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

Bradley, Shirley E; Johnson, Alyssa E.; Le, Isabelle P.; Oosterhouse, E.; Hledin, Michael P.; Marquez, Gabriel A.; Burnatowska-Hledin, Maria

doi:10.1074/jbc.m109.085225

Cited by 15 publications

(14 citation statements)

References 46 publications

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“…In addition to the important mechanistic studies of CUL5 that have been reported from the laboratory of Dr Burnatowska-Hledin since their initial discovery of CUL5 in 2000 ( 18 – 20 ), two other studies have demonstrated a regulatory role for two miRNAs, one, for miR-19a and miR-19b, in cervical cancer and another, for miR-7, in hepatocellular cancer ( 4 , 5 ). We searched miRNA target prediction algorithms for CUL5 and discovered that several, including TargetScan and PicTar, indicated that miR-182 was an even higher confidence prediction than either of the others.…”

Section: Discussionmentioning

confidence: 99%

Cullin-5, a ubiquitin ligase scaffold protein, is significantly underexpressed in endometrial adenocarcinomas and is a target of miR-182

et al. 2016

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Abstract. Altered expression of cullin-5 (CUL5), a member of the cullin-RING E3 ubiquitin ligase family, has been implicated in a number of types of cancers including breast, cervical and hepatocellular cancers. In the present study, we found that CUL5 expression was significantly decreased in both endometrioid and serous endometrial adenocarcinomas with the more aggressive serous type displaying a higher reduction (-4.3-fold) than the less aggressive endometrioid type (-2.9-fold). Overexpression of CUL5 mRNA and protein in Ishikawa H endometrial cancer cells resulted in decreased cell proliferation and in a reduction in CUL5-RING E3 ligase downstream clients JAK2 and FAS-L. Finally, we demonstrated for the first time that CUL5 is a direct target of miR-182 that we previously showed to be significantly overexpressed in endometrial adenocarcinomas and we provided evidence that increased miR-182 expression is, at least in part, a result of demethylation of its upstream promoter. These data suggest a cascade in which miR-182 expression is epigenetically increased leading to decreased CUL5 expression and increased cellular proliferation. The final step in the cascade may be operating through a decrease in ubiquitination of pro-growth CUL5 ubiquitin ligase clients. This cascade offers a series of potential interventional steps involving epigenetic modification, miRNA and/or gene targeting and ubiquitination.

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Section: Discussionmentioning

confidence: 99%

Cullin-5, a ubiquitin ligase scaffold protein, is significantly underexpressed in endometrial adenocarcinomas and is a target of miR-182

et al. 2016

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“…Cul5(S730A) accelerates cellular proliferation and induces angiogenic growth in rat adrenal medullary endothelial cells (RAMECs) [ 19 ]. Cul5 neddylation is increased by the S730A mutation, and activation of PKA by forskolin suppresses the neddylation of Cul5 [ 20 ]. Furthermore, PKC-induced RAMEC proliferation is enhanced by Cul5(S730A) [ 20 ].…”

Section: Identification and Regulation Of Cullinmentioning

confidence: 99%

“…Cul5 neddylation is increased by the S730A mutation, and activation of PKA by forskolin suppresses the neddylation of Cul5 [ 20 ]. Furthermore, PKC-induced RAMEC proliferation is enhanced by Cul5(S730A) [ 20 ]. Cul5(S730A) expression in RAMECs increases the levels of phosphorylated MAPK and the translocation of the transcription factor EGR-1, a tumor suppressor, to the nucleus; it also causes morphological alterations mediated by actin rearrangement [ 19 ].…”

