G-protein-coupled receptor (GPCR) kinases, GRKs, are known as serine/threonine kinases that regulate GPCR signaling, but recent findings propose functions for these kinases besides receptor desensitization. Indeed, GRK5 can translocate to the nucleus by means of a nuclear localization sequence, suggesting that this kinase regulates transcription events in the nucleus. To evaluate the effect of GRK5-IB␣ interaction on NFB signaling, we induced the overexpression and the knockdown of GRK5 in cell cultures. GRK5 overexpression causes nuclear accumulation of IB␣, leading to the inhibition of NFB transcriptional activity. Opposite results are achieved by GRK5 knockdown through siRNA. A physical interaction between GRK5 and IB␣, rather than phosphorylative events, appears as the underlying mechanism. We identify the regulator of gene protein signaling homology domain of GRK5 (RH) and the N-terminal domain of IB␣ as the regions involved in such interaction. To confirm the biological relevance of this mechanism of regulation for NFB, we evaluated the effects of GRK5-RH on NFB-dependent phenotypes. In particular, GRK5-RH overexpression impairs apoptosis protection and cytokine production in vitro and inflammation and tissue regeneration in vivo. Our results reveal an unexpected role for GRK5 in the regulation of NFB transcription activity. Placing these findings in perspective, this mechanism may represent a therapeutic target for all those conditions involving excessive NFB activity.angiogenesis ͉ gene transcription ͉ inflammation ͉ signal transduction G -protein-coupled receptor (GPCR) kinases, GRKs, constitute a large family of serine/threonine protein kinases that regulate GPCR signaling (1-3), consisting of 7 isoforms that share structural and functional similarities (4). A central catalytic domain is flanked by an N-terminal domain that includes a region of homology to regulators of G-protein signaling (RH) and a C-terminal domain of variable length (5, 6). The catalytic domain of GRKs is relatively well-conserved among the members of different subfamilies (Ϸ45% sequence identity), whereas N-terminal RH domains display weak homology (Ϸ27%) and C termini have little or no sequence homology. GRKs have different tissue distribution, subcellular localization, and kinase activity regulation (7,8). GRKs mostly localize at the plasma membrane (2), but recently Johnson et al. (7) demonstrated that GRK4-6 (but not other GRKs) can shuttle between cytosol and nucleus through functional nuclear localization sequence (NLS) and nuclear exporting sequence (NES), thus suggesting a nuclear effect for the GRK4-6 subfamily.NFB is an ubiquitously expressed and highly regulated dimeric transcription factor (3) regulating the expression of genes responsible for innate and adaptive immunity, tissue regeneration, stress responses, apoptosis, cell proliferation, and differentiation (9-14). Within the canonical view of the regulation of the NFB activity, the inactive NFB/IB␣ complex localizes in the cytosol until an extracellular stimulus, ...