bond with ubiquitin in an ATP-dependent manner. E1 then passes the attached ubiquitin to an E2 (ubiquitin-conjugating enzyme), which also forms a thioester bond with ubiquitin. Subsequently, the E3, also called a ubiquitin ligase, either accepts this activated ubiquitin before transfer to the substrate or serves as a scaffold to bring the substrate and E2-ubiquitin together. After ubiquitin is linked to the substrate, additional ubiquitin moieties can be conjugated to the previously attached ubiquitin, forming polyubiquitin chains. Ubiquitinated proteins have a number of fates, with the most characterized being degradation by the proteasome (5-7). Additionally, nonproteasomal fates have been described (8 -10).In the ubiquitin pathway, the E3 is an important contributor to substrate specificity. Currently, there are ϳ1300 E3 ligases predicted in the annotated proteome of Arabidopsis thaliana, which includes ϳ480 RING-type E3 ligases (11-13). The RING domain, consisting of eight conserved cysteine or histidine residues...