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Query: UNIPROT:P50502 (Hip)
 7,003 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent evidence indicates that the transactivation of estrogen receptor alpha (ERalpha) requires estrogen-dependent receptor ubiquitination and degradation. Here we show that estrogen-unbound (unliganded) ERalpha is also ubiquitinated and degraded through a ubiquitin-proteasome pathway. To investigate this ubiquitin-proteasome pathway, we purified the ubiquitin ligase complex for unliganded ERalpha and identified a protein complex containing the carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP preferentially bound to misfolded ERalpha and ubiquitinated it to induce degradation. Ligand binding to the receptor induced the dissociation of CHIP from ERalpha. In CHIP-/- cells, the degradation of unliganded ERalpha was abrogated; however, estrogen-induced degradation was observed to the same extent as in CHIP+/+ cells. Our findings suggest that ERalpha is regulated by two independent ubiquitin-proteasome pathways, which are switched by ligand binding to ERalpha. One pathway is necessary for the transactivation of the receptor and the other is involved in the quality control of the receptor.
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PMID:Ligand-dependent switching of ubiquitin-proteasome pathways for estrogen receptor. 1553 84

The TGF-beta superfamily signaling pathway regulates many important biological processes, including cell growth, differentiation and embryonic pattern formation. Smad1, a member of this signaling pathway that functions downstream of serine/threonine kinase receptors, has ability to interact with carboxyl terminus of Hsc70-interacting protein (CHIP), which is an E3 ubiquitin ligase in other cases. It has been reported that Smurf1, a member of the Hect family E3 ubiquitin ligases, can target Smad1 to 26S proteasome for degradation. In this paper, we studied the interaction of Smad1 and CHIP by combination of surface plasmon resonance and supported monolayer approach. The specific binding of Smad1 to CHIP indicates that the degradation of Smad1 may also be mediated by CHIP, and CHIP may play an essential role in the TGF-beta signaling pathway.
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PMID:Specific interaction between Smad1 and CHIP: a surface plasmon resonance study. 1570 1

Transforming growth factor-beta (TGF-beta) signaling is critical in a variety of biological processes such as cell proliferation, differentiation, and apoptosis. TGF-beta signaling is mediated by a group of proteins including TGF-beta receptors and Smads. It is known that different cells can exhibit different sensitivities to TGF-beta. Several molecular mechanisms, such as the differential expression of the receptor levels, have been suggested as contributing to these differences. Here, we report evidence for a novel mechanism of regulating TGF-beta sensitivity that depends on the role of CHIP (carboxyl terminus of Hsc70-interacting protein) in regulating the basal level of Smad3 via the ubiquitin-dependent degradation pathway. First, using a luciferase assay we found that overexpression of CHIP inhibited TGF-beta signaling, whereas silencing CHIP expression by small interfering RNAs led to increased TGF-beta signaling sensitivity. Second, based on the results of cell proliferation assays and JunB expression, we found that TGF-beta signaling could be abolished by stably overexpressing CHIP. Third, in those cell lines with stably expressed CHIP, we observed that the Smad3 protein level was dramatically decreased. Finally, we demonstrated that CHIP served as a U-box dependent E3 ligase that can directly mediate ubiquitination and degradation of Smad3 and that this action of CHIP was independent of TGF-beta signaling. Taken together, these findings suggest that CHIP can modulate the sensitivity of the TGF-beta signaling by controlling the basal level of Smad3 through ubiquitin-mediated degradation.
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PMID:CHIP controls the sensitivity of transforming growth factor-beta signaling by modulating the basal level of Smad3 through ubiquitin-mediated degradation. 1578 69

