UC San Diego

It is well known that nearly half of all NF-κB subunits are inhibited by the precursor inhibitors, p100 and p105, whereas the other half are by classical IκBs (IκBα, IκBβ and IκBε). Our laboratory showed, through experiments done in vivo, that inhibition by these precursors requires their association with multiple NF-κB molecules including their processed products resulting in the formation large complexes of MW about 600 kDa to 800 kDa. These large complexes are known as kappaBsomes (κBsomes). p100-centric κBsomes are intriguing in that they must bind to RelB, a member of the NF-κB family. In contrast, p105-centric κBsomes do not interact with RelB. However, effort to form p100-centric κBsome by co-expressing p100, RelB and RelA were not successful. Although they associated and formed a complex, neither the size nor the stability matched in vivo results. This led to the identification of PRMT5 as a possible partner of p100-κBsome. To further study this association,I co-expressed p100, RelB, RelA, PRMT5 and MEP50 (partner of PRMT5) in the baculovirus expression system and tested the stability of their association through multiple chromatographic purification steps. I observed that the p100-κBsome and PRMT5:MEP50 complexes associate with each other, but this association is strong. We observed that the complex tends to disassemble as the purification progresses. This resulted in very little complex yield after the final step of purification. I conclude that the association between the two complexes is dynamic involving weak, transient interactions. To test the functional relevance of this possible association, I tested phosphorylation of p100 by NIK:IKK1 in the presence or absence of its NF-κB and PRMT5 partners. I found that the PRMT5:MEP50 complex partly inhibits p100 phosphorylation and this inhibition is enhanced in the presence of RelB. p100 is not phosphorylated in the five-protein complex (p100, RelA, RelB, PRMT5 and MEP50). These results suggest that the dynamic association of PRMT5:MEP50 complex is necessary to prevent aberrant phosphorylation of p100 in unstimulated cells.