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

Abstract

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. IntroductionThe disease with the second highest rate of mortality, for women, is human cervical carcinoma (HCC), which is prevalent in developing countries (1,2). The majority of HCCs derive from epithelial cells that have been persistently infected with high-risk oncogenic human papillomaviruses (HPVs), which express the E6 and E7 mRNAs, and proteins binding/neutralizing anti-oncogenic p53 and pRb. Furthermore, other co-factors, which have been less thoroughly studied, also promote HCC development (3,4). Notably, a small percentage (10%) of HCCs develops independently of a persistent high-risk oncogenic HPV infection (5). The complex molecular mechanisms responsible for the development of HCCs thus remain partially unclear, and this hinders the identification of predictive markers for the early detection of malignancy development and the discovery of novel, effective therapies.Proteomics and bioinformatics analysis have allowed researchers to identify differential protein expression patterns and the molecular mechanisms responsible for the development of HCC (6). A previous study identified a consensus of 66 proteins termed the 'central core of HCC protein expression̓ which was deduced from the proteomes of 6 HCC cell lines (not comprising the C4-I cell line); this differed from the proteomic profile...

Over Expressed TKTL1, CIP-2A, and B-MYB Proteins in Uterine Cervix Epithelium Scrapings as Potential Risk Predictive Biomarkers in HR-HPV-Infected LSIL/ASCUS Patients

“…Finally, to achieve total cell lysis samples were subjected to one freeze/thaw cycle at −20°C and ice-homogenized with an UltraTurrax™. The protein contents of the extracts were assayed according to Chiarini et al (39, 40) using bovine serum albumin (BSA) as standard.…”

“…WB was performed as detailed in Chiarini et al (39, 40). Equal amounts of protein lysates (15 μg for the analysis of TKTL1 and CIP-2a and 20 μg for B-MYB) were heat–denatured for 10-min at 70°C in an appropriate volume of 1X NuPAGE LDS Sample Buffer supplemented with 1X NuPAGE Reducing Agent (Life Technologies, Italia).…”

PDK1 regulates the survival of the developing cortical interneurons

“…In this study, we report that PDK1 is required for the survival of cortical interneurons but has no obvious effects on cell proliferation and tangential migration. Deletion of Pdk1 in the PDK1 serves as an antiapoptotic factor in a variety of cell types, such as cancer cells, endothelial cells, and skeletal muscle cells [28,61,62,[77][78][79][80][81]. Recently, it was reported that the ablation of Pdk1 in the dorsal telencephalon leads to the increased apoptosis of cortical excitatory neurons at postnatal rather than embryonic stages [30,31].…”

Vitamin D receptor activation influences the ERK pathway and protects against neurological deficits and neuronal death

“…Of the various subtypes of caspases, caspases-3 is the principal caspase actively causing neuronal cell death ( 23 ). Numerous studies have demonstrated that caspase-3 activity increases after cerebral ischemia/reperfusion injury ( 24 , 25 ). In the present study, caspase-3 was induced by GCI.…”