Anti-BCL10 monoclonal antibody, clone 5B9 [PE]

To determine the impact of B cell leukemia/lymphoma (BCL) 10 on the phosphorylation of crucial mediators in NF-kappa B-mediated inflammatory pathways, human colonic epithelial cells were exposed to carrageenan (CGN), a sulfated polysaccharide commonly used as a food additive and known to induce NF-kappa B nuclear translocation by both canonical and noncanonical pathways. Phosphorylations of intermediates in inflammatory cascades, including NF-kappa B-inducing kinase (NIK) at Thr(559), transforming growth factor-beta-activating kinase (TAK) 1 at Thr(184), Thr(187), and Ser(192), and inhibitory factor kappa B alpha (I kappa B alpha) at Ser(32), were examined following mutation of BCL10 at Ser(138) and at Ser(218). Specific phosphoantibodies were used for detection by enzyme-linked immunosorbent assay, immunoblot, and confocal microscopy of differences in phosphorylation following transfection by mutated BCL10. Both mutations demonstrated dominant-negative effects, with inhibition of phospho(Ser(32))-I kappa B alpha to less than control levels. Both of the BCL10 mutations reduced the CGN-induced increases in nuclear RelA and p50, but only the Ser(138) mutation inhibited the CGN-induced increases in nuclear RelB and p52 and in NIK Thr(559) phosphorylation. Hence, the phosphorylation of BCL10 Ser(138), but not Ser(218), emerged as a critical event in activation of the noncanonical pathway of NF-kappa B activation. Either BCL10 Ser(138) or Ser(218) mutation inhibited the phosphorylation of TAK1 at Thr(184) and at Thr(187), but not at Ser(192). These findings indicate that BCL10 phosphorylations act upstream of phosphorylations of NIK, TAK1, and I kappa B alpha and differentially affect the canonical and noncanonical pathways of NF-kappa B activation.

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-C zeta (PKC zeta) overexpression. Via proteomic, bioinformatic, and biochemical approaches we identified 31 and 33 proteins co-immunoprecipitating with PKC zeta 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, PKC zeta center dot Bcl10 complexes increasingly accrued at NMs, where PKC zeta phosphorylated Bcl10, as PKC zeta also did in vitro and in cell-free systems, both processes being thwarted by interfering RNA (iRNA) PKC zeta depletion. Caspase-3 was associated with PKC zeta center dot Bcl10 complexes and proteolyzed PKC zeta leading to its inactivation/destruction; both events were prevented by Bcl10 iRNA suppression. Thus, PKC zeta's molecular interactions and functional roles changed strikingly according to the untreated or apoptogen-treated cells context, and by complexing with Bcl10, PKC zeta surprisingly favored its own demise, which suggests both proteins as HCCs therapeutic targets.