In estrogen target cells, estrogen receptor-alpha (ERalpha) protein levels are strictly regulated. Although receptor turnover is a continuous process, dynamic fluctuations in receptor levels, mediated primarily by the ubiquitin-proteasome pathway, occur in response to changing cellular conditions. In the absence of ligand, ERalpha is sequestered within a stable chaperone protein complex consisting of heat shock protein 90 (Hsp90) and cochaperones. However, the molecular mechanism(s) regulating ERalpha stability and turnover remain undefined. One potential mechanism involves CHIP, the carboxyl terminus of Hsc70-interacting protein, previously shown to target Hsp90-interacting proteins for ubiquitination and proteasomal degradation. In the present study, a role for CHIP in ERalpha protein degradation was investigated. In ER-negative HeLa cells transfected with ERalpha and CHIP, ERalpha proteasomal degradation increased, whereas ERalpha-mediated gene transcription decreased. In contrast, CHIP depletion by small interference RNA resulted in increased ERalpha accumulation and reporter gene transactivation. Transfection of mutant CHIP constructs demonstrated that both the U-box (containing ubiquitin ligase activity) and the tetratricopeptide repeat (TPR, essential for chaperone binding) domains within CHIP are required for CHIP-mediated ERalpha down-regulation. In addition, coimmunoprecipitation assays demonstrated that ERalpha and CHIP associate through the CHIP TPR domain. In ERalpha-positive breast cancer MCF7 cells, CHIP overexpression resulted in decreased levels of endogenous ERalpha protein and attenuation of ERalpha-mediated gene expression. Furthermore, the ERalpha-CHIP interaction was stimulated by the Hsp90 inhibitor geldanamycin (GA), resulting in enhanced ERalpha degradation; this GA effect was further augmented by CHIP overexpression but was abolished by CHIP depletion. Finally, ERalpha dissociation from CHIP by various ERalpha ligands, including 17beta-estradiol, 4-hydroxytamoxifen, and ICI 182,780, interrupted CHIP-mediated ERalpha degradation. These results demonstrate a role for CHIP in both basal and GA-induced ERalpha degradation. Furthermore, based on our observations that CHIP promotes ERalpha degradation and attenuates receptor-mediated gene transcription, we suggest that CHIP, by modulating ERalpha stability, contributes to the regulation of functional receptor levels, and thus hormone responsiveness, in estrogen target cells.
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PMID:CHIP (carboxyl terminus of Hsc70-interacting protein) promotes basal and geldanamycin-induced degradation of estrogen receptor-alpha. 1603 32

The study of protein ubiquitination, a post-translational modification by ubiquitin, has emerged as one of the most active areas in biology because of the important role of this type of modification on the regulation of various cellular proteins. Advances in techniques for the determination and site mapping of protein ubiquitination can facilitate the elucidation of molecular mechanisms of this modification. We have recently described a novel method for identifying peptides containing ubiquitinated amino acid residues, based on the MALDI-MS/MS analysis of tryptic peptide derivatives. In particular, we have utilized N-terminal sulfonation of these peptides to provide a unique fragmentation pattern that leads to the direct identification and sequencing of ubiquitin modified peptides. Here we present an application of this new method on the characterization of ubiquitin conjugated C-terminal Hsc70-interacting protein (CHIP), a recently identified U-box containing E3 enzyme. Three peptides bearing ubiquitination sites have been identified from the digest of ubiquitinated CHIP; one of these was a site on CHIP, while the other two were found on the ubiquitin molecules, demonstrating that sulfonation of tryptic peptides is a general and efficient method for characterizing protein ubiquitination.
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PMID:Direct identification of ubiquitination sites on ubiquitin-conjugated CHIP using MALDI mass spectrometry. 1621 6

It is notable that both chaperone and ubiquitin-proteasome systems are required for the removal of aberrant cellular proteins to ensure protein homeostasis in cells. However, the entity that links the two systems had remained elusive. The carboxyl terminus of Hsc70-interacting protein (CHIP), originally identified as a cochaperone of Hsc70, has both a TPR motif and a U-box domain. The TPR motif associates with Hsp70 and Hsp90, whereas the U-box domain executes ubiquitin ligase activity. Thus, CHIP is an ideal molecule, acting as a protein quality control ubiquitin ligase that selectively leads abnormal proteins recognized by molecular chaperones to degradation by the proteasome. This chapter describes methods of analyzing chaperone-dependent ubiquitin ligase activity of CHIP using firefly luciferase as a model substrate.
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PMID:Purification and assay of the chaperone-dependent ubiquitin ligase of the carboxyl terminus of Hsc70-interacting protein. 1627 35

The ubiquitin-proteasome system (UPS) is involved in the pathogenetic mechanisms of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Dorfin is a ubiquitin ligase (E3) that degrades mutant SOD1 proteins, which are responsible for familial ALS. Although Dorfin has potential as an anti-ALS molecule, its life in cells is short. To improve its stability and enhance its E3 activity, we developed chimeric proteins containing the substrate-binding hydrophobic portion of Dorfin and the U-box domain of the carboxyl terminus of Hsc70-interacting protein (CHIP), which has strong E3 activity through the U-box domain. All the Dorfin-CHIP chimeric proteins were more stable in cells than was wild-type Dorfin (Dorfin(WT)). One of the Dorfin-CHIP chimeric proteins, Dorfin-CHIP(L), ubiquitylated mutant SOD1 more effectively than did Dorfin(WT) and CHIP in vivo, and degraded mutant SOD1 protein more rapidly than Dorfin(WT) does. Furthermore, Dorfin-CHIP(L) rescued neuronal cells from mutant SOD1-associated toxicity and reduced the aggresome formation induced by mutant SOD1 more effectively than did Dorfin(WT).
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PMID:Dorfin-CHIP chimeric proteins potently ubiquitylate and degrade familial ALS-related mutant SOD1 proteins and reduce their cellular toxicity. 1715 13

