Bcl10 crucially nucleates the pro-apoptotic complexes comprising PDK1, PKCζ and caspase-3 at the nuclear envelope of etoposide-treated human cervical carcinoma C4-I cells

Chiarini,Anna; Liu,Daisong; Armato,Ubaldo; Dal Prà,Ilaria

doi:10.3892/ijmm.2015.2290

Bcl10 crucially nucleates the pro-apoptotic complexes comprising PDK1, PKCζ and caspase-3 at the nuclear envelope of etoposide-treated human cervical carcinoma C4-I cells

Authors:
- Anna Chiarini
- Daisong Liu
- Ubaldo Armato
- Ilaria Dal Prà
View Affiliations

Affiliations: Histology and Embryology Section, Department of Life and Reproduction Sciences, University of Verona Medical School, I‑37134 Verona, Venetia, Italy, Chongqing Key Laboratory for Disease Proteomics, Institute of Burn Research, Southwest Hospital, Third Military Medical University, Chongqing 400038, P.R. China
Published online on: July 20, 2015 https://doi.org/10.3892/ijmm.2015.2290
Pages: 845-856

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Protein kinase (PK)Cζ signaling at various subcellular levels affects cell survival, differentiation, growth and/or apoptosis. However, the mechanisms modulating PKCζ activity at the nuclear membrane (NM) are not yet fully understood. Previously, we demonstrated that PKCζ interacts with the B‑cell lymphoma 10 (Bcl10) protein at the NM of human cervical carcinoma (HCC) C4‑I cells. In the present study, we aimed to further clarify the interactions between PKCζ, Bcl10 and other proteins co-immunoprecipitated from NMs isolated from untreated and etoposide (also known as VP‑16; 2.0 µg/ml)‑treated C4‑I cells using biochemical and proteomics analyses. Aside from the Bcl10 protein, 3‑phosphoinositide‑dependent protein kinase‑1 (PDK1) also co-immunoprecipitated with PKCζ from NMs of C4‑I cells, indicating the assembly of a heterotrimeric complex, which increased with time in VP‑16‑exposed cells, as did the activity of PDK1‑phosphorylated‑PKCζ. In turn, PKCζ‑phosphorylated‑Bcl10 straddled an enlarged complex which comprised caspase‑3. Subsequently, activity‑enhanced caspase‑3 cleaved and inactivated PKCζ. Finally, the suppression of Bcl10 using specific siRNA or lentiviral transduction prevented the increase in the PDK1•PKCζ association, the increase in the activity of PKCζ and caspase‑3, as well as the caspase‑3‑mediated PKCζ proteolysis and inactivation from occurring at the NMs of the VP‑16‑exposed C4‑I cells. Our observations provide evidence that Bcl10 acts as a pivotal pro-apoptotic protein which crucially nucleates complexes comprising PDK1, PKCζ and active caspase‑3 at the NMs of VP‑16‑exposed C4‑I cells. Hence, our data suggest that Bcl10 and PKCζ are potential therapeutic targets in the treatment of HCC.