Section: Identification and Regulation Of Cullinmentioning

confidence: 99%

The role of cullin 5-containing ubiquitin ligases

et al. 2016

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The suppressor of cytokine signaling (SOCS) box consists of the BC box and the cullin 5 (Cul5) box, which interact with Elongin BC and Cul5, respectively. SOCS box-containing proteins have ubiquitin ligase activity mediated by the formation of a complex with the scaffold protein Cul5 and the RING domain protein Rbx2, and are thereby members of the cullin RING ligase superfamily. Cul5-type ubiquitin ligases have a variety of substrates that are targeted for polyubiquitination and proteasomal degradation. Here, we review the current knowledge on the identification of Cul5 and the regulation of its expression, as well as the signaling pathways regulated by Cul5 and how viruses highjack the Cul5 system to overcome antiviral responses. Identification and regulation of cullin 5Cullin 5 (Cul5) was originally identified as a vasopressin-activated calcium-mobilizing (VACM-1) protein, an arginine vasopressin (AVP) receptor [1]. AVP is a nonapeptide that regulates body fluid and blood pressure homeostasis. VACM-1 is recognized as Cul5 because of its homology to the Caenorhabditis elegans gene Cul5 [2,3]. Cul5 is expressed in many cells and organs, including endothelial cells, brain, kidney collecting tubule cells, and vascular endothelial cells [2,[4][5][6]7]. Cul5 inhibits cyclic AMP production, and this effect is reversed by staurosporin, a protein kinase A (PKA) inhibitor, or by mutating S730A, the PKA-dependent phosphorylation site in the Cul5 sequence in COS-1 cells [8]. The inhibitory effect of Cul5 on AVP-stimulated cAMP production is enhanced by a protein kinase C inhibitor [8]. CUL-5 expression is downregulated in 82 % (41/50) of breast tumors compared with matched normal tissues [9]. Overexpression of Cul5 in T47D breast cancer cells decreases cell growth and mitogen activated protein kinase (MAPK) phosphorylation [10], and Cul5 overexpression downregulates early growth response 1 (EGR-1) protein expression and upregulates Fas-L mRNA expression [10]. The regulation of both MAPK and EGR-1 pathways by 17β-estradiol led to the examination of estrogen-dependent T47D cell growth, which showed that Cul5 inhibits basal and 17β-estradiol-dependent cell growth and MAPK phosphorylation [11].Resveratrol (trans-3,5,4′-trihydroxystilbene), which inhibits tumor initiation and promotion, is a natural component of the human diet, and its wide range of biological activities has been demonstrated in vivo and in vitro [12][13][14][15]. The antiproliferative effect of resveratrol is significantly enhanced by Cul5 overexpression in T47D cells [16].The expression of Cul5 is regulated by several stimuli and pathways (Fig. 1). Resveratrol upregulates Cul5 expression and decreases T47D cell growth, suggesting that the antiproliferative effect of resveratrol is mediated by Cul5 [16]. Cul5 is a flexible scaffold protein with a preferred distribution of conformational states [17], and NEDD8 modification (neddylation) alters the conformation of Cul5 and activates it [18]. Cul5(S730A) accelerates cellular proliferation and induces an...

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“…Although not studied here, the possibility that resveratrol regulates VACM-1 protein modification and/or stability should also be considered. It is now accepted that in order to activate the E3 ligases all cullins must be modified by a ubiquitin like protein called Nedd8 (review in Saha and Deshaies, 2008) and we have shown that the antiproliferative effect of VACM-1/cul5 is clearly affected by such modification (Buchwalter et al 2008, Bradley et al, 2010. Unfortunately, the role of Nedd8 in VACM-1/cul5-dependent regulation of resveratrol activity was not studied here.…”

Section: Resultsmentioning

confidence: 82%

Resveratrol enhances anti-proliferative effect of VACM-1/cul5 in T47D cancer cells

et al. 2010

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Vasopressin-activated calcium-mobilizing (VACM-1) protein is a cul-5 gene product that forms complexes with a subclass of ubiquitin E3 ligases involved in proteasomal protein degradation. The expression of VACM-1 cDNA in the T47D breast cancer cell line inhibits growth and decreases phosphorylation of mitogen activated protein kinase. Factors that regulate expression or stability of VACM-1 protein have not been identified, however. In our search to identify drugs/substances that may control VACM-1 protein expression, we examined the effects of resveratrol (trans-3,5,4'-trihydroxystilbene), a natural component in the human diet which inhibits tumor initiation and promotion. CMV vector and VACM-1 cDNA stably transfected T47D breast cancer-derived cells were treated with resveratrol and cell growth and VACM-1 protein concentrations were measured. Since the cellular mechanism of resveratrol-dependent inhibition of cell growth also involves the regulation of estrogen receptors, the effect of 17-β-estradiol and resveratrol on ERα levels and on cell growth was examined in control and in VACM-1 cDNA transfected cells. Our results demonstrate that antiproliferative effect of resveratrol observed in the control T47D cancer cells was significantly enhanced in VACM-1 cDNA transfected T47D cells. Western blot results indicated that resveratrol increased VACM-1 protein concentration. Finally, treatment with resveratrol for 24 and 48 h attenuated 17-β-estradiol induced increase in cell growth both in control and in VACM-1 cDNA transfected cells. The effect was significantly higher in the VACM-1 cDNA transfected cells when compared to controls. These results indicate that the antiproliferative effect of resveratrol may involve induction of VACM-1/cul5.

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

Cited by 15 publications

References 46 publications

Cullin-5, a ubiquitin ligase scaffold protein, is significantly underexpressed in endometrial adenocarcinomas and is a target of miR-182

Cullin-5, a ubiquitin ligase scaffold protein, is significantly underexpressed in endometrial adenocarcinomas and is a target of miR-182

The role of cullin 5-containing ubiquitin ligases

Resveratrol enhances anti-proliferative effect of VACM-1/cul5 in T47D cancer cells

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