While wild-type p53 is normally a rapidly degraded protein, mutant forms of p53 are stabilized and accumulate to high levels in tumor cells. In this study, we show that mutant and wild-type p53 proteins are ubiquitinated and degraded through overlapping but distinct pathways. While Mdm2 can drive the degradation of both mutant and wild-type p53, our data suggest that the ability of Mdm2 to function as a ubiquitin ligase is less important in the degradation of mutant p53, which is heavily ubiquitinated in an Mdm2-independent manner. Our initial attempts to identify ubiquitin ligases that are responsible for the ubiquitination of mutant p53 have suggested a role for the chaperone-associated ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein), although other unidentified ubiquitin ligases also appear to contribute. The contribution of Mdm2 to the degradation of mutant p53 may reflect the ability of Mdm2 to deliver the ubiquitinated mutant p53 to the proteasome.
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PMID:Ubiquitination and degradation of mutant p53. 1790 90

Smad1, a downstream regulator of the bone morphogenetic protein (BMP) receptors, is tightly regulated by the ubiquitin-proteasomal degradation system. To dissect the mechanisms that underlie the regulation of Smad1, it is important to investigate the specific ubiquitination site(s) in Smad1. Here we report that the alpha-NH(2) group of the N terminus and the epsilon-NH(2) groups of internal lysine residues 116, 118 and 269 (K116, K118 and K269) of Smad1 are ubiquitin acceptor sites mediated by the carboxyl terminus of Hsc70-interacting protein (CHIP). The in vitro degradation assay indicates that ubiquitination at the N terminus partially contributes to the degradation of Smad1. Furthermore, we demonstrate that the ubiquitination level of pseudo-phosphorylated Smad1 by CHIP is stronger than that of wild-type Smad1 and can be strongly inhibited by a phosphorylated tail of Smad1, PIS(pS)V(pS). Third, our results indicate that Hsp70 facilitates CHIP-mediated poly-ubiquitination of Smad1 whereas it attenuates CHIP-meditated mono-ubiquitination of Smad1. Finally, consistent with the in vitro observation, we show that CHIP preferentially mediates the degradation of phospho-Smad1/5 in vivo. Taken together, these results provide us a hint that CHIP might preferentially regulate phosphorylated Smad1 and thus the BMP signaling.
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PMID:Differential ubiquitination of Smad1 mediated by CHIP: implications in the regulation of the bone morphogenetic protein signaling pathway. 1796 81

Cellular levels of estrogen receptor-alpha (ERalpha) protein are regulated primarily by the ubiquitin-proteasome pathway. Dynamic interactions between ERalpha and the protein degradation machinery facilitate the down-regulation process by targeting receptor lysine residues for polyubiquitination. To date, the lysines that control receptor degradation have not been identified. Two receptor lysines, K302 and K303, located in the hinge-region of ERalpha, serve multiple regulatory functions, and we examined whether these might also regulate receptor polyubiquitination, turnover, and receptor-protein interactions. We used ERalpha-negative breast cancer C4-12 cells to generate cells stably expressing wild-type (wt)ERalpha or ERalpha with lysine-to-alanine substitutions at K302 and K303 (ERalpha-AA). In the unliganded state, ERalpha-AA displayed rapid polyubiquitination and enhanced basal turnover, as compared with wtERalpha, due to its elevated association with the ubiquitin ligase carboxy terminus of Hsc70-interacting protein (CHIP) and the proteasome-associated cochaperone Bag1. Treatment of C4-12 cells with either 17beta-estradiol (E2) or the pure antiestrogen ICI 182,780 (ICI) induced rapid degradation of wtERalpha via the ubiquitin-proteasome pathway; however, in the presence of these ligands, ERalpha-AA was less efficiently degraded. Furthermore, ERalpha-AA was resistant to ICI-induced polyubiquitination, suggesting that these lysines are polyubiquitinated in response to the antiestrogen and demonstrate a novel role for these two lysines in the mechanism of action of ICI-induced receptor down-regulation. The reduced stability of ERalpha-AA in the unliganded state and the increased stability of ERalpha-AA in the liganded state were concordant with reporter gene assays demonstrating that ERalpha-AA has lower basal activity but higher E2 inducibility than wtERalpha. These data provide the first evidence that K302/303 protect ERalpha from basal degradation and are necessary for efficient E2- and ICI-induced turnover in breast cancer cells.
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PMID:Estrogen receptor-alpha hinge-region lysines 302 and 303 regulate receptor degradation by the proteasome. 1838 50


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