In-cell chemical crosslinking identifies hotspots for p62-IκBα interaction that underscore a critical role of p62 in limiting NF-κB activation through IκBα-stabilization

We have previously documented that in liver cells, the multifunctional protein scaffold p62/SQSTM1 is closely associated with I{kappa}B, an inhibitor of the transcriptional activator NF-{kappa}B. Such an intimate p62-I{kappa}B association we now document leads to a marked 18-fold proteolytic I{kappa}B-stabilization, enabling its nuclear entry and termination of the NF-{kappa}B-activation cycle. In p62-/--cells, such termination is abrogated resulting in the nuclear persistence and prolonged activation of NF-{kappa}B following inflammatory stimuli. Utilizing various approaches both classic (structural deletion, site-directed mutagenesis) as well as novel (in cell chemical crosslinking), coupled with proteomic analyses, we have defined the precise structural hotspots of p62-I{kappa}B association. Accordingly, we have identified such I{kappa}B hotspots to reside around N-terminal (K38, K47 and K67) and C-terminal (K238/C239) residues in its 5th ankyrin repeat domain. These sites interact with two hotspots in p62: One in its PB-1 subdomain around K13, and the other comprised of a positively charged patch (R183/R186/K187/K189) in the intervening region between its ZZ- and TB-subdomains. APEX proximity analyses upon I{kappa}B co-transfection of cells with and without p62 have enabled the characterization of the p62 influence on I{kappa}B-protein-protein interactions. Interestingly, consistent with p62s capacity to proteolytically stabilize I{kappa}B, its presence greatly impaired I{kappa}Bs interactions with various 20S/26S proteasomal subunits. Furthermore, consistent with p62-interaction with I{kappa}B on an interface opposite to that of its NF-{kappa}B-interacting interface, p62 failed to significantly affect I{kappa}B-NF-{kappa}B interactions. These collective findings together with the known dynamic p62 nucleocytoplasmic shuttling, leads us to speculate that it may be involved in "piggy-back" nuclear transport of I{kappa}B following its NF-{kappa}B-elicited transcriptional activation and de novo synthesis, required for the termination of the NF-{kappa}B-activation cycle. Consequently, mice carrying a liver specific deletion of p62-residues 68-252 harboring its positively charged patch, reveal age-dependent enhanced liver inflammation. Our findings reveal yet another mode of p62-mediated pathophysiologically relevant regulation of NF-{kappa}B. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/512146v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@a2cce1org.highwire.dtl.DTLVardef@38da91org.highwire.dtl.DTLVardef@95847aorg.highwire.dtl.DTLVardef@2c829b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIp62 binds to and stabilizes I{kappa}B by preventing its proteolytic degradation C_LIO_LIIn-cell chemical crosslinking/LC-MS/MS identified the inter-crosslinked sites C_LIO_LIHotspots of p62-I{kappa}B association are defined C_LIO_LIAPEX proximity labeling revealed p62 impaired I{kappa}B-interaction with proteasome C_LIO_LIp62 chaperones newly synthesized I{kappa}B to terminate NF-{kappa}B activation. C_LI In BriefThe transcriptional activator NF-{kappa}B inhibitor, I{kappa}B is proteolytically unstable when uncomplexed. How newly synthesized I{kappa}B escapes degradation to terminate nuclear NF-{kappa}B-activation is unknown. Using in-cell chemical crosslinking and proximity labeling MS analyses, we uncovered a novel association of p62 with I{kappa}B via well-defined structural hotspots, which impairs its interaction with the 26S/20S proteasome, extending its life-span and enabling termination of NF-{kappa}B-activation. Mice carrying liver-specific genetic deletion of p62-I{kappa}B hotspot exhibit enhanced liver inflammation upon aging, validating this novel p62 role.