View Figures

View References

1	Mukherjee S, Dey S, Bhattacharya RK and Roy M: Isothiocyanates sensitize the effect of chemotherapeutic drugs via modulation of protein kinase C and telomerase in cervical cancer cells. Mol Cell Biochem. 330:9–22. 2009. View Article : Google Scholar : PubMed/NCBI
2	Scarinci IC, Garcia FA, Kobetz E, Partridge EE, Brandt HM, Bell MC, Dignan M, Ma GX, Daye JL and Castle PE: Cervical cancer prevention: new tools and old barriers. Cancer. 116:2531–2542. 2010.PubMed/NCBI
3	Molano M, Van den Brule A, Plummer M, Weiderpass E, Posso H, Arslan A, Meijer CJ, Muñoz N and Franceschi S; HPV Study Group: Determinants of clearance of human papillomavirus infections in Colombian women with normal cytology: a population-based, 5-year follow-up study. Am J Epidemiol. 158:486–494. 2003. View Article : Google Scholar : PubMed/NCBI
4	Perez-Plasencia C, Duenas-Gonzalez A and Alatorre-Tavera B: Second hit in cervical carcinogenesis process: involvement of wnt/beta catenin pathway. Int Arch Med. 1:102008. View Article : Google Scholar : PubMed/NCBI
5	Kwasniewska A, Postawski K, Gozdzicka-Jozefiak A, Kwasniewski W, Grywalska E, Zdunek M and Korobowicz E: Estrogen and progesterone receptor expression in HPV-positive and HPV-negative cervical carcinomas. Oncol Rep. 26:153–160. 2011.PubMed/NCBI
6	Zhu X, Lv J, Yu L, Zhu X, Wu J, Zou S and Jiang S: Proteomic identification of differentially-expressed proteins in squamous cervical cancer. Gynecol Oncol. 112:248–256. 2009. View Article : Google Scholar
7	Higareda-Almaraz JC, Enríquez-Gasca MR, Hernández-Ortiz M, Resendis-Antonio O and Encarnación-Guevara S: Proteomic patterns of cervical cancer cell lines, a network perspective. BMC Syst Biol. 5:962011. View Article : Google Scholar : PubMed/NCBI
8	Carlson MW, Iyer VR and Marcotte EM: Quantitative gene expression assessment identifies appropriate cell line models for individual cervical cancer pathways. BMC Genomics. 8:117–129. 2007. View Article : Google Scholar : PubMed/NCBI
9	Dempsey EC, Newton AC, Mochly-Rosen D, Fields AP, Reyland ME, Insel PA and Messing RO: Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol. 279:L429–L438. 2000.PubMed/NCBI
10	Reyland ME: Protein kinase C isoforms: multi-functional regulators of cell life and death. Front Biosci (Landmark Ed). 14:2386–2399. 2009. View Article : Google Scholar
11	Breitkreutz D, Braiman-Wiksman L, Daum N, Denning MF and Tennenbaum T: Protein kinase C family: on the crossroads of cell signaling in skin and tumor epithelium. J Cancer Res Clin Oncol. 133:793–808. 2007. View Article : Google Scholar : PubMed/NCBI
12	Diaz-Meco MT, Dominguez I, Sanz L, Dent P, Lozano J, Municio MM, Berra E, Hay RT, Sturgill TW and Moscat J: zeta PKC induces phosphorylation and inactivation of I kappa B-alpha in vitro. EMBO J. 13:2842–2848. 1994.PubMed/NCBI
13	Filomenko R, Poirson-Bichat F, Billerey C, Belon JP, Garrido C, Solary E and Bettaieb A: Atypical protein kinase C zeta as a target for chemosensitization of tumor cells. Cancer Res. 62:1815–1821. 2002.PubMed/NCBI
14	Malhas AN and Vaux DJ: The nuclear envelope and its involvement in cellular stress responses. Biochem Soc Trans. 39:1795–1798. 2011. View Article : Google Scholar : PubMed/NCBI
15	Dal Pra I, Whitfield JF, Chiarini A and Armato U: Changes in nuclear protein kinase C-delta holoenzyme, its catalytic fragments, and its activity in polyomavirus-transformed pyF111 rat fibroblasts while proliferating and following exposure to apoptogenic topoisomerase-II inhibitors. Exp Cell Res. 249:147–160. 1999. View Article : Google Scholar : PubMed/NCBI
16	Chiarini A, Whitfield JF, Armato U and Dal Pra I: Protein kinase C-beta II Is an apoptotic lamin kinase in polyomavirus-transformed, etoposide-treated pyF111 rat fibroblasts. J Biol Chem. 277:18827–18839. 2002. View Article : Google Scholar : PubMed/NCBI
