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

Abstract

Many features of deadly human cervical cancers (HCCs) still require elucidation. Among HCC-derived cell lines, here we used the C4-I one since its quantitative gene expression pattern most closely mimics invasive HCCs, including protein kinase-Cζ (PKCζ) overexpression. Via proteomic, bioinformatic, and biochemical approaches we identified 31 and 33 proteins co-immunoprecipitating with PKCζ from nuclear membranes (NMs) of, respectively, untreated or VP-16-exposed C4-I cells. Such proteins belonged to eight functional groups, whose compositions and relative sizes changed with either context. Of the 56 proteins identified, only eight were shared between the two subproteomes, including Bcl10. Surprisingly, proteins known to associate with Bcl10, like Carma1/3 and Malt1, in so-called CBM signalosomes were absent. Notably, in VP-16-treated C4-I cells, PKCζ•Bcl10 complexes increasingly accrued at NMs, where PKCζ phosphorylated Bcl10, as PKCζ also did in vitro and in cell-free systems, both processes being thwarted by interfering RNA (iRNA) PKCζ depletion. Caspase-3 was associated with PKCζ•Bcl10 complexes and proteolyzed PKCζ leading to its inactivation/destruction; both events were prevented by Bcl10 iRNA suppression. Thus, PKCζ's molecular interactions and functional roles changed strikingly according to the untreated or apoptogen-treated cells context, and by complexing with Bcl10, PKCζ surprisingly favored its own demise, which suggests both proteins as HCCs therapeutic targets.

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

“…The detected interaction of PDK1 with Bcl10 was particularly noteworthy, as we had previously reported that increasing amounts of assembled PKCζ•Bcl10 complexes accrued at the NMs of VP16-treated C4-I cells (20,31). Therefore, in this study, we aimed to establish whether the specific knockdown of Bcl10 expression would hinder the assembly of PDK1•PKCζ complexes and, consequently, hinder the increase in PKCζ activity at the NMs of apoptotic C4-I cells.…”

“…Equal amounts (10-20 µg) of protein from each subcellular fraction were determined by means of Bio-rad Protein Assay (Bio-rad Laboratories, Milan, Italy) and processed for western blot analysis as previously described (15)(16)(17)(18)20 ]-PKCζ (sc-101778), anti-Bcl10 (331.3 sc-5273 mouse monoclonal or H-197 sc-5611 rabbit polyclonal), anti-PDK1 (A-10; sc-17766) and anti-Lamin B1 (C-20; sc-6216; this antibody was used to reveal equal loading of proteins) (all from Santa Cruz Biotechnology, Inc.). The blots were then incubated with alkaline phosphatase-conjugated anti-mouse, anti-rabbit or anti-goat IgGs (Santa Cruz Biotechnology, Inc.) and stained with BCIP/NBT liquid substrate reagent or CDP-Star (both from Sigma-Aldrich).…”

“…Of the proteins detected, only 8 proteins, including B-cell lymphoma 10 (Bcl10) were belonged to both subproteomes. Therefore, given the highly dynamic complexity of the NM-linked interactors/substrates, the molecular interactions and functional roles of PKCζ changed remarkably, in keeping with the untreated or apoptogen-treated cellular contexts (20). Moreover, we unexpectedly found that at the NMs of VP-16-treated C4-I cells, PKCζ formed complexes with and phosphorylated the Bcl10 protein, which prompted the caspase-3-mediated pro-apoptotic inactivation of PKCζ (20).…”

“…It has been reported that the enforced overexpression of wt Bcl10 protein weakly induces apoptosis and activates NF-κB in cells; yet, the expression of a Bcl10 mutant protein, in which the CARD domain is truncated, does not trigger apoptosis but activates 28,29). Evidence to date suggests that the Bcl10 protein is involved in the autoproteolytic activation of pro-caspase-9 (28), in dissociating caspases from their inhibitors, cellular inhibitor of apoptosis proteins (cIAPs) (30), and in the formation of pro-apoptotic complexes with PKCζ at the NMs of HCC C4-I cells (20,31).In our previous study, of the proteins that co-immunoprecipitated with PKCζ from the NMs, we identified the 3-phosphoinositide-dependent protein kinase-1 (PDK1) (20), a master regulator of the AGC family of PKs. PDK1 mediates various important cellular functions, and it phosphorylates and activates several AGC-family members involved in the modulation of cell growth, proliferation, survival and metabolism (32).…”

“…It has been reported that the enforced overexpression of wt Bcl10 protein weakly induces apoptosis and activates NF-κB in cells; yet, the expression of a Bcl10 mutant protein, in which the CARD domain is truncated, does not trigger apoptosis but activates 28,29). Evidence to date suggests that the Bcl10 protein is involved in the autoproteolytic activation of pro-caspase-9 (28), in dissociating caspases from their inhibitors, cellular inhibitor of apoptosis proteins (cIAPs) (30), and in the formation of pro-apoptotic complexes with PKCζ at the NMs of HCC C4-I cells (20,31).…”