T47D breast cancer cell growth is inhibited by expression of VACM-1, a cul-5 gene

Burnatowska-Hledin, Maria; Kossoris, J.; Dort, Christa J. Van; Shearer, R. L.; Zhao, Ping; Murrey, Douglas A.; Abbott, J.L.; Kan, Charlene E.; Barney, Christopher C.

doi:10.1016/j.bbrc.2004.05.057

Cited by 29 publications

(17 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Heterologous CUL5 expression has been reported to exert an antiproliferative effect (32,33). Furthermore, a clinical study showed a significant decrease in CUL5 expression in breast tumors versus matched normal tissue (34).…”

Section: Discussionmentioning

confidence: 99%

E3 ubiquitin ligase Cullin-5 modulates multiple molecular and cellular responses to heat shock protein 90 inhibition in human cancer cells

Samant

Clarke

Workman

2014

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The molecular chaperone heat shock protein 90 (HSP90) is required for the activity and stability of its client proteins. Pharmacologic inhibition of HSP90 leads to the ubiquitin-mediated degradation of clients, particularly activated or mutant oncogenic protein kinases. Client ubiquitination occurs via the action of one or more E3 ubiquitin ligases. We sought to identify the role of Cullin-RING family E3 ubiquitin ligases in the cellular response to HSP90 inhibition. Through a focused siRNA screen of 28 Cullin-RING ligase family members, we found that CUL5 and RBX2 were required for degradation of several HSP90 clients upon treatment of human cancer cells with the clinical HSP90 inhibitor 17-AAG. Surprisingly, silencing Cullin-5 (CUL5) also delayed the earlier loss of HSP90 client protein activity at the same time as delaying cochaperone dissociation from inhibited HSP90-client complexes. Expression of a dominant-negative CUL5 showed that NEDD8 conjugation of CUL5 is required for client degradation but not for loss of client activity or recruitment of clients and HSP90 to CUL5. Silencing CUL5 reduced cellular sensitivity to three distinct HSP90 inhibitors, across four cancer types driven by different protein kinases. Our results reveal the importance of CUL5 in multiple aspects of the cellular response to HSP90 inhibition.eat shock protein 90 (HSP90) is a molecular chaperone that facilitates the stabilization and activation of around 350 client proteins (see www.picard.ch/downloads/Hsp90interactors. pdf for an updated client protein list) (1). As well as being involved in a wide range of normal cellular processes, many HSP90 clients are oncogenic kinases that are hyperactivated by mutation or amplified/overexpressed in malignancies (2, 3). HSP90-mediated activation and stabilization of client proteins requires an ATP-driven chaperone cycle regulated by a number of cochaperones (4, 5). Pharmacologic inhibition of HSP90 disrupts this cycle and leads to the ubiquitin-mediated proteasomal degradation of client proteins (3, 6, 7).The proposed model is that clients are ubiquitinated and thus targeted to the proteasome by the action of one or more E3 ubiquitin ligases (8). Some evidence suggests that the U boxcontaining ligase CHIP is involved in the degradation of certain HSP90 clients (9, 10). However, the stability of protein kinase clients such as ERBB2 is not increased in CHIP −/− cells treated with the first-in-class pharmacologic HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin [17-AAG, tanespimycin (11)] (10), suggesting that other E3 ubiquitin ligases are also involved.One study showed that the Cullin-RING ligase Cullin-5 (CUL5) is recruited to HSP90-containing complexes and is involved in the ubiquitination and degradation of the client ERBB2 following HSP90 inhibition (12). Cullin-RING ligases function as modular, multisubunit complexes that consist of a Cullin scaffold, a RING-H2 finger protein, a substrate-recognition subunit, and, in most cases, an adaptor that links the Cullin to the substrate reco...

show abstract

Section: Discussionmentioning

confidence: 99%

E3 ubiquitin ligase Cullin-5 modulates multiple molecular and cellular responses to heat shock protein 90 inhibition in human cancer cells

Samant

Clarke

Workman

2014

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…In the present study, Cul5 mRNA and protein expression increased 1.6-fold and 6.5-fold, respectively, in ATRA-treated HL-60 cells versus vehicle-control cells, and this increase was associated with granulocytic differentiation. An increase in Cul5 expression with granulocytic differentiation is not totally unexpected, as overexpression of Cul5 inhibits proliferation in CHO, COS-1, and T47D cell lines (Van Dort et al 2003;Burnatowska-Hledin et al 2004), and Cul5 plays a role in cell fate determination in Drosophila (Ayyub et al 2005). Additional studies also support a putative tumor suppressor role for Cul5 in breast cancer, as the chromosomal location (11q22-23) for Cul5 is associated with loss of heterozygosity (Carter et al 1994;Hampton et al 1994;Gudmundsson et al 1995;Negrini et al 1995;Tomlinson et al 1995;Winqvist et al 1995;Byrd et al 1997;Driouch et al 1998) and there is decreased expression of Cul5 in breast tumor tissues versus matched normal tissues (Fay et al 2003).…”

Section: Discussionmentioning

confidence: 99%

Granulocytic differentiation of HL-60 promyelocytic leukemia cells is associated with increased expression of Cul5

Baxter

Carlson

Mayer

et al. 2009

In Vitro Cell.Dev.Biol.-Animal

View full text Add to dashboard Cite

The human HL-60 promyelocytic leukemia cell line has been widely used as a model for studying granulocytic differentiation. All-trans retinoic acid (ATRA) treatment of HL-60 cells promotes granulocytic differentiation and is effective as differentiation therapy for patients with acute promyelocytic leukemia. The identification of genes that are transcriptionally regulated by ATRA has provided insight into granulocytic differentiation and differentiation therapy. The Asb-2 (ankyrin repeat SOCS box 2) gene has previously been identified as a transcriptional target in ATRA-treated HL-60 cells. The ASB-2 protein forms an E3 ubiquitin ligase complex with the proteins, Cul5, regulator of cullin 2 (ROC2), and elongin B and C. The purpose of this study was to determine if there is increased expression of Cul5 during granulocytic differentiation of HL-60 cells. To induce granulocytic differentiation, HL-60 cells were treated for 5 d with ATRA and differentiation was confirmed by examining superoxide anion production, nuclear morphology, and changes in the expression of CD11b, CD13, and CD15. Quantitative real-time RT-PCR was used to measure Cul5 mRNA expression and also the expression of other components of the E3 ubiquitin ligase (ASB-2, ROC2, elongin B and C). Granulocytic differentiation of HL-60 cells was associated with a 1.6-, 1.7-, and 23-fold statistically significant (P

show abstract

“…Transfection of various cell lines with VACM-1/cul5 cDNA attenuates cellular growth by a mechanism that involves inhibition of cAMP production, decreased phosphorylation of MAPK, 3 and a decrease in nuclear localization of early growth response gene (egr-1) product (5)(6)(7). In vivo, VACM-1/Cul5 protein expression is specific to established endothelial cells (8) but is absent in sprouting capillaries (9), suggesting its involvement in the regulation of endothelium-specific growth.…”

mentioning

confidence: 99%

Phosphorylation of VACM-1/Cul5 by Protein Kinase A Regulates Its Neddylation and Antiproliferative Effect

Bradley

Johnson

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

Expression of the VACM-1/cul5 gene in endothelial and in cancer cell lines in vitro inhibits cellular proliferation and decreases phosphorylation of MAPK. Structure-function analysis of the VACM-1 protein sequence identified consensus sites specific for phosphorylation by protein kinases A and C (PKA and PKC) and a Nedd8 protein modification site. Mutations at the PKA-specific site in VACM-1/Cul5 ( S730A VACM-1) sequence resulted in increased cellular growth and the appearance of a Nedd8-modified VACM-1/Cul5. The aim of this study was to examine if PKA-dependent phosphorylation of VACM-1/ Cul5 controls its neddylation status, phosphorylation by PKC, and ultimately growth. Our results indicate that in vitro transfection of rat adrenal medullary endothelial cells with anti-VACM-1-specific small interfering RNA oligonucleotides decreases endogenous VACM-1 protein concentration and increases cell growth. Western blot analysis of cell lysates immunoprecipitated with an antibody directed against a PKAspecific phosphorylation site and probed with anti-VACM-1-specific antibody showed that PKA-dependent phosphorylation of VACM-1 protein was decreased in cells transfected with S730A VACM-1 cDNA when compared with the cytomegalovirustransfected cells. This change was associated with increased modification of VACM-1 protein by Nedd8. Induction of PKA activity with forskolin reduced modification of VACM-1 protein by Nedd8. Finally, rat adrenal medullary endothelial cells transfected with S730A VACM-1/cul5 cDNA and treated with phorbol 12-myristate 13-acetate (10 and 100 nM) to induce PKC activity grew significantly faster than the control cells. These results suggest that the antiproliferative effect of VACM-1/Cul5 is dependent on its posttranslational modifications and will help in the design of new anticancer therapeutics that target the Nedd8 pathway.VACM-1 (vasopressin-activated calcium-mobilizing) protein (1), now identified as a cul5 gene product (2-4), is a 780-amino acid protein with a calculated M r of 91 kDa. Transfection of various cell lines with VACM-1/cul5 cDNA attenuates cellular growth by a mechanism that involves inhibition of cAMP production, decreased phosphorylation of MAPK, 3 and a decrease in nuclear localization of early growth response gene (egr-1) product (5-7). In vivo, VACM-1/Cul5 protein expression is specific to established endothelial cells (8) but is absent in sprouting capillaries (9), suggesting its involvement in the regulation of endothelium-specific growth. Interestingly, the human homolog of VACM-1 differs only in six amino acids from the rabbit VACM-1 and has been proposed to be a candidate for a tumor suppressor (2). Although expression of VACM-1/cul5 cDNA in a cancer-derived cell line, T47D, decreased nuclear concentration of estrogen receptor, ER␣, and inhibited cellular growth (6), the precise mechanism by which VACM-1/Cul5 may regulate cell growth is not known. Like other cullins, however, VACM-1/Cul5 may serve as scaffold protein that allows the assembly of E3 ubiquitin ligase comp...

show abstract

T47D breast cancer cell growth is inhibited by expression of VACM-1, a cul-5 gene

Cited by 29 publications

References 29 publications

E3 ubiquitin ligase Cullin-5 modulates multiple molecular and cellular responses to heat shock protein 90 inhibition in human cancer cells

E3 ubiquitin ligase Cullin-5 modulates multiple molecular and cellular responses to heat shock protein 90 inhibition in human cancer cells

Granulocytic differentiation of HL-60 promyelocytic leukemia cells is associated with increased expression of Cul5

Phosphorylation of VACM-1/Cul5 by Protein Kinase A Regulates Its Neddylation and Antiproliferative Effect

Contact Info

Product

Resources

About