17	Chiarini A, Whitfield JF, Armato U and Dal Pra I: VP-16 (etoposide) and calphostin C trigger different nuclear but akin cytoplasmic patterns of changes in the distribution and activity of protein kinase C-betaI in polyomavirus-transformed pyF111 rat fibroblasts. Int J Mol Med. 17:111–120. 2006.
18	Chiarini A, Whitfield JF, Pacchiana R, Armato U and Dal Pra I: Photoexcited calphostin C selectively destroys nuclear lamin B1 in neoplastic human and rat cells - a novel mechanism of action of a photodynamic tumor therapy agent. Biochim Biophys Acta. 1783:1642–1653. 2008. View Article : Google Scholar : PubMed/NCBI
19	Watanabe Y, Hoshiai H, Nakanishi T, Kawamura N, Tanaka N, Isaka K, Kamiura S, Ohmichi M, Hatae M and Ochiai K: Evaluation of oral etoposide in combination with cisplatin for patients with recurrent cervical cancer: long-term follow-up results of a Japanese multicenter study. Anticancer Res. 31:3063–3067. 2011.PubMed/NCBI
20	Chiarini A, Marconi M, Pacchiana R, Dal Prà I, Wu J and Armato U: Role-shifting PKCζ fosters its own proapoptotic destruction by complexing with Bcl10 at the nuclear envelope of human cervical carcinoma cells: a proteomic and biochemical study. J Proteome Res. 11:3996–4012. 2012. View Article : Google Scholar : PubMed/NCBI
21	Willis TG, Jadayel DM, Du M-Q, Peng H, Perry AR, Abdul-Rauf M, Price H, Karran L, Majekodunmi O, Wlodarska I, et al: Bcl10 is involved in t(1;14)(p22;q32) of MALT B cell lymphoma and mutated in multiple tumor types. Cell. 96:35–45. 1999. View Article : Google Scholar : PubMed/NCBI
22	Chen M, Li LY and Qi Y-P: Bcl10 protein can act as a transcription activator in yeast. Mol Cell Biochem. 246:97–103. 2003. View Article : Google Scholar : PubMed/NCBI
23	Liu Y, Dong W, Chen L, Zhang P and Qi Y: Characterization of Bcl10 as a potential transcriptional activator that interacts with general transcription factor TFIIB. Biochem Biophys Res Commun. 320:1–6. 2004. View Article : Google Scholar : PubMed/NCBI
24	Thome M and Weil R: Post-translational modifications regulate distinct functions of CARMA1 and BCL10. Trends Immunol. 28:281–288. 2007. View Article : Google Scholar : PubMed/NCBI
25	Ruland J, Duncan GS, Elia A, del Barco Barrantes I, Nguyen L, Plyte S, Millar DG, Bouchard D, Wakeham A, Ohashi PS and Mak TW: Bcl10 is a positive regulator of antigen receptor-induced activation of NF-kappaB and neural tube closure. Cell. 104:33–42. 2001. View Article : Google Scholar : PubMed/NCBI
26	Zhang Q, Siebert R, Yan M, Hinzmann B, Cui X, Xue L, Rakestraw KM, Naeve CW, Beckmann G, Weisenburger DD, et al: Inactivating mutations and overexpression of BCL10, a caspase recruitment domain-containing gene, in MALT lymphoma with t(1;14)(p22;q32). Nat Genet. 22:63–68. 1999. View Article : Google Scholar : PubMed/NCBI
27	Lambers AR, Gumbs C, Ali S, Marks JR, Iglehart JD, Berchuck A and Futreal PA: Bcl10 is not a target for frequent mutation in human carcinomas. Br J Cancer. 80:1575–1576. 1999. View Article : Google Scholar : PubMed/NCBI
28	Yan M, Lee J, Schilbach S, Goddard A and Dixit V: mE10, a novel caspase recruitment domain-containing proapoptotic molecule. J Biol Chem. 274:10287–10292. 1999. View Article : Google Scholar : PubMed/NCBI
29	Koseki T, Inohara N, Chen S, Carrio R, Merino J, Hottiger MO, Nabel GJ and Núñez G: CIPER, a novel NF kappaB-activating protein containing a caspase recruitment domain with homology to Herpesvirus-2 protein E10. J Biol Chem. 274:9955–9961. 1999. View Article : Google Scholar : PubMed/NCBI
30	Yui D, Yoneda T, Oono K, Katayama T, Imaizumi K and Tohyama M: Interchangeable binding of Bcl10 to TRAF2 and cIAPs regulates apoptosis signaling. Oncogene. 20:4317–4323. 2001. View Article : Google Scholar : PubMed/NCBI
31	Pacchiana R, Abbate M, Armato U, Dal Prà I and Chiarini A: Combining immunofluorescence with in situ proximity ligation assay: a novel imaging approach to monitor protein-protein interactions in relation to subcellular localization. Histochem Cell Biol. 142:593–600. 2014. View Article : Google Scholar : PubMed/NCBI
32	Bayascas JR: Dissecting the role of the 3-phosphoinosi-tide-dependent protein kinase-1 (PDK1) signalling pathways. Cell Cycle. 7:2978–2982. 2008. View Article : Google Scholar : PubMed/NCBI
33	Casamayor A, Morrice NA and Alessi DR: Phosphorylation of Ser-241 is essential for the activity of 3-phosphoin-ositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo. Biochem J. 342:287–292. 1999. View Article : Google Scholar
34	Kikani CK, Dong LQ and Liu F: 'New̓-clear functions of PDK1: beyond a master kinase in the cytosol? J Cell Biochem. 96:1157–1162. 2005. View Article : Google Scholar : PubMed/NCBI
35	Sephton CF, Zhang D, Lehmann TM, Pennington PR, Scheid MP and Mousseau DD: The nuclear localization of 3′-phosphoinositide-dependent kinase-1 is dependent on its association with the protein tyrosine phosphatase SHP-1. Cell Signal. 21:1634–1644. 2009. View Article : Google Scholar : PubMed/NCBI
36	Lee KY, D'Acquisto F, Hayden MS, Shim JH and Ghosh S: PDK1 nucleates T cell receptor-induced signaling complex for NF-kappaB activation. Science. 308:114–118. 2005. View Article : Google Scholar : PubMed/NCBI
37	Hodgkinson CP and Sale GJ: Regulation of both PDK1 and the phosphorylation of PKC-zeta and -delta by a C-terminal PRK2 fragment. Biochemistry. 41:561–569. 2002. View Article : Google Scholar : PubMed/NCBI
38	Yu LR, Lv JQ, Jin LY, Ding SD, Ma XY, Wang JJ and Zhu XQ: Over-expression of protein kinase C isoforms (α, δ, θ and ζ) in squamous cervical cancer. Neoplasma. 58:491–498. 2011.
39	Hirai T and Chida K: Protein kinase Czeta (PKCzeta): Activation mechanisms and cellular functions. J Biochem. 133:1–7. 2003. View Article : Google Scholar : PubMed/NCBI
40	Xin M, Gao F, May WS, Flagg T and Deng X: Protein kinase Czeta abrogates the proapoptotic function of Bax through phosphorylation. J Biol Chem. 282:21268–21277. 2007. View Article : Google Scholar : PubMed/NCBI
41	Nazarenko I, Jenny M, Keil J, Gieseler C, Weisshaupt K, Sehouli J, Legewie S, Herbst L, Weichert W, Darb-Esfahani S, et al: Atypical protein kinase C zeta exhibits a proapoptotic function in ovarian cancer. Mol Cancer Res. 8:919–934. 2010. View Article : Google Scholar : PubMed/NCBI
42	Yoneda T, Imaizumi K, Maeda M, Yui D, Manabe T, Katayama T, Sato N, Gomi F, Morihara T, Mori Y, et al: Regulatory mechanisms of TRAF2-mediated signal transduction by Bcl10, a MALT lymphoma-associated protein. J Biol Chem. 275:11114–11120. 2000. View Article : Google Scholar
43	Rosebeck S, Rehman AO, Lucas PC and McAllister-Lucas LM: From MALT lymphoma to the CBM signalosome: three decades of discovery. Cell Cycle. 10:2485–2496. 2011. View Article : Google Scholar : PubMed/NCBI
44	Yeh PY, Kuo S-H, Yeh K-H, Chuang SE, Hsu C-H, Chang WC, Lin H-I, Gao M and Cheng A-L: A pathway for tumor necrosis factor-alpha-induced Bcl10 nuclear translocation. Bcl10 is up-regulated by NF-kappaB and phosphorylated by Akt1 and then complexes with Bcl3 to enter the nucleus. J Biol Chem. 281:167–175. 2006. View Article : Google Scholar
45	Kuo SH, Chou CH, Cheng AL, Wang CW, Chen YH and Chen RJ: Expression of BCL10 in cervical cancer has a role in the regulation of cell growth through the activation of NF-κB-dependent cyclin D1 signaling. Gynecol Oncol. 126:245–251. 2012. View Article : Google Scholar : PubMed/NCBI
46	Parrish AB, Freel CD and Kornbluth S: Cellular mechanisms controlling caspase activation and function. Cold Spring Harb Perspect Biol. 5:a0086722013. View Article : Google Scholar : PubMed/NCBI
47	Kurokawa M and Kornbluth S: Caspases and kinases in a death grip. Cell. 138:838–854. 2009. View Article : Google Scholar : PubMed/NCBI
48	Voss OH, Kim S, Wewers MD and Doseff AI: Regulation of monocyte apoptosis by the protein kinase Cdelta-dependent phosphorylation of caspase-3. J Biol Chem. 280:17371–17379. 2005. View Article : Google Scholar : PubMed/